Annex D. Toxicological Studies 

Table D-1. Cardiovascular effects. 

Study Pollutant Exposure Effects 

Reference: Anselme et 

al. (2007, 097084) 

Species: Rat 

Gender: Male 

Strain: Wistar Kyoto 

Age: Adult 

Weight: 200-225g 

DE: monocylinder Diesel engine using 

Euro 4 ELF 85A reference gasoline 

Particle Size: DE: 10-650 nm (85 nm 

mean mobility diameter) 

Route: Whole-body Inhalation 

Dose/Concentration: DE: 0.5 mg/m3; Other 

emissions measured: non-methane 

hydrocarbons (7.7 ppm), NO2 (1.1 ppm), CO 

(4.3 ppm) 

Time to Analysis: Experiments started 3 mo 

after L coronary artery ligation. ECG started at 

t0 and the DE exposure at t30 min for a 3-h 

period; ventricular premature beats (VPBs) and 

RMSSD calculated every 30 min during clean 

room air exhaust and PE periods. Early (t210-

300 min) and late (t480-540 min) PE were 

analyzed. 

Immediate decrease in RMSSD was observed in 

both healthy and CHF rats PE. Immediate 

increase in VPBs observed in CHF rats only; 

which lasted 4-5 h after exposure ceased. 

Whereas HRV progressively returned to baseline 

values within 2.5 h post-exposure (PE), the 

proarrhythmic effect persisted as late as 5 h PE 

termination in CHF rats 

Reference: Bagate et 

al. (2004, 055638) 

Species: Rat 

Gender: Male 

Strain: SH 

Age: 13-15 wk 

LPS and EHC-93 (PM): Urban Air 

collected at the Health Effects Institute 

Ottawa, Canada 

Particle Size: EHC-93: 0.8-0.4 μm 

(mean) (range: <3 μm) 

Route: IT Instillation 

Dose/Concentration: PM: 10 mg/kg; LPS- 350 

EU/animal 

Time to Analysis: Sacrificed 4 or 24 h postinstillation 

PM and LPS elicited a significant increase in 

receptor-dependent vasorelaxation of the aorta 

compared to saline-instilled rats. 

Reference: Bagate et 

al. (2004, 055638) 

Species: Rat 

Gender: Male 

Strain: SH 

Age: 13-15 wk 

EHC-93 (PM), CB-V or CB-Fe, LPS 

Particle Size: EHC-93: 0.8-0.4 μm 

(mean) (range: <3 μm) 

Route: Aortic Suspension Fluid 

Dose/Concentration: Cumulative 

concentrations of EHC-93, CB-V and CB-Fe 

(10, 25, 50, 75, 100 μg/mL) 

CB 1.5-2.0 nm (mean) (range <5 μm) 

Time to Analysis: Immediately post-exposure 

of aortic rings to cumulative concentrations of 

EHC-93, CB-V, CB-Fe and LPS. 

CB-V particles induced more relaxation than CBFe particles or EHC-93 in a dose-dependent 

manner. PM and LPS had an acute transient 

effect on the receptor dependent vasorelaxation. 

PM and LPS attenuated ACh-elicited 

vasocontraction in denuded aortic rings (DARs). 

 

Note: Hyperlinks to the reference citations throughout this document will take you to the NCEA HERO database (Health and 

Environmental Research Online) at http://epa.gov/hero. HERO is a database of scientific literature used by U.S. EPA in the process of 

developing science assessments such as the Integrated Science Assessments (ISA) and the Integrated Risk Information System (IRIS). 

December 2009 D-1 
Study Pollutant Exposure Effects 

Reference: Bagate et 

al. (2004, 055638) 

Species: Rat 

Gender: Male 

Strain: Wistar Kyoto 

Age: 13-15 wk 

EHC-93 (PM): Urban Air collected at 

the Health Effects Institute Ottawa, 

Canada. 

EHC-93 filtrate (PMF) 

Zn2+ and Cu2+ particles (10,000 and 

845 μg PM respectively) 

Particle Size: PM: 4.6 μm (GSD = 3.2)

Route: In Vitro 

Dose/Concentration: PM Suspensions: 10-100 

μg/mL; CuSO4/ZnSO4 1-100 μmol; Phe 2 μm; 

arbacol: 10 μm 

Time to Analysis: Measured immediately after 

maximum response for each cumulative dose 

was achieved. 

PM-Induced Contraction: No effect of 

suspension or filtrate seen on resting tension of 

aorta and SMRA. 

PM- and Metal-Induced Vasorelaxation:

Cumulative concentrations (10-100 μg/mL) of PM 

suspension and its water soluble components 

(PMF) elicited dose-dependent relaxation in 

aorta. Relaxation induced by particle suspension 

was higher than relaxation induced by free 

filtrate. The difference was significant at 100 

μg/mL. In SMRA, vasorelaxation similar to aorta’s 

was observed, and the activity of the particle suspension was stronger than the filtrate, with the 

difference being significant starting at 30 μg/mL. 

Both Zn2+ and Cu2+ in sulfate salts (10-100 μmol) 

induced relaxation in pre-contracted aortic rings, 

with Cu2+ having a greater effect than Zn2+ at the 

same concentration. Ions didn’t affect ACh 

relaxation. 

Effect of PM on α-Adrenergic Contraction:

Phenylephrine-induced dose-response 

contraction, starting at 1μM with max at 100 

μmol. Pre-treatment of SMRA did not change the 

phenylephrine-induced contraction. 

Reference: Bagate et 

al. (2006, 097608) 

Species: Rat 

Gender: Male 

Strain: Wistar Kyoto 

and SH 

Age: 13-15 wk 

EHC-93 (PM) 

EHC-93 (Filtrate) 

Cu2+ and Zn2+ solutions 

Particle Size: PM: 4.6 μm (GSD = 3.2)

Route: In Vitro 

Dose/Concentration: PM and PMF 

Suspensions: 10-100 μg/mL; CuSO4 or 

ZnSO4:10-100 μmol; Phenylephrine: 2 μm; 

Carbacol: 10 μm 

Time to Analysis: Responses evaluated at 

maximum of each dose-response. 

PM and its soluble components elicited 

endothelium-independent vasodilation in rat aorta 

rings. This response is a result of the activation of 

sGC since its inhibition by NS2028 practically 

eliminated relaxation. PM suspensions stimulated 

cGMP production in purified isolated sGC. 

Neither receptor nor their signaling pathways 

played a significant role in the direct relaxation by 

PM or metals. Vasodilation responses were 

significantly higher in SH than WKY control rats. 

Reference: Bagate et 

al. (2006, 096157) 

Species: Rat 

Gender: Male 

Strain: SH/NHsd 

Age: 11-12 wk 

Weight: 250-350 g 

EHC-93 (PM): Urban Air collected at 

the Health Effects Institute Ottawa, 

Canada. 

EHC-93 (Filtrate), 

Zinc (in PM), LPS 

Particle Size: PM: 4.6 μm (GSD = 3.2)

Route: IT Instillation 

Dose/Concentration: PM: 10 mg/kg; LPS: 350 

EU/animal (0.5 mL) 

Time to Analysis: 4 h post-exposure 

Effect of Pretreatment on Baseline 

Parameters of Isolated Perfused Heart: After 

PM exposure a slight increase of baseline coronary flow (CF) and heart rate (HR) was noted. In 

contrast, a significant decrease of left developing 

ventricular pressure (LDVP) was observed in SH. 

LPS also elicited a non-significant decrease in 

LVDP. 

Effect of Pretreatment and Ischemia on 

Cardiac Function: When SH rats were 

pretreated with PM or LPS the isolated heart had 

a reduced ability to recover to baseline levels 

after occlusion, in comparison with saline treated 

rats. After occlusion was released CF went back 

to baseline values. Saline and LPS treated rats, 

showed a gradual decrease in CF noted during 

the reperfusion period. Isolated hearts from PMexposed SH showed a complete restoration of 

CF and no gradual decrease. The increase of 

Zn2+ elicited a rapid decrease of LDVP and HR. 

The impairment of cardiac function measured by 

LDVP and HR started immediately upon Zn2+ 

infusion and remained the same during the 

perfusion period (no Zn2+ was present in the 

perfusate). 

Reference: Bagate et 

al. (2006, 096157)

Species: Rat 

Strain: H9c2 (EACC), 

cardiomyocyte cells 

EHC-93 (PM) Filtrate: Urban Air collected at the Health Effects Institute 

Ottawa, Canada, 

ZnSO4

Particle Size: PM: 4.6 μm 

(GSD = 3.2); Carbon Particles: 44 nm 

Route: In Vitro 

Dose/Concentration: PM: 1, 50, 100 μg/mL; 

ZnSO4: 50 μmol 

Time to Analysis: 30 min incubation 

Effect of EHC-93 filtrate on Ca2+ Uptake in 

Cardiomyocytes: Both PMF and Zn2+ inhibited 

ATP or ionophore-stimulated Ca2+ influx in 

cardiomyocytes. 

December 2009 D-2 
Study Pollutant Exposure Effects 

Reference: Bartoli et 

al. (2009, 156256) 

Species: Dog 

Gender: Female 

Strain: Mixed breed 

Age: 2-12 yr 

Weight: 

Average: 15.7 kg, 

Range: 13.6-18.2 kg

CAPs (Boston; Harvard Ambient 

Particle Concentrator) 

Particle Size: Diameter: 0.15-2.5 μm 

Route: Permanent Tracheostomy 

Dose/Concentration: Concentration range and 

mean: CAPs: 94.1-1557(358.1 ± 306.7) μg/m3, 

BC: 1.3-32(7.5 ± 6.1) μg/m3

, Particle count: 

3000-69300(18230 ± 13.151) particles/cm3

 

Time to Analysis: Preanesthetized. 

Tracheostomy. 5 h exposures separated by 

minimum 1wk. Prazosin administered in 8 of 13 

dogs 30-60 min before exposure. 55 exposure 

days.

CAPs significantly increased SBP, DBP, mean 

arterial pressure, HR and rate-pressure product. 

Prazosin (α-adrenergic antagonist) decreased 

these CAPs-induced effects. CAPs mass, BC, 

particle number concentrations were positively 

and significantly associated with each of the 

cardiovascular parameters except for pulse 

pressure.

Reference: Bartoli et 

al. (2009, 179904) 

Species: Dog 

Gender: Female 

Strain: Mixed breed 

Age: Adult 

Weight: 14-18 kg

CAPs (Boston; Harvard Ambient 

Particle Concentration) 

Particle Size: Diameter: ≤2.5 μm 

Route: Permanent Tracheostomy 

Dose/Concentration: Concentration range and 

mean: CAPs: 94.1-1556.8 (349 ± 282.6) μg/m3

, 

BC: 1.3-32 (7.5 ± 5.6) μg/m3, Particle number: 

3000-69300 (20381 ± 13075) particles/cm3

Time to Analysis: Tracheostomy. Minimum 3 

wk recovery. Acclimatized. Exposed 5 h. 2 5 

min occlusions of LAD coronary artery 

separated by 20 min rest. Exposure days 

separated by 1wk minimum.

During coronary artery occlusion, CAPs exposure 

reduced myocardial blood flow and increased 

coronary vascular resistance, SBP and DBP. 

CAPs effects were greater in ischemic tissue 

than nonischemic. Increases in CAPs mass, 

particle number and BC concentrations were 

significantly associated with decreased 

myocardial blood flow and increased coronary 

vascular resistance.

Reference: Campen et 

al. (2005, 083977) 

Species: Mouse 

Gender: Male 

Strain: C57BL/6J and 

Apo E-/-

Age: 10-12 wk 

High Whole DE (HWDE); Low Whole 

DE (LWDE); High PM Filtered (HPMF); 

Low PM Filtered (LPMF) 

Particle Size: NR 

Route: Whole-body Inhalation and Ex-vivo 

Exposures (isolated, pressurized septal 

coronary arteries) 

Dose/Concentration: HWDE: PM = 3.6 mg/m3; 

NOX = 102 ppm 

LWDE: PM = 0.512 mg/m3

; NOX = 19 ppm; 

PM = 0.770 mg/m3

; NOX = 105 ppm 

LPMF: PM = 0.006 mg/m3

; NOX = 26 ppm 

Time to Analysis: Whole-body Exposures: DE 

or PFDE for 6 h/day for 3 days, euthanized at 

the end of last exposure. 

Coronary Vessels Exposure: PSS bubbled 

with DE to expose coronary vessels to the 

soluble contents of DE. Analysis occurred 

immediately post exposure. 

Whole-body Exposure on ApoE-/-: During DE 

exposure, ApoE-/- mice HR consistently 

decreased during high concentration exposures, 

compared to the C57BL/6J strain. 

Coronary Vascular Effects on ApoE-/-: DE had 

no significant effects on the resting myogenic 

tone of isolated septal coronary arteries. Control 

coronary arteries showed constrictive responses 

to ET-1 and dilatory responses to SNP. DE 

exposed PSS vessels responses to ET-1 

enhanced compared to control. SNP-induced 

dilation blunted in vessels resting in dieselexposed saline. 

Reference: Campen et 

al.(2003, 055626) 

Species: Rat 

Gender: Male and 

Female 

Strain: SH 

Age: 4 mo 

DE: generated by either of two 

Cummins (2000 model) 5.9-L ISB turbo 

engines fueled by Number 2 Diesel 

Certification Fuel. 

Particle Size: 0.1-0.2 μm aerodynamic 

diameter 

Route: Whole-body exposure 

Dose/Concentration: 0, 30, 100, 300, 1000 

μg/m3

Time to Analysis: 6 h/day for 7 days; ECG 

measurements taken 4 days post-exposure. 

HR: Significantly higher in exposed animals and 

not concentration-dependent. More substantial 

results seen in male rats. 

ECG: The PQ interval was significantly prolonged 

among exposed animals in a concentrationdependent manner. 

Reference: Campen et 

al. (2006, 096879) 

Species: Mouse 

Gender: Male 

Strain: ApoE-/-

Age: 10 wk 

Road dust from paved surfaces (Reno, 

NV) 

Gasoline engine emissions, containing 

PM, NOX, CO and HC 

Particle Size: Road dust: 1.6 μm 

(Standard Deviation 2.0) 

Gasoline engine emissions: Average 

particle diameter of 15 nm 

Route: Whole-body inhalation 

Dose/Concentration: Road dust: 0.5 and 3.5 

mg/m3

Gasoline engine emissions: 5 to 60 μg/m3

 (at 

dilutions of 10:1, 15:1, and 90:1) 

Mean concentrations of PM: 61 μg/m3

; NOX: 

18.8 ppm; CO: 80 ppm. 

Time to Analysis: 6 h/days for 3 days. 

Sacrificed 18 h post-exposure. 

ET-1: Gasoline exhaust significantly upregulated 

ET-1 in a dose-dependent manner. ET-1 increased levels in the PM filtered group and 

decreased in the low levels of road dust. 

ECG: HR consistently decreased from beginning 

to end of exposure in all groups. No significant 

HR effects on road dust or gasoline exposure 

was observed. No significant effects on P-wave, 

PQ-interval, QRS-interval, or QT-interval were 

observed in either treatment. 

T-wave: Mice exposed to whole gasoline exhaust 

displayed significant increases in T-wave 

morphology from the beginning of exposures; this 

effect was consistent on all exposure days. 

December 2009 D-3 
Study Pollutant Exposure Effects 

Reference: Cascio et 

al. (1987, 007583) 

Species: Mouse 

Gender: Male 

Strain: ICR 

Age: 6-10 wk 

UFPM: Ultra fine PM, EPA Chapel Hill, 

NC 

Particle Size: <0.1 μm 

Route: IT Instillation 

Dose/Concentration: 100 μg in 100 μl 

Time to Analysis: 24 h post-exposure (single 

exposure) 

UFPM exposure double the size of myocardial infarction attendant to an episode of ischemia and 

reperfusion while increasing post ischemic 

oxidant stress. UFPM alters endotheliumdependent/independent regulation of systemic 

vascular tone; increases platelet number, plasma 

fibrinogen, and soluble P-selectin levels; reduces 

bleeding time. 

Reference: Chang et 

al. (2007, 155720) 

Species: Rat 

Gender: Male 

Strain: SH 

Age: 60 days 

UfCB: Ultra fine carbon black Ferric 

sulfate Fe2(SO4)3

Nickel sulfate NiSO4

Particle Size: UfCB 

Route: IT Instillation 

Dose/Concentration: UfCB: 415 and 830 μg 

Ferric Sulfate: 105 and 210 μg 

Nickel Sulfate: 263 and 526 μg 

Combined UfCB and ferric sulfate: 830 μg UfCB 

+ 105 μg ferric sulfate 

Combined UfCB with Nickel Sulfate: 830 μg 

UfCB + 263 μg Nickel Sulfate 

Time to Analysis: Single dose, radiotelemetry 

readings recorded for 72 h post- exposure. 

Both high/low-dose UfCB decreased ANN 

(normal-to-normal intervals) slightly around the 

30th h, concurrent increases of LnSDNN. 

LnRMSSD returned to baseline levels after small 

initial increases. Minor effects observed after lowdose Fe and Ni instillation; biphasic changes 

occurred after high-dose instillations. Combined 

exposures of UfCB and either Fe or Ni resulted in 

HRV trends different from values estimated from 

individual-component effects. 

Reference: Chang et 

al. (2007, 155720) 

Species: Rat 

Gender: Male 

Strain: SH 

Age: 10 wk 

CAPs: collected during a dust storm 

from Chung-Li, Taipei 

Particle Size: PM2.5

Route: Nose-only Inhalation 

Dose/Concentration: 315.55 μg/m3

Time to Analysis: 6 h 

A linear mixed-effects model revealed sigmoid 

increases in HR and a sigmoid decrease of QAI 

during exposure, after an initial incubation period.

Reference: Chang et 

al. (2004, 055637) 

Species: Rat 

Gender: Male 

Strain: SH 

Age: 60 days 

CAPs collected in Chung-Li, Taipei 

(spring and summer periods) 

Particle Size: PM2.5

Route: Nose-only Inhalation 

Dose/Concentration: Spring exposure: 202.0 ± 

68.8 μg/m3

; Mean number concentration: 2.30 × 

105 particles/cm3 (range: 7.12 ×103 - 8.26 ×105) 

Summer exposure: 141.0 ± 54.9 μg/m3

; Mean 

number concentration: 2.78 ×105

 particles/cm3

(range: 7.76 ×103 - 8.87 × 105) 

Time to Analysis: 4 days of spring exposure 

and days of summer exposure for 5 h each 

exposure. Parameters measured throughout 

duration of exposures. 

During spring exposures, the maximum increase 

of heart rate (HR) and blood pressure (BP) were 

51.6 bpm and 8.5 mmHg respectively. The 

maximum decrease of QAI (measures cardiac 

contractility) noted at the same time was 1.6 ms. 

Similar pattern was observed during summer 

exposure, however., the responses were less 

prominent. 

Reference: Chang, et 

al. (2005, 097776) 

Species: Rat 

Gender: Male 

Strain: SH 

Weight: 200 g 

CAPs collected in Chung-Li, Taipei 

Particle Size: PM2.5 (0.1-2.5 μm) 

Route: Nose-only Inhalation 

Dose/Concentration: 202.0 ± 68.8 μg/m3

Time to Analysis: 5 h/days for 4 days 

During the inhalation stage, crude effects of both 

LnSDNN and LnRMSSD for exposure and 

control groups decreased from the baseline 

values. Immediately after the experiments, both 

LnSDNN and LNRMSSD decreased due to 

stresses produced by release from the exposure 

system, then returned to the baseline values. 

Reference: Chauhan 

et al. (2005, 155722) 

Tumor Cell Line: A549 

derived from alveolar 

type II epithelial cells 

SRM-1879 (SiO2) and SRM-154b 

(TiO2) from the NIST 

EHC-93 from Ontario, Canada 

(EHCsol, EHCinsol) 

Particle Size: EHC-93 median 

physical diameter: 0.4 μm; TiO2 and 

SiO2 particle size distribution: 0.3-0.6 

μm 

Route: Cell Culture 

Dose/Concentration: 0, 1, 4, and 8 mg EHC 

total equivalent per 5 mL 

Time to Analysis: Culture medium was 

removed from flasks and replaced w/ 5 mL of 

the particle suspension media. Plates were 

incubated for 24 h. After 24 h cell culture 

supernatants were collected and analyzed. 

The decreased expression of preproET-1 in A549 

cells suggests that epithelial cells may not be the 

source of higher pulmonary ET-1 spillover in the 

circulation measured in vivo in response to 

inhaled urban particles. However, higher levels 

ECE-1 in A549 post-exposure to particles suggests an increased ability to process bigET-1 into 

mature ET-1 peptide, while increased receptor 

expression implies responsiveness. The 

increased release of Il-8 and VEGF by epithelial 

cells in response to particles could possibly up 

regulate ET-1 production in the adjacent pulmonary capillary endothelial cells, with concomitant 

increased ET-1 spillover in the systemic 

circulation. 

December 2009 D-4 
Study Pollutant Exposure Effects 

Reference: Chen et al. 

(2005, 087218) 

Species: Mouse 

Strain: C57 and ApoE-/-

CAPs (NYU, NY) 

Particle Size: PM2.5 

Route: Whole-body Inhalation 

Dose/Concentration: 19.7 μg/m3 average 

concentration over 5 mo (daily average exposure concentration was 110 μg/m3

) 

Time to Analysis: 6 h/days, 5 days/wk, for 5 

mo. Parameters measured continuously 

throughout. 

Significant decreasing patterns of HR, body 

temperature, and physical activity for ApoE-/-

mice, with nonsignificant changes for C57 mice. 

SDNN and RMSSD in the late afternoon and 

overnight for ApoE-/- mice showed a gradual 

increase for the first 6 wk, a decline for about 12 

more weeks, and a slight turn upward at the end 

of the study period. For C57 mice, there were no 

chronic effect changes in SDNN or RNSSD in the 

late afternoon, and a slight increase after 6 wk for 

the overnight period. 

Reference: Chen and 

Nadziejkov(2005, 

087219)(Chen and 

Nadziejko, 2005, 

087219) 

Species: Mouse 

Strain: C57, ApoE-/-

Age: 26-28 wk (C57), 

39-41 wk (ApoE-/-), and 

18-20 wk (LDLr-/- [DK]) 

CAPs (NYU, NY) 

Particle Size: PM2.5

Route: Whole-body Inhalation 

Dose/Concentration: Mean exposure 

concentration: 110 μg/m3

Time to Analysis: 6 h/days, 5 days/wk for up to 

5mo. Sacrificed 3-6 days after last exposure. 

All DK mice developed extensive lesions in the 

aortic sinus regions. In male DK mice, the lesion 

areas appeared to be enhanced by CAPs 

exposure. Plaque cellularity was increased, but 

there were no CAPs-associated changes in the 

lipid content. ApoE-/- and DK mice showed 

prominent areas of severe atherosclerosis. 

Quantitative measurements showed that CAPs 

increased the percentage of aortic intimal surface 

covered by grossly discernible atherosclerotic 

lesion. 

Reference: Corey LM 

et al. (2006, 156366)

(2006, 156366)(Corey 

et al., 2006, 156366) 

Species: Mouse 

Gender: Male 

Strain: ApoE-/-

Age: 11-12 mo 

Weight: 32.84 g (avg) 

PM collected November - March 

between 1996-1999 (Seattle, WA) 

Silica (U.S. Silica Company, Berkeley 

Springs, WV) 

Particle Size: PM2.5

Route: Nasal Instillation 

Dose/Concentration: PM: 1.5 mg/kg; Saline: 

50 ul; Silica: Min-u-Sil 5 in 50 μl saline 

Time to Analysis: Mice monitored for 1 day 

baseline prior to and for 4 days following 

exposure. 

After an initial increase in both HR and activity in 

all groups, there was delayed bradycardia with no 

change in activity of the animals in the PM and 

silica exposed groups. In addition, with PM and 

silica exposure, there was a decrease in HRV 

parameters. 

Reference: Cozzi et al. 

(2006, 091380) 

Species: Mouse 

Strain: ICR 

Age: 6-10 wk 

Ultrafine PM (collected continuously 

over 7-day periods in Oct 2002 in 

Chapel Hill, NC) 

Particle Size: <150 nm 

Route: IT Instillation 

Dose/Concentration: 100 μg of PM in vehicle 

Time to Analysis: 24 h post-exposure 

Ischemia-Reperfusion: PM exposure doubled 

the relative size of myocardial infarction compared with the vehicle control. No difference was 

observed in the percentage of the vehicle at the 

risk of ischemia. PM exposure increased the 

level of oxidative stress in the myocardium after IR. The density of neutrophils in the reperfused 

myocardium was increased by PM exposure, but 

differences in the numbers of blood leukocytes, 

expression of adhesion molecules on circulating 

neutrophils, and activation state of circulating 

neutrophils, 24 h after PM exposure, could not be 

correlated to the increase I-R injury observed. 

Isolated Aortas: Aortas isolated from PMexposed animals exhibited a reduced 

endothelium-dependent relaxation response to 

ACh. 

Reference: Dvonch JT 

et al. (2004, 055741) 

Species: Rat 

Gender: Male 

Strain: Brown Norway 

CAPs, Detroit, MI 

Particle Size: PM2.5

Route: Whole-body Inhalation 

Dose/Concentration: Average concentration 

354 μg/m3

Time to Analysis: 8 h/days for 3 consecutive 

days; plasma samples collected 24 h postexposure. 

Plasma concentrations of asymmetric 

dimethylarginine (ADMA) were significantly 

elevated in rats exposed to CAPs versus filtered 

air. 

December 2009 D-5 
Study Pollutant Exposure Effects 

Reference: Elder et al. 

(2004, 055642) 

Species: Rat 

Gender: Male 

Strain: Fischer 344 

and SH 

Age: 23 mo (Fischer); 

11-14 mo (SH) 

Weight: NR 

UFP - Ultrafine carbon particles; 

LPS (Sigma) 

Particle Size: UFP: 36 nm (median 

size) 

Route: Whole-body inhalation; intraperitoneal 

injection (ip) for saline and LPS 

Dose/Concentration: Particles: 150 μg/m3; 

LPS: 2 mg/kg bw

Time to Analysis: Single 6-h exposure to 

particles. Sacrificed 24 h after ip LPS exposure. 

BAL Fluid Cells: Neither inhaled UFP nor ip LPS 

cause a significant increase in BAL fluid total 

cells or the percentage of neutrophils in either rat 

strain. No significant exposure-related alteration 

in total protein concentration or the activities of 

LDH and b-glucuronidase. 

Peripheral Blood: In both rat strains ip LPS 

induced significant increase in the number and 

percentage of circulating PMNs. When combined 

with inhaled UFP, PMNs decreased, significantly 

for F-344 rats. Plasma fibrinogen increased with 

ip LPS in both rat strains with the magnitude of 

change greater in SH rats. UFP alone decreased 

plasma fibrinogen in SH rats. Combined UFP and 

LPS response was blunted, but significantly 

higher than controls. Hematocrit was not altered 

in either rat strain by any treatment. 

TAT Complexes: With all exposure groups 

averaged, plasma TAT complexes in SH rats 

were 6.5 times higher than in F-344 rats. LPS 

caused an overall increase in TAT complexes for 

F-344 rats that was further augmented by inhaled 

UFP. UFP alone decreased response. In SH rats, 

UFP alone significant increased response and 

LPS decreased response. 

ROS in BAL Cells: In F-344 rats both UFP and 

LPS have independent and significant effects on 

DCFD oxidation. Effects were in opposite 

directions; particles decreased ROS, LPS 

increased ROS. 

Reference: Finnerty et 

al. (2007, 156434) 

Species: Mouse 

Gender: Male 

Strain: C57BL/6 

Age: 9 wk 

Weight: 22-2 7g 

Coal Fly Ash (U.S. EPA), Analysis: 

(PM2.5 samples) low unburned carbon 

(0.53 wt%), moderate levels of 

transition metals, including (in μg/g): 

Fe (30, 400), Mg (31, 200), Ti (6, 180 ), 

Mn (907), and V (108). 

Particle Size: 1.8 and 2.5 μm 

Route: IT Instillation 

Dose/Concentration: PM: 200 μg; 200 μg 

PM+10 μg LPS; 200 μg PM+100 μg LPS 

Time to Analysis: 18 h after IT instillation 

Plasma: TNF-α significantly increased in both 

PM+LPS10 and PM+LPS100 treatments. For 

plasma IL-6, all groups tended to rise with a 

significant increase in the PM+LPS100 group. 

Reference: Floyd et al. 

(2009, 190350) 

Species: Mouse 

Gender: Male 

Strain: ApoE-/-

Age: 20 wk 

CAPs: PM2.5 concentrated from 

Tuxedo, NY (April-Sept 2003) 

Particle Size: NR 

Route: Whole-body Inhalation 

Dose/Concentration: Avg 120 μg/m3

(n=6/group) 

Time to Analysis: 6 h/days × 5 days/wk × 5 mo

Gene Expression: Microarry gene expression 

identified 395 genes downregulated and 216 

genes upregulated in the aortic plaques. 

Ontologic analysis identified a list of functional 

processes associated with gene expression and 

included: inflammation, tissue development, 

cellular movement, cellular growth and 

proliferation, hematological system development 

and function, lipid metabolism, cardiovascular 

system function, cellular assembly and 

organization, and cell death. 

Reference: Folkmann 

et al. (2007, 097344) 

Species: Mouse 

Gender: Female 

Strain: Wild type and 

ApoE-/-

Age: 11-13 wk 

Weight: 21 g (avg) 

DEP: SRM2975 (particulate fraction of 

exhaust from a filtering system 

designed for diesel-powered forklifts). 

Particle Size: DEP: NR 

Route: Intraperitoneal Iinjection 

Dose/Concentration: 0, 50, 500, 5,000 μg 

DEP/kg 

Time to Analysis: 6 or 24 h post-ip injection 

The expression of inducible nitric oxide synthase 

(iNOS) mRNA was increased in the liver 6 h postip injection. The level of oxidized purine bases, 

determined by formamidopyrimidine DNA 

glycosylase sites increased significantly in the 

liver after 24 h in mice injected w/ 50μg/kg. There 

was no indication of systemic inflammation 

determined as the serum concentration of nitric 

oxide and iNOS expression, and DNA damage 

was not increased in the aorta. 

December 2009 D-6 
Study Pollutant Exposure Effects 

Reference: Furuyama 

et al. (2006, 097056) 

Species: Rat 

Cell Type: Heart Micro 

vessel Endothelial 

(RHMVE) Cells 

OE-DEP, OE-UFP (from Urawa, 

Saitama, Japan) 

OE = Organic Extracts 

Particle Size: NR 

Route: Cell Culture 

Dose/Concentration: 0, 5, 10, 25 μg/mL of 

OE-DEP or OE-UFP 

Time to Analysis: Exposed for 0, 6, 12, 24, or 

36 h 

The cell monolayer exposed to 10 μg/mL OEUFP produced a larger amount of HO-1 than 

cells exposed to 10 μg/mL OE-UFP. OE-DEP and 

OE-UFP exposure reduced PAI-1 production by 

the cells but did not affect the production of 

thrombomodulin, tissue-type PA, or urokinasetype PA. Increased PAI-1 synthesis in response 

to treatment with 1 ng/mL TNF-α or 0.5ng/mL 

TGF-β1 was reduced by OE-DEP exposure. 

Suppression of PAI-1 production by OE-DEP 

exposure was mediated through oxidative stress 

and was independent of HO-1 activity. 

Reference: GerlofsNijland et al. (2009, 

190353) 

Species: Rat 

Gender: Male 

Strain: SH 

Age: 12 wk 

Weight: 200-300 g

PM (Prague, Czech Republic; 

Duisburg, Germany; Barcelona, Spain) 

(Prague and Barcelona coarse PM 

organic extracts) 

Particle Size: Coarse: 2.5-10 μm, 

Fine: 0.2-2.5 μm 

Route: IT Instillation 

Dose/Concentration: 7 mg /kg 

Time to Analysis: DTPA added to some PM 

samples preinstillation. Instilled with PM. 

Necropsy 24 h post-exposure. 

Inflammation (LDH, protein, albumin), cytotoxicity 

(NAG, MPO, TNF-α), and fibrinogen were 

increased by PM, and were greatest in the 

coarse PM fraction. Metal-rich PM had greater 

inflammatory and cytotoxic effects, and increased 

fibrinogen and vWF and decreased ACE. PAH 

content influenced greater inflammation 

(including neutrophils), cytotoxicity, and 

fibrinogen. Generally, whole PM and coarse PM 

were more potent than organic extracts and fine 

PM, respectively. 

Reference: GerlofsNijland et al. (2007, 

097840) 

Species: Rat 

Gender: Male 

Strain: SH/NHsd 

Age: 13 wk 

Weight: 250-350 g

PM samples collected from: 

1. MOB high traffic density 

2. HIA high traffic density 

3. ROM high traffic density 

4. DOR moderate traffic density 

5 MGH low traffic density 

6 LYC low traffic density 

Particle Size: Coarse: 2.5-10 μm; 

Fine: 0.1-2.5 μm 

Route: IT Instillation 

Dose/Concentration: 3, 10 mg/kg 

Time to Analysis: 24 h

Hematology: Fibrinogen responses of SH rats 

increased significantly at the high dose of both 

fractions of all PM samples, except fine PM from 

DOR. 

Location-Related Differences: Coarse PM from 

LYC lowered fibrinogen values more than PM 

from location MOB, HIA, and MGH. Fine PM 

showed less differences among the various sites.

Reference: GerlofsNijland et al. (2005, 

088652) 

Species: Rat 

Gender: Male 

Strain: SH/NHsd 

Age: 11-12 wk 

Weight: 250-350 g

RTD: road tunnel dust (obtained from a 

Motorway tunnel in Hendrik-IdoAmbacht, Netherlands) 

EHC-93 

(Ottawa, Canada) 

Particle Size: Coarse: 2.5-10 μm; fine: 

0.1-2.5 μm 

Route: IT Instillation 

Dose/Concentration: 0.3, 1, 3, 10 mg/kg; 

EHC-93: 10 mg/kg

Time to Analysis: 4, 24, 48 h 

Hematology: No significant changes in plasma 

for bigET-1 or von Willebrand factor were 

observed. At the highest dose, fibrinogen levels 

significantly increased at 24 and 4 h for both PM 

types. 

Reference: Ghelfi et al. 

(2008, 156468) 

Species: Rat 

Strain: SD 

Age: Adult 

CAPs 

CPZ (Capsazepine) (Axxora LLC, San 

Diego, CA) 

Particle Size: PM2.5

Route: CAPs: Whole-body Inhalation; CPZ: IP 

Injection or Aerosol 

Dose/Concentration: CAPs: mean mass 

concentration: 218 ± 23 μg/m3

; CPZ: 10 mg/kg 

(ip), 500 μmol (aerosol) 

Time to Analysis: Experiment 1: CPZ ip or 20 

min aerosol pretreatment immediately prior to 

CAPs exposure. Single CAPs exposure for 5 h. 

Parameters measured immediately following 

exposure. 

Experiment 2: CPZ ip pretreatment prior to 

CAPs exposure. Exposed to CAPs for 5 h/day 

for 4 mo. Parameters measured throughout 

duration of experiment. 

CPZ (ip or aerosol) decreased CAPs-induced 

chemiluminescence (CL), lipid thiobarbituric acid 

reactive substances (TBARS), and edema in the 

heart, indicating that blocking TRP receptors, 

systemically or locally, decreases heart CL. CAPs 

exposure led to significant decreases in HR and 

in the length of QT, RT, Pdur and Tpe intervals. 

These changes were observed immediately upon 

exposure, and were maintained throughout the 

5-h period of CAPs inhalation. Changes in 

cardiac rhythm and ECG morphology were prevented by CPZ. 

December 2009 D-7 
Study Pollutant Exposure Effects 

Reference: Gilmour et 

al. (2004, 054175) 

Species: Rat 

Gender: Male 

Strain: Wistar 

ufCB (Printex 90 from Frankfurt, 

Germany) 

fCB (Huber 990 from UK) 

Particle Size: ufCB: 114 nm (MMAD); 

fCB: 268 nm (MMAD). 

Route: Whole-body Inhalation 

Dose/Concentration: ufCB: 1.66 mg/m3; fCB: 

1.40 mg/m3

Time to Analysis: Single exposure for 7 h. 

Sacrificed and samples taken at 0, 16, and 48 h 

post-exposure. 

Exposure to ultrafine, but not fine, CB particles 

was also associated with significant increases in 

the total number of blood leukocytes. Plasma 

fibrinogen factor VIII and vWF were unaffected by 

particle treatments as was plasma Trolox 

equivalent antioxidant status. 

Reference: Gilmour et 

al. (2005, 087410) 

Species: Human 

Cell Types: Primary 

Human Monocyte 

Derived Macrophages 

(MP); Human Umbilical 

Vein Endothelial Cells 

(HUVEC); A549 cells; 

16HBE 

PM10: (Carbon Black from Degussa 

Ltd, Frankfurt, Germany) 

Particle Size: PM10

Route: Cell Culture 

Dose/Concentration: PM10: 50 and 100 μg/mL 

Time to Analysis: 6 and 20 h 

The culture media from MPs and 16HBE cells but 

not A549 cells, exposed to PM10 had an 

enhanced ability to cause clotting. H2O2 also 

increased clotting activity. Apoptosis was 

significantly increased in MPs exposed to PM10

and LPS as shown by annexin V binding. TF 

gene expression was enhanced in MPs exposed 

to PM10 and HUVEC tissue factor. tPA gene and 

protein expression were inhibited. 

Reference: Gilmour et 

al. (2006, 156472) 

Species: Rat 

Gender: Male 

Strain: Wistar Kyoto 

Age: 12-14 wk 

Weight: 280-340 g 

Zinc Sulfate 

(ZnSO4 in saline solution) 

Particle Size: NR 

Route: IT Instillation 

Dose/Concentration: 131 μg/kg (2 μmol/kg) 

Time to Analysis: 1, 4, 24, 48 h 

Zinc levels in plasma and tissue: At 1-24 h 

post-exposure, zinc plasma levels increased to 

nearly 20% above baseline. 

mRNA expression: Cardiac tissues demonstrated similar temporal increases in expressions 

of TF, PAI-1 and thrombomodulin mRNA, 

following pulmonary instillation of Zn. 

Cardiac histopathology: Mild and focal acute, 

myocardial lesions developed in a few Zn exposed rats. No changes in fibrin deposition or 

troponin disappearance were observed. At 24 

and 48h PE to Zn, increases occurred in levels of 

systemic fibrinogen an the activated partial 

thromboplastin time. 

Reference: Gong et al. 

(2007, 091155) 

Species: Mouse 

Cell Type: Human 

Microvascular 

Endothelial Cells 

(HMEC) 

Strain: C57BL/6J 

Gender: Male 

Age: 2 mo 

Organic DEP extract: collected from 

exhaust in a 4JB1-type LD, 2.74 liter, 

4-cylinder Isuzu diesel engine 

(provided by Masaru Sagai, Tsukuba, 

Japan) 

ox-PAPC: (provided by Judith Berliner, 

UCLA, CA) 

In vivo validation: Ultrafine (ufp) and 

fine (fp) particulate matter 

Particle Size: DEP <1 μm (diameter)

Route: Cell culture; In vivo validation via 

Whole-body inhalation 

Dose/Concentration: ox-PAPC: 10, 20, and 40 

μg/mL; DEP: 5, 15, and 25 μg/mL; DEP (5 

μg/mL)+ox-PAPC: 10 or 20 μg/mL 

In Vivo Validation: Ufp: 3.2 4×105

/cm3; fp: 2.7 

×105

/cm3 

In vivo validation: Ufp: <0.18 μm; fp: <2.5 μm 

Time to Analysis: 4 h 

In vivo validation: Exposed to CAPs for 5 h/day, 

3 days/wk for 8 wk. Sacrificed 24 h after last 

CAPs exposure. 

Gene-expression profiling showed that both DEP 

extract and ox-PAPC co-regulated a large 

number of genes. Network analysis to identify coexpressed gene modules, led to the discovery of 

three modules that were highly enriched in genes 

that were differentially regulated by the stimuli. 

These modules were also enriched in 

synergistically co-regulated genes and pathways 

relevant to vascular inflammation. 

In vivo validation: Results were validated by 

demonstrating that hypercholesterolemic mice 

exposed to ambient ultrafine particles inhibited 

significant upregulation of the module genes in 

the liver. 

December 2009 D-8 
Study Pollutant Exposure Effects 

Reference: Goto et al. 

(2004, 088100) 

Species: Rabbit 

Gender: Female 

Strains: New Zealand 

White 

Age: NR 

Weight: 2.3 kg 

EHC-93 (Ottawa, ON,Canada) 

CC: Coilloidal Carbon (obtained from 

Hamburg, Germany) 

Particle Size: EHC-93: PM10; CC: <1 

μm 

Route: Intrabronchial Instillation 

Dose/Concentration: AMs incubated with 

EHC-93 or CC: 0.6 ml/kg 

EHC-93 alone: 1 mL (500 μg/ mL) 

CC alone: 1mL (1% CC) 

Time to Analysis: WBC counts measured 

4-168 h after BrdU injection. Sacrificed 7days 

post instillation. 

Lung Distribution of PM10: PM-containing AMs 

were distributed diffusely. PM-containing AMs 

were more prevalent in the PM exposed animals. 

There was no AM-containing particle difference 

between the CC-exposed and EHC-93-exposed 

groups. 

Monocyte Release from Bone Marrow: EHC 

exposure increased WBC and band cell counts 

from 12 h after instillation. Monocyte count was 

not affected. Labeled monocytes peaked more 

quickly after DEP exposure (12 vs 16 h for 

control). There was no observed change in BM 

monocyte pool. 

Cytokine Release: EHC stimulation increased 

the release of GM-CSF, IL-6, IL-1β, TNF-α, IL-8 

and MCP-1. No effect on m-CSF and MIP-1β. CC 

particles induced increases in IL-6 and TNF-α; 

other cytokine levels did not differ from control. 

Supernatant Instillation: AM+EHC increased 

circulating WBC and band cell counts. Circulating 

monocyte counts were unaffected. AM+EHC 

showed a major increase in fraction and amount 

of monocyte released as well as faster clearance 

when compared to control. The BM monocyte 

pool was similar in all groups. 

Monocyte Transit Time Through BM: Exposure 

to EHC, CC only shortened the transit time of 

monocytes as compared to controls. AM+EHC 

also shortened monocyte transit time whereas 

AM+CC had a nonsignificant effect. 

Reference: Gottipolu 

et al. (2009, 190360) 

Species: Rat 

Gender: Male 

Strain: Wistar Kyoto, 

SH 

Age: 14-16 wk 

Weight: NR

DE (30-kW (40hp) 4-cylinder indirect 

injection Deutz diesel engine) (O2- 

20%, CO- 1.3-4.8 ppm, NO- <2.5-5.9 

ppm, NO2- <0.25-1.2ppm, SO2

-

 0.2-0.3 

ppm, OC/EC- 0.3 ± 0.03) 

Particle Size: Number Median 

Diameter: Low- 83 ± 2 nm, High- 88. 2 

nm; Volume Median Diameter: Low207 ± 2 nm, High- 225 ± 2 nm

Route: Inhalation 

Dose/Concentration: Low: 507 ± 4 μg/m3; 

High: 2201 ± 14 μg/m3

Time to Analysis: Exposed 4 h/days, 5 

days/wk, 4 wk. Necropsied 1day post-exposure.

DE dose-dependently inhibited mitochondrial 

aconitase activity. DE caused 377 genes to be 

differentially expressed within WKY rats, most of 

which were downregulated, but none in SH rats. 

However, WKY rats had an expression pattern 

shift that mimicked baseline expression of SH 

rats without DE. These genes regulated 

compensatory response, matrix metabolism, 

mitochondrial function, and oxidative stress 

response. 

Reference: Graff et al. 

(2005, 087956) 

Species: Rat 

Cell Type: Ventricular 

Myocytes

Zn; V 

Particle Size: NR 

Route: Cell Culture 

Dose/Concentration: 0, 6.25, 12.5, 25, or 50 

μm 

Time to Analysis: Toxicity: 24 h post- exposure

Beat Rate: 0.5, 1, 2, 4, and 24 h PE 

PCR: 6 and 24 h PE 

Beat Rate: There were statistically significant 

reductions in spontaneous beat rate 4 and 24 h 

post-exposure (greater reductions were observed 

with Zn). 

Inflammation: Exposure to Zn or V (6.25-50 μm) 

for 6 h produced significant increases in IL-6, ILα, heat shock protein 70, and connexin 43 

(Cx43). 

Impulse Conduction: 24 h post-exposure, Zn 

induced significant changes in the gene expression of Kv4.2 and KvQLt, α-1 subunit of Ltype Ca channel, Cx43, IL-6, and IL-1α. V produced a greater effect on Cx43 and affected only 

KvLQT1. 

Reference: Gunnison 

and Chen (2005, 

087956) 

Species: Mouse 

Gender: Male 

Strain: F2 generation 

DK (ApoE-/-, LDLr-/-) 

Age: 18-20 wk 

CAPs (Tuxedo, NY) 

Copollutants measured: O3 and NO2. 

Particle Size: 389 ± 2 nm 

Route: Whole-body Inhalation 

Dose/Concentration: CAPs: 131 ± 99 μg/m3

(range 13-441 μg/m3

) 

O3: 10 ppb 

NO2: 4.4 ppb 

Time to Analysis: 6 h/days, 5 days/wk for 

approximately 4 mo. Tissue collection was 

performed 3-4 days after the last day of 

exposure. 

Gene Expression: In CAPs-exposed heart 

tissue, the expression of Limd1 and Rex3 were 

the most consistently affected genes among the 

exposed mice. Limd1 was down regulated by 

1.5-fold or greater from moderate baseline 

expression. Rex3 showed a relatively small 

increase in absolute expression. 

December 2009 D-9 
Study Pollutant Exposure Effects 

Reference: Gurgueira 

et al. (2002, 036535) 

Species: Rat 

Gender: Male 

Strain: SD 

Weight: 250-300 g 

CAPs; 

Carbon Black (CB from Fisher 

Scientific, Pittsburgh, PA): C (85.9 ± 

0.2%); O (13 ± 0.2%); S (1.17 ± 

0.02%) 

ROFA: obtained from an oil-fired power 

plant (Boston, MA) 

Particle Size: CAPs size range: 0.1-

2.5 μm; CB and ROFA (PM2.5) 

Route: Whole-body Inhalation 

Dose/Concentration: CAPs: average mass 

concentration: 300 ± 60 μg/m3; ROFA: 1.7 

mg/m3

; CB: 170 μg/m3

Time to Analysis: CAPs: 1, 3, and 5 h; ROFA: 

30 min; CB: 5 h 

Oxidative Stress: Rats breathing CAPs aerosols 

for 5 h showed significant oxidative stress, 

determined as in situ chemiluminescence (CL) in 

the lung, heart, but not in the liver. ROFA also 

triggered increases in oxidant levels but not 

particle-free air or CB. Increases in CL showed 

strong associations with the CAPs content of Fe, 

Al, Si and Ti in the heart. The oxidant stress 

imposed by 5 h exposure to CAPs was 

associated with slight, but significant increases in 

the lung and heart water content, with increased 

serum levels of lactate dehydrogenase, indicating 

mild damage to tissues. CAPs inhalation also led 

to tissue-specific increases in the activities of 

SOD and catalase. 

Reference: Gursinsky 

et al. (1976, 015607) 

Species: Rat 

Cell Type: Fibroblasts 

isolated from adult 

male Wistar rats hearts 

Fly ash (TAF98) 

Particle Size: NR 

Route: In Vitro 

Dose/Concentration: TAF98: 0, 1, 2 3, 10, 25, 

50, 100, 200 μg/mL 

Time to Analysis: 0, 5, 10, 30, 60, 120 min 

Brief treatment of fibroblasts with fly ash triggered 

the immediate formation of ROS. Using 

phosphospecific antibodies the activation of p38 

MAP kinase, p44/42 MAP kinase (ERK1/2) and 

p70S6 kinase. Prolonged incubation with fly ash 

increased the expression of collagen 1 and TGFβ1, but decreased mRNA levels of MMP9 and 

TNF-α. Cell proliferation was inhibited at high 

concentrations of fly ash. An increase in the level 

of advanced glycation end product modification 

of various cellular proteins was observed. 

Reference: Hansen et 

al. (2007, 090703) 

Species: Mouse 

Gender: Female 

Strain: ApoE-/- and 

C57BL/6J ApoE+/+

Age: 11-13 wk 

DEP: SRM-2975 (NIST) 

Particle Size: DEP: 215 nm 

(geometric mean diameter) 

Route: Intraperitoneal Injection 

Dose/Concentration: DEP: 0, 0.5 and 5 mg/kg; 

Aorta segments incubated with 0, 10 and 100 

μg DEP/mL 

Time to Analysis: Sacrificed 1 h after ip 

injection. 

Exposure to 0.5 mg/kg DEP caused a decrease 

in the endothelium-dependent Ach elicited 

vasorelaxation in ApoE-/- mice, whereas the response was enhanced in ApoE+/+ mice. No 

significant changes were observed after 

administration of 5 mg/kg DEP. K+

 or 

phenylephrine induced constriction was not affected. 

Reference: Hansen et 

al. (2007, 090703) 

Species: Mouse 

Gender: Female 

Strain: ApoE-/- and 

C57BL/6J ApoE+/+

Use: Aorta rings used 

for in-vitro studies 

DEP: SRM-2975 (NIST) 

Particle Size: DEP: 215 nm 

(geometric mean diameter) 

Route: Cell Culture 

Dose/Concentration: 0, 10 and 100 μg 

DEP/mL

Time to Analysis: Basal tone measured at 5 

different points throughout experiment.

Exposure to 100 μg DEP/mL enhanced AChinduced relaxation and attenuated 

phenylephrine-induced constriction. 

Vasodilatation induced by sodium nitroprusside 

was not affected by any DEP exposure.

Reference: Harder et 

al. (2005, 087371) 

Species: Rat 

Gender: Male 

Strain: Wistar Kyoto 

Age: 12-15 wk 

Carbon UFPs ((generated by Electric 

Spark Generator GFG 1000; Palas, 

Karlsruhe, Germany) 

Particle Size: 37.6 ± 0.7 nm (mean) 

Route: Whole-body Inhalation 

Dose/Concentration: 180 μg/m3

Time to Analysis: Days 1-3: baseline reading, 

Day 4: exposure to UFPs or filtered air for 4 or 

24 h then sacrificed immediately following 

exposure period OR 

Sacrificed following 1-3 days recovery period. 

Cardiovascular Performance: Mild but 

consistent increase in HR, which was associated 

with a significant decrease in HR variability 

during exposure (particle-induced alteration of 

cardiac autonomic balance, mediated by a 

pulmonary receptor activation). 

Lung Inflammation and Acute-Phase Response: BALF revealed significant but low-grade 

pulmonary inflammation. 

Effects on Blood: There was no evidence of an 

inflammation-mediated increase in blood 

coagulability; no changes in plasma fibrinogen or 

factor VIIa. 

Pulmonary and Cardiac Histopathology: Sporadic accumulation of particle-laden 

macrophages found in the alveolar region. No 

signs of cardiac inflammation or cardiomyopathy. 

mRNA Expression Levels: No significant 

changes in the lung or heart. 

December 2009 D-10 
Study Pollutant Exposure Effects 

Reference: Hirano et 

al. (2003, 097345) 

Species: Rat 

Cell Types: Heart 

Microvessel Endothelial 

Cells (RHMVE)

Organic Extracts of DEP (DEP) and 

Organic Extracts of Ultra Fine Particles 

(UFP). 

(Urawa City, Saitama, Japan) 

Particle Size: DEP and UFP: <2.0 μm 

Route: Cells Culture 

Dose/Concentration: NAC effects on viability: 

DEP: 25 μg/ml; UFP: 50 μg/ml 

mRNA levels for DEP and UFP: 0,1,3,10 μg/ml 

cell monolayer exposed to DEP and UFP: 

1,10,100 μg/ml 

Time to Analysis: mRNA levels measured after 

6 h incubation with DEP or UFP. Other 

parameters measured after 24 h. 

Cytotoxicity and Oxidative Stress: LC50 

values were 17 and 34 μg/mL for DEP and UFP 

respectively. The viability of DEP and UFP 

exposed cells was ameliorated by N-acetyl-Lcysteine (NAC). 

mRNA Levels: mRNA levels increased dosedependently with DEP and HO-1 mRNA showed 

the most marked response to DEP. mRNA levels 

of antioxidant enzymes and heat shock protein 

72 (HSP72) in DEP-exposed cells were higher 

than UFP exposed cells at the same 

concentration. The transcription levels of HO-1 

and HSP72 in DEP and UFP-exposed cells were 

also reduced by NAC. 

Reference: Hwang et 

al. (2005, 089454) 

Species: Mouse 

Strain: C57 and ApoE-/-

CAPs (Tuxedo, NY) 

Particle Size: 389 ± 2 nm 

Route: Whole-body Inhalation 

Dose/Concentration: CAPs Range: 5-627 

μg/m3

. Mean CAPs Concentration: 133μg/m3. 

Mean Concentrations of O3 and NO2 in CAPs: 

10 and 4.4 ppb respectively. 

Time to Analysis: 6 h/day, 5 days/wk for 5 mo. 

Long-term Analysis: Significant decreasing 

patterns of HR, body temperature, and physical 

activity in ApoE-/- mice. Nonsignificant changes 

for C57 mice. The chronic effect changes for 

ApoE-/- mice were maximal in the last three wk. 

Short-term Analysis: Dose-dependent relationship for HR variations in ApoE-/- mice. 

Heart Rate Fluctuation: HR fluctuations in 

ApoE-/- mice during the period of 3-6 h increased 

by 1.35 fold at the end of the exposure and 

during a 15 min period increases by 0.7 fold at 

the end of the exposure. 

Reference: Inoue et al. 

(2006, 190142) 

Species: Mouse 

Gender: Male 

Strain: ICR 

Age: 6- 7 wk 

DEP (obtained from a 4Jb1-type lightduty, 4-cylinder, 2.74-L Isuzu diesel 

engine) 

Washed DEP (carbonaceous nuclei of 

DEP after extraction) and DEP-OC 

(organic chemicals in DEP extracted 

with CH2Cl2); Washed DEP+LPS and 

DEP-OC+LPS 

Particle Size: PM2.5

Route: IT Instillation 

Dose/Concentration: Washed DEP: 4 mg/kg. 

DEP-OC: 4 mg/kg. LPS: 2.5 mg/kg. Washed 

DEP+LPS and DEP-OC+LPS: respective additions of LPS to each component priorexperimentation. 

Time to Analysis: Sacrificed 24 h post single 

dose instillation. 

Both DEP components exacerbated vascular 

permeability. The increased fibrinogen and Eselectin levels induced by LPS. This exacerbation 

was more prominent with washed DEP than with 

DEP-OC. Washed DEP+LPS significantly 

decreased protein C and antithrombin-III and 

elevated circulatory levels of IL-6, KC and LPs 

without significance. 

Reference: Inoue et al. 

(2006, 097815) 

Species: Mouse 

Gender: Male 

Strains: C3H/HeJ 

(TLR-4 point mutant) 

and C3H/HeN (Control) 

Age: 6 wk

DEP (derived from 4 cyl, 2.74l light 

duty diesel engine) 

Particle Size: NR 

Route: IT Instillation 

Dose/Concentration: 12 mg/kg 

Time to Analysis: 24 h

Hematology: DEP increased plasma fibrinogen 

in both strains but with a greater increase in the 

knockout mice than the wild type.

Reference: Ito et al. 

(2008, 096823) 

Species: Rat 

Gender: Male 

Strain: Wistar Kyoto 

(Specific pathogenfree) 

Age: 13-14 wk 

CAPs (f-PM), Yokohama City, Japan. 

Particle Size: 0.1-2.5 μm 

Route: Whole-body Inhalation 

Dose/Concentration: 0.6-1.5 mg/m3

Time to Analysis: Three groups exposed to: 

(1) filtered air for 4 days, (2) filtered air for 3 

days and CAPs for 1 day or (3) CAPs for 4 

days. All groups exposed for a maximum of 4.5 

h/days for 4 consecutive days. 

mRNA Expression and Cardiovascular 

Function: In samples of heart tissue, the mRNA 

of cytochrome P450 (CYP) 1B1, heme 

oxygenase-1 (HO-1), and endothelin A (ETA) 

receptor were up-regulated by CAPs; their levels 

were significantly correlated with the cumulative 

weight of CAPs in the exposure chamber. The 

up-regulation of ETA receptor mRNA was significantly correlated with the increase in HO-1 

mRNA and weakly with the increase in MBP. 

December 2009 D-11 
Study Pollutant Exposure Effects 

Reference: Khandoga 

A et al. (2004, 087928) 

Species: Mouse 

Gender: Female 

Strain: C57B1/6 

Age: 5-7 wk 

UFPs: Ultra fine carbon black particles 

(Printex 90) 

Particle Size: 14 nm diameter (60% 

<100 nm) 

Route: Aortic Infusion 

Dose/Concentration: 1×107 and 5 ×107 total 

particles infused 

300 m2

/g surface area 

Time to Analysis: Single exposure, analysis 

 2 h post-exposure 

Platelet Effects: Application of UFPs caused 

significantly enhanced platelet accumulation on 

endothelium of postsinusodal venules and 

sinusoids in healthy mice. UFP-induced platelet 

adhesion was not preceded by platelet rolling but 

was strongly associated with fibrin deposition and 

an increase in vWF expression on the endothelial 

surface. 

Inflammatory Effects: In contrast, inflammatory 

parameters such as the number of 

rolling/adherent leukocytes, P-selectin expression/translocation, and the number of apoptotic 

cells were not elevated. UFPs did not affect 

sinusoidal perfusion and Kupffer cell function. 

Reference: Knuckles 

et al. (2007, 156652) 

Species: Rat 

Gender: Female 

(Pregnant, purchased 

at GD19) 

Strain SD 

Age: 60-90 days 

Weight: 300 g 

Use: RMCs were 

harvest from 1 day-old 

neonatal pups 

ROFA-L: Leachate 

Particle Size: <0.2 μm 

Route: Cell Culture 

Dose/Concentration: 3.5 μg/mL 

Time to Analysis: 1 h 

ROFA-L Induced Alterations to the RCM 

Transcriptosome: 38 genes were suppressed 

and 44 genes were induced PE. Genomic 

alterations in pathways related to IGF-1, VEGF, 

IL-2, PI3/AKT, CVD, and free radical scavenging 

were detected. Global gene expression was 

altered in a manner consistent with cardiac myocyte electrophysiological remodeling, cellular 

oxidative stress and apoptosis. 

ROFA-L Induced Alterations to the RCM 

Transcription Factor Proteome: ROFA-L 

altered the transcription factor proteome by suppressing activity of 24 and activating 40 transcription factors out of 149. 

Reference: Knuckles 

et al. (2008, 191987) 

Species: Mouse 

Gender: Male 

Strain: C57BL/6 

Age: 8-10 wk 

Weight: NR

DE (single cylinder Yanmar diesel 

generator burning #2 certified diesel 

fuel (Chevron-Phillips, Borger, TX) 

under 100% load) 

Particle Size: PM2.5 

Route: Whole-body Inhalation. Ex Vivo. 

Dose/Concentration: In vivo: 350 μg/m3; Ex 

vivo: PM2.5 concentration 2-3 mg/m3

 flow rate 

500 mL/min 

Time to Analysis: Exposed 4 h. Ex vivo 

assays. 

Veins: DE increased vascular reactivity to ET-1. 

Ex vivo exposed vessels had greater 

vasoconstriction. L-NAME (an arginine blocker) 

did not promote constriction in DE-exposed rats 

but did so in controls. 

Arteries: DE did not significantly alter vascular 

reactivity. Carbonyls or alkanes alone or with DE 

did not alter vasoconstriction.

Reference: Kodavanti 

et al. (2008, 155907) 

Species: Rat 

Gender: Male 

Strain: Wistar Kyoto 

Age: 12-14 wk 

G1: saline (control); G2: Mount Saint 

Helen’s ash (SH); G3: whole suspension of oil combustion PM at high 

concentration (PM-HD); G4: whole 

suspension of oil combustion PM at 

low concentration (PM-LD); G5: salineleachable fraction of PM highconcentration suspension; G6: zinc 

sulfate 

Particle Size: PM2.5

Route: IT Instillation 

Dose/Concentration: Doses (mg/kg/wk) are for 

8 and 16 wk (PM-solid and soluble Zn) 

respectively. G1: 0.00-0.00 and 0.00-0.00; G2: 

4.60-0.00 and 2.30-0.00; G3: 4.60-66.8 and 

2.30-33.4; G4: 2.30-33.4 and 1.15-16.7; G5: 

0.00-66.8 and 0.00-33.4; G6: 0.00-66.8 and 

0.00-33.4 

Time to Analysis: 1 x/wk for 8 or 16 wk; 

analyzed 48 h after last instillation. 

DNA Damage (left ventricular tissue): All 

groups except MSH caused varying degrees of 

damage relative to control. Total cardiac 

aconitase activity was inhibited in rats receiving 

soluble Zn. Analysis of heart tissue revealed modest changes in mRNA for genes involved in signaling, ion channels function, oxidative stress, 

mitochondrial fatty acid metabolism, and cell 

cycle regulation in Zn, but not MSH-exposed rats.

Reference: Kooter et 

al. (2006, 097547) 

Species: Rat 

Gender: Male 

Strain: SH 

Age: 12-14 wk

CAP-F = fine (Site I) 

CAP-UF = fine + ultrafine (Site II) 

(Netherlands) 

Some measured components: Ammonium, nitrate, sulfate ions: 56 ± 16% 

CAP-F mass, 17 ± 6% CAP-UF mass 

Particle Size: 0.15<CAP-F<2.5 

0.65-0.75 μm 

CAP-UF<2.5 

0.58-1.41 μm 

Route: Nose-only Inhalation 

Dose/Concentration: CAP-F 399- 3613 μg/m3

CAP-UF 269-556 μg/m3

Time to Analysis: 6 h/days for 2 days 

consecutive, 18 h

Hematology: WBC and lymphocytes decreased 

with both CAP-F and CAP-UF. MPV and MPC 

(mean platelet volume and component) 

increased with CAP-UF. 

December 2009 D-12 
Study Pollutant Exposure Effects 

Reference: Kyoso et 

al. (2005, 186998) 

Species: Rat 

Gender: NR 

Strain: NR 

Age: 15 mo 

DE 

PM and NOX exposures 

Particle Size: NR 

Route: Whole-body Inhalation 

Dose/Concentration: PM (mg/m3): 0.01, 0.109, 

0.54, 1.09, 0.01 (from 1.09 concentration w/o 

PM) 

NOX (ppm): 0.19, 0.59, 2.60, 5.53, 5.47 (w/o 

PM) 

Time to Analysis: Exposed 16 h/days (from 

5pm-9am) for 7 mo 

All of the resting R-R intervals before exposure 

were lower at night than during the day, but few 

changes were found after exposure. 

Reference: Lei et al. 

(2004, 087884) 

Species: Rat 

Gender: Male 

Strain: SD 

Weight: 300-350 g

CAPs from Asian dust storm (Taiwan) 

Measured Components: Si, Al, S, Ca, 

K, Mg, Fe, As, Ni, W, V, OC, EC, SO2, 

NO2, nitrate, sulfate 

Particle Size: 0.01- 2.5 μm 

Route: Nose-only Inhalation 

Dose/Concentration: 315.6 μg/m3 (Low) 

or 684.5 μg/m3

 (High) 

Time to Analysis: Low: Exposed for 6 h. 

Sacrificed 36 h post-exposure 

High: Exposed for 4.5 h. Sacrificed 36 h postexposure 

Pulmonary hypertension induced 2 wk preexposure. 

Hematology: PM induced a dose-dependent 

increase in WBCs. No change was seen in 

RBCs. Platelet results were highly variable.

Reference: Lei et al. 

(2005, 088660)

Species: Rat 

Gender: Male 

Strain: SD 

Weight: 200-250 g 

Use: ip STZ (60 mg/kg) 

dissolved in citric acid 

buffer administered to 8 

rats to induce diabetes; 

ip citric acid buffer 

administered to 8 nondiabetic rats 

CAPs: Hsin-Chuang, Taipei 

Particle Size: PM: 0.01-2.5 μm 

Route: IT Instillation 

Dose/Concentration: PM2.5: 200 μg in 0.5 mL 

saline. Components (μg/m3

): (9.8-SD 2.4)), EC 

(3.6-SD 3.2), Sulfate (4.8-SD 1.2), Nitrate (6.3-

SD 3.4) 

Time to Analysis: Single dose. Animals 

sacrificed 24 h post instillation. 

Effects of Diabetes: Body weight (bw) of 

diabetic (D) rats (397.5 g) was lower than nondiabetic (ND) rats (483.1 g). Mean plasma 

glucose level was 163 mg/daysL in ND rats and 

448.2 mg/daysL in D rats. D rats had significant 

greater levels of 8-OHdG in plasma compared to 

ND rats. D rats had significantly increased levels 

of plasma [nitrate+nitrite]. No observable 

changes in TNF-α for D and ND rats. 

Effects of PM Exposure ND Rats: Increase in 

plasma levels of 8-OHdG and plasma IL-6, TNFα, and serum CRP. Significant reduction of 

plasma [nitrate+nitrite]. No significant effect on 

plasma ET-1. 

Effects of PM Exposure STZ-D Rats: 

Significant elevation of plasma ET-1. Decrease in 

plasma [nitrate+nitrite] Plasma 8-OHdG and 

TNF-α significantly increased. No significant 

alterations in IL-6 and CRP. 

Reference: Lemos et 

al. (2006, 088594) 

Species: Mouse 

Gender: NR 

Strain: BALB/c 

Age: 1day (neonatal) 

n: 10 

Weight: 4-6 g 

PM10, CO, NO2, and SO2 from 

Universidade de Sao Paulo, Brazil. 

Particle Size: PM10

Route: Whole-body Inhalation 

Dose/Concentration: Mean (± SD) 

concentrations were: CO2: 2.06 ± 0.08 ppm (8h 

mean); NO2: 104.75 ± 42.62 μg/m3

 (24 h mean); 

SO2: 11.07 ± 5.32 μg/m3

 (24 h mean); 

PM10: 35.52 ± 12.84 μg/m3

 (24 h mean) 

Time to Analysis: 24 h/days, 7 days/wk for 4 

mo 

Morphometric measurements of the ratio 

between the lumen and the wall (L/W) areas 

were performed on transverse sections of renal, 

pulmonary and coronary arteries. A significant 

decrease of L/W with exposure to air pollution 

was detected in pulmonary and coronary arteries, 

whereas no effects of air pollution were observed 

in renal vessels. 

Reference: Li et al. 

(2005, 088647) 

Species: Rat 

Strain: SD 

Tissues/Cell Types: 

Cultured HPAECs; 

Pulmonary Artery Rings 

(PARs) 

Urban Particles (UPs SRM 1648) 

Major Constituents (mass fraction in 

%): Al (3.4), Fe (3.9), K (1.1). 

Minor Constituents (mass fraction in 

%): Na (0.43), Pb (0.66), Zn (0.48). 

Trace Constituents (ng/mg): As (115), 

Cd (75), Cr (403), Cu (609), Mn (786), 

Ni (82), Se (27), U (5.5), V (127). 

Particle Size: NR 

Route: PARs: In vitro organ model HPAECs: 

grown to 80% confluence 

Dose/Concentration: PARs and HPAECs: 1 to 

100 μg/mL; Losartan treatment: 0.2 μmol 

Captopril treatment: 100 μmol 

Time to Analysis: PARs were exposed to 

increasing doses of UPs from 1 to 100 μg/mL. 

Maximum tension was recorded within 5 min 

after each UPs dose. HPAECs: exposed to UPs 

from 1 to 100 μg/mL for up to 2 min 

Effects of UPs on the constriction of isolated rat 

pulmonary PARs and the activation of 

extracellular signal-regulated kinases 1 and 2 

(ERK1/2) and p38 mitogen-activated protein 

kinases (MAPKs) in HPAECs with or without 

Losartan at 1-100 μg/mL induced acute 

vasoconstriction. UPs also produced a time- and 

dose-dependent increase in phosphorylation of 

ERK1/2 and p38 MAPK. Losartan pre-treatment 

inhibited both vasoconstriction and activation of 

ERK1/2 and p38. The water soluble fraction of 

UPs was sufficient for inducing ERK1/2 and p38 

phosphorylation, which was also inhibited by 

Losartan. Cu (CuSO4) and V (VOSO4), induced 

pulmonary vasoconstriction and phosphorylation 

of ERK1/2 and p38, but only phosphorylation of 

p38 was inhibited by Losartan. UPs induced 

activation of ERK1/2 and p38 was attenuated by 

Captopril. 

December 2009 D-13 
Study Pollutant Exposure Effects 

Reference: Li et al. 

(2006, 156693) 

Species: Rat, Rabbit, 

and Human 

Tissues/Cell Types: 

Pulmonary Artery Rings 

(PARs) (rat); isolated 

buffer-infused lungs 

(rabbits) and cultured 

HPAECs 

Strain: SD Rats, New 

Zealand White Rabbits 

Weight: Rat: 200-350 

g; Rabbit: 2.5-3.0 kg 

Urban Particles (UPs SRM 1648). 

Major Constituents (mass fraction in 

%): Al (3.4), Fe (3.9), K (1.1). 

Minor Constituents (mass fraction in 

%): Na (0.43), Pb (0.66), Zn (0.48). 

Trace Constituents (ng/mg): As (115), 

Cd (76), Cr (403), Cu (609), Mg (786), 

Ni (82), Se (27), U (5.5), V (127). 

Particle Size: NR 

Route: In Vitro 

Dose/Concentration: PARs and HPAECs: 1 to 

100 μg/mL 

Time to Analysis: PARs: treatment given 15 

min prior to exposure. Exposed to increasing 

doses of UPs from 1 to 100 μg/mL. Maximum 

tension was recorded within 5 min after each 

UPs dose. HPAECs: exposed to UPs from 1 to 

100 μg/mL for 20 and 120 min. 

Effects of UP on H202 Release: Within minutes 

after UPs treatment, HPAEC increased H2O2

production that could be inhibited by DPI, APO, 

and NaN3. The water soluble fraction of UPs as 

well as its two transition metal components Cu 

and V, also stimulated H2O2 production. NaN3

inhibited H2O2 production stimulated by Cu and 

V, whereas DPI and APO inhibited only Cu-stimulated H2O2 production. Inhibitors of other H2O2-

producing enzymes, including N-methyl-Larginine, indomethacin, allopurinol, cimetidine, 

rotenone, and antimycin, had no effects. 

Effects of UP-induced H202 on MAPK 

Activation: DPI but not NaN3 attenuated UPsinduced pulmonary vasoconstriction and 

phosphorylation of ERK1/2 and p38 MAPKs. 

Knockdown of p47phox gene expression by 

small interfering RNA attenuated UPs-induced 

H2O2 production and phosphorylation of ERK1/2 

and p38 MAPKs. 

Reference: Lippmann 

et al. (2005, 087453) 

Species: Mouse 

Strain: C57 and ApoE-/-

(March-September 2003). Chemical 

Composition: regional secondary 

sulfate (SS) characterized by high S, 

Si, and organic C; resuspended soil 

(RS) characterized by high 

concentrations of Ca, Fe, Al, and Si; 

RO-fired powered emissions of the 

Eastern U.S. identified by the presence 

of V, Ni, and Se; and motor vehicle 

(MV) traffic and other sources. 

Contributors to Average Mass: SS 

(56.1%), RS (11.7%), RO combustion 

(1.4%), MV traffic and other sources 

(30.9%) 

Particle Size: PM2.5

Route: Whole-body Inhalation 

Dose/Concentration: PM2.5 concentrated tenfold, producing an average of 113 μg/m3

Time to Analysis: 6 h/days, 5 days/wk for 5 

mo. Parameters measured daily: during 

exposure, the afternoon after exposure, and 

late at night 

Associations Between Sources and Shortterm Heart Rate Changes: There were no 

significant associations between SS, RS, RO, 

and MV factors and HR in C57 mice at any of the 

three intervals. There were significant associations between PM2.5 and the RS source factor 

and decreases in HR for the ApoE-/- mice during 

the daily CAPs exposures but no associations 

with the other factors. There was no residual 

association of HR with PM2.5 or the RS factor 

later in the afternoon or late at night. In the 

afternoon, there was a significant association 

between decreases in HR and the SS factor for 

the ApoE-/- mice that had not been present during 

exposure and did not persist into the night time 

period. MV traffic and others were not 

significantly associated with HR during any of 

these three time periods. For the C57 mice, there 

were no significant associations of HR with PM2.5

or any of its components during any of the three 

daily time periods. 

Associations Between Sources and Shortterm HRV Changes: Signal noise during 

exposures did not permit reliable analyses of 

HRV changes during the hours of CAP exposure.

Reference: Lippmann 

et al. (2005, 087453) 

Species: Mouse 

Gender: NR 

Strain: ApoE-/-, ApoE-/-

LDLr-/-, C57BL/6 

Age: NR 

Weight: NR

CAPs (Sterling Forest, spring-summer 

2003) 

Particle Size: PM2.5

Route: Inhalation 

Dose/Concentration: PM2.5 average 

concentration: 110 μg/m3

, Long-term average: 

19.7 μg/m3

 

Time to Analysis: Exposed 6 h/days, 5 

days/wk, 5 or 6 mo. Semicontinuous EKG 

recordings.

HR increased in ApoE-/- mice but not C57 mice. 

HRF increased over the duration of the 

experiment. Atherosclerotic plaque deposits and 

coronary artery disease lesions occurred in both 

CAPs-exposed mice and controls, but invasive 

lesions were only present in CAPs-exposed 

mice. A gene affecting circadian rhythm was 

upregulated in double knockout mice. CAPs 

activated NF-κB. No inflammation occurred in the 

pulmonary system. 

Reference: Lippman M 

et al. (2006, 091165) 

Species: Mouse 

Gender: Male 

Strain: ApoE-/-

Age: 6 wk 

CAPs from Tuxedo, NY. Component of 

interest: Ni. 

Particle Size: PM2.5

Route: Whole-body Inhalation 

Dose/Concentration: Average daily CAPs: 

85.6 μg/m3; Average daily Ni: 43 ng/m3

Time to Analysis: 6 h/day, 5 days/wk, for 6 mo 

(July 2004-January 2005). 10-s ECG, HR, 

activity, and body temperature data were 

sampled every 5 min for the duration of the 

experiment. 

For the CAPs-exposed mice, on 14 days there 

were Ni peaks at approximately 175 ng/m3

 and 

usually low CAPs and V. For those days backtrajectory analysis identified a remote Ni point 

source. ECG measurements on CAPs-exposed 

and sham-exposed mice showed Ni to be 

significantly associated with acute changes in HR 

and HRV. 

December 2009 D-14 
Study Pollutant Exposure Effects 

Reference: Lund et al. 

(2007, 125741) 

Species: Mouse 

Gender: Male 

Strain: ApoE-/-

Age: 10 wk 

Use: Mice were placed 

on a high fat at the 

beginning of the 

exposure. 

Varying dilutions of gasoline emissions: (generated using two 1996 

model 4.3L General Motors V-6 

engines, fueled with conventional, 

unleaded, non-oxygenated gasoline, 

equipped with stock exhaust systems). 

Composition for Hi, Med, and Lo 

dilutions: 

PM, NOX, CO, and Total Hydrocarbons 

(THC) 

Particle Size: NR 

Route: Whole-body Inhalation 

Dose/Concentration: FA: PM (2 μg/m3), NOX

(0 ppm), CO (0.1 ppm), HC (0.1 ppm); 

Low (1: 90 dilution of exhaust): PM (8 μg/m3

), 

NOX (2 ppm), CO (9 ppm), HC (0.9 ppm); 

Mid (1: 20): PM (39 μg/m3

), NOX (12 ppm), CO 

(50 ppm), HC (8.4 ppm); 

High (1: 12): PM (61 μg/m3

), NOX (19 ppm), CO 

(80 ppm), HC (12 ppm); 

High-filtered (1:12): PM (2 μg/m3

), NOX (18 

ppm), CO (80 ppm), HC (12.7 ppm). 

Time to Analysis: 6 h/day, 7 days/wk for 7 wk. 

Mice were sacrificed within 16 h PE. During the 

study period all animals concurrently exposed 

to the following: FA: 8 μg/m3

 and 40 μg/m3; PM 

Whole Exhaust: 60 μg/m3

; or Filtered Exhaust 

w/ gases matching the 60 μg/m3

 concentration. 

Inhalation exposure to gasoline engine emissions 

resulted in increased aortic mRNA expression of 

matrix metalloproteinase-3 (MMP-3), MMP-7, and 

MMP-9, tissue inhibitor of MMP-2, ET-1 and HO1 in ApoE-/- mice; increased aortic MMP-9 protein 

levels were confirmed through immunochemistry. 

Elevated ROS were also observed in arteries 

from exposed animals, despite absence of 

plasma markers. Similar findings were also 

observed in the aortas ApoE-/- mice exposed to 

particle filtered atmosphere, implicating the 

gaseous components of the whole exhaust in 

mediating the expression of markers associated 

with vasculopathy. 

Reference: Lund et al. 

(2007, 125741) 

Species: Mouse 

Gender: Male 

Strain: ApoE-/-

Age: 10 wk 

Weight: NR

GEE (conventional unleaded, 

nonoxygenated, nonreformulated 

gasoline- ChevronPhillips Specialty 

Fuels Division) 

Particle Size: 0.150 μm (MMAD) 

Route: Inhalation 

Dose/Concentration: PM: 60 μg/m3, NO2: 2 

ppm, NO: 16 ppm, CO: 80 ppm, THC: 12.7 ppm

Time to Analysis: Mice fed high-fat diet 30 

days before exposure. Exposed 6 h/day, 1 or 7 

days. Some groups dosed with Tempol or BQ123. Killed within 18 h of last exposure.

Aorta gelatinase activity increased with GEE 

exposure time. MMP-2/9 activity spread 

throughout the vasculature by day 7. 7 day GEE 

exposure significantly increased the aorta protein 

expression of MMP-9, MMP-2, TIMP-2, and 

plasma MMP-9. Generally, in GEE-exposed 

mice, Tempol decreased TBARS and vascular 

ET-1, and BQ-123 decreased vascular ROS, ET1, MMP-9, and gelatinase activity. 

Reference: McQueen 

et al. (2007, 096266) 

Species: Rat 

Gender: Male 

Strain: Wistar Kyoto 

Weight: 228-500 g

DEP: SRM 2975 (NIST) 

Particle Size: NR 

Route: IT Instillation 

Dose/Concentration: 0.5 mL/rat of 1 mg/mL; 

1-2.2 mg/kg

Time to Analysis: 6 h. 

Pre-exposure: Vagotomy (sectioning of vagus 

nerve) or atropine, 1mg/kg i.p. administered 30 

min prior, 2 and 4 h post.

Cardiovascular Response: Blood pressure and 

heart rate were unaffected. Average arterial O2

increased after DEP, but not when compared for 

each animal. CO2 and pH were not affected 

Reference: Medeiros 

et al. (2004, 096012) 

Species: Mouse 

Gender: Male 

Strain: BALB/c 

Age: 60 days 

Weight: 20-30 g

CP: Carbon particles 

PSA: ROFA (solid waste incinerator 

hospital Sao Paulo, Brazil) 

PSB: electric precipitator, steel plant, 

Brazil) 

PSA/PSB Characteristics: Generally, 

PSB had greater component 

concentrations than PSA: Br (100+x), 

Cr (3x), Fe (10+x), Mn (2x), Rb (60+x), 

Se (7x), Zn (4x). PMA>PMB: Ce (3x), 

Co (10+x), La (100x), Sb (15x), V 

(50x). 

Particle Size: CP: 1.7 ± 2.5 μm 

(78%<2.5 μm) 

PMA: 1.2 ± 2.2 μm(98 %<2.5 μm) 

PMB: 1.2 ± 2.2 μm (98%<2.5 μm) 

Reference: Intranasal Instillation 

Dose/Concentration: CP: 10 μg/mouse; 

0.5mg/kg 

PSA: 0.1, 1 or 10 μg/mouse; 0.005, 0.05, 0.5 

mg/kg 

PSB: 0.1, 1 or 10 μg/mouse; 0.005, 0.05, 0.5 

mg/kg

Time to Analysis: Single, 24 h 

Hematology: PSA and PSB decreased 

leukocyte count (all 3 doses) and platelet count 

(2 high doses). No effect on hemoglobin, 

erythrocytes and reticulocytes was observed. 

Fibrinogen levels increased for both PSB and 

PSA with PSB seeing a higher increase. None of 

the effects were dose-dependent. 

Bone Marrow: Erythroblasts increased for PSA 

at all dose levels and PSB at mid and high dose 

levels (high variability). 

December 2009 D-15 
Study Pollutant Exposure Effects 

Reference: MontielDavalos et al.(2007, 

156778) 

Species: Human 

Cell Types: HUVEC 

(from primary human 

endothelial cells) and 

U937 (human leukemia 

pro-monocytic) cell 

cultures. 

PM2.5 and PM10 from Mexico City 

Particle Size: PM2.5, PM10

Route: In Vitro 

Dose/Concentration: HUVEC TNF-α (10 

ng/mL), and a PM range of 5, 10, 20, and 40 

μg/cm2 concentrations. 

Time to Analysis: 6 or 24 h (early and late 

adhesion molecules respectively) 

Results showed that both PM2.5 and PM10

induced the adhesion of U937 cells to HUVEC, 

and their maximal effect was observed at 20 

μg/cm2

. This adhesion was associated with an increase in the expression of all adhesion 

molecules evaluated for PM10, and E-selectin, Pselectin, and ICAM-1 for PM2.5. In general the 

maximum expression of adhesion molecules induced by PM2.5 and PM10 was obtained with 20 

μg/cm2

; however PM10-induced expression was 

observed from 5 μg/cm2

. E-selectin and ICAM-1 

had the strongest expression in response to 

particles. 

Reference: Moyer et 

al. (2002, 052222) 

Species: Mouse 

Gender: Male and 

Female 

Strain: B6C3F1 

In phosphide (InP), Co sulfate heptahydrate (CoSO4 ·7H2O), Vanadium 

pentoxide(V2O5) Gallium arsenide 

(GaAs), Ni oxide (NiO), Ni subsulfide 

(Ni3S2), Ni sulfate hexahydrate 

(NiSO4 · 6H2O), talc, and Mo trioxide 

(MoO3) 

Particle Size: MMAD particle size 

(μm): InP (1.1-1.3), CoSO4 ·7H2O (1.5-

1.8), V2O5: (1.0), GaAs: (1.0) 

Route: Inhalation 

Dose/Concentration: High-Dose Concentration in Chronic Studies, Male (μg/m3

): 

InP: 0.3, CoSO4 ·7H2O: 3.0, V2O5: 4.0, GaAs: 

1.0 

High-Dose Concentration in Sub-Chronic 

Studies, Male or Female (μg/m3

): InP: 100, 

CoSO4 ·7H2O: 30, V2O5: 16, GaAs: 75 

Time to Analysis: Phase One: Evaluation of 

heart, kidney and lung tissues from all control 

and high dose male B6C3F1 mice exposed by 

inhalation to 9 particulate compounds for a 2yr 

period. Phase Two: evaluated heart, lung, 

kidney and mesentery tissues of control and 

high dose male and female B6C3F1 mice from 

the 90-day studies of the 4-compounds demonstrating arteritis after a 2-yr period. 

Phase One: High-dose males developed significantly increased incidences of arteritis over 

controls in 2 of the 9 studies (InP and CoSO4 

·7H2O), while marginal increases of arteritis were 

detected in 2 additional studies (V2O5 and GaAs). 

In contrast, arteritis of the muscular arteries of 

the lung was not observed. Morphological 

features of arteritis in these studies included an 

influx of mixed inflammatory cells including 

neutrophils, lymphocytes, and macrophages. 

Partial and complete effacement of the normal 

vascular wall architecture, often with the extension of the inflammatory process into the 

periarterial connective tissue, was observed. 

Phase Two: Results showed that arteritis did not 

develop in the 90-day studies, suggesting that 

long-term chronic exposure to lower-dose 

metallic PM may be necessary to induce or 

exacerbate arteritis. 

Reference: Mutlu et al. 

(2007, 121441) 

Species: Mouse 

Gender: Male 

Strain: 57BL/6 (IL-6+/+

and IL-6-/-) 

Age: 6-8 wk 

Weight: 20-25 g 

PM10 from ambient air in Düsseldorf, 

Germany 

Particle Size: PM10

Route: IT Instillation 

Dose/Concentration: PM10: 10 μg; Clodronate: 

120 mg 

Time to Analysis: For alveolar macrophage 

depletion, clodronate instilled into mice lungs 

following endotracheal intubation 48 h prior to 

instillation of PM. Parameters measured 24 h 

post-exposure. 

Mice treated with PM10 exhibited a shortened 

bleeding time, decreased prothrombin and partial 

thromboplastin times (decreased plasma clothing 

times), increased levels of fibrinogen, and 

increased activity of factors II, VIII, and X. This 

prothrombotic tendency was associated with 

increased generation of intravascular thrombin, 

an acceleration of arterial thrombosis, and an 

increase in BALF concentration of prothrombotic 

IL-6. IL-6-/- mice were protected against PMinduced intravascular thrombin formation and the 

acceleration of arterial thrombosis. Depletion of 

macrophages by the IT administration of 

liposomal clodronate attenuated PM-induced IL-6 

production and the resultant prothrombotic 

tendency. 

Reference: Nadziejko 

et al. (2002, 087460) 

Species: Rat 

Gender: Male 

Strain: Wistar Kyoto 

Age: 16 wk 

CAPs (PM2.5) from Tuxedo, NY. (SO2, 

NO2 O3 and NH3 were removed prior to 

exposure). 

H2SO4 (fine and ultrafine) 

Particle Size: Ultrafine H2SO4 50-75 

nm (MMAD) 

Route: Nose-only Inhalation 

Dose/Concentration: CAPs: 80 and 66 μg/m3

(avg 73); Fine H2SO4: 299, 280, 119, and 203 

μg/m3

 (avg 225); Ultrafine H2SO4: 140, 565, 

416, 750 μg/m3 (avg 468) 

Time to Analysis: 4 h/exposure 

Exposure to CAPs caused a striking decrease in 

respiratory rate that was apparent soon after the 

start of exposure and stopped when exposure to 

CAPs ceased. The decrease in respiratory rate 

was accompanied by a decrease in HR. 

Exposure of the same animals to fine-particlesize H2SO4 aerosol also caused a significant 

decrease in respiratory rate similar to the effect of 

CAPs. Ultrafine H2SO4 had the opposite effect on 

respiratory rate compared to CAPs. 

Reference: Nadziejko 

et al. (2004, 055632) 

Species: Rat 

Gender: Male 

Strain: F344 

Age: 18 mo 

PM/CAPs (Tuxedo, NY) 

UFC (lab generated) 

SO2 

Particle Size: PM (Size Range): 0.5-

2.5μm; UFC (MMAD): 30-50 nm 

Route: Nose-only Inhalation 

Dose/Concentration: PM (μg/m3): 161-200, 

avg. 180; UFC (μg/m3): 500-1280, avg. 890; 

SO2 (ppm): 1.2, 1.2, avg. 1.2 

Time to Analysis: A total of 8 exposures were 

performed: 2 exposures to CAPs, 2 exposures 

to UFC, 4 exposures to SO2. All three pollutants 

were tested w/ a crossover design so that each 

group alternated exposure to air and to 

pollutant. Exposures lasted 4 h and were 

performed at least 1wk apart. Parameters 

measured throughout duration of experiment. 

Old F344 rats had many spontaneous 

arrhythmias. There was a significant increase in 

the frequency of irregular and delayed beats after 

exposure to CAPs. The same rats were 

subsequently exposed to UFC, SO2 or air with 

repeated crossover design. In these experiments 

there was no significant change in the frequency 

of any category of spontaneous arrhythmia 

following exposure to UFC or SO2. 

December 2009 D-16 
Study Pollutant Exposure Effects 

Reference: Nemmar et 

al. (2008, 096566) 

Species: Rat 

Gender: Male 

Strain: Wistar Kyoto 

Weight: 440 ± 14 g 

DEP (SRM 2975) 

Particle Size: <1 μm 

Route: Intravenous via the tail vein 

Dose/Concentration: DEP: 0.02mg or 0.1mg 

DEP/kg (corresponding to about 8 μg or 44 μg 

DEP/rat) 

Time to Analysis: 48 h following systemic 

administration of saline or DEP 

Intravenous administration of DEP (0.1 mg/kg) 

triggered systemic inflammation characterized by 

an increase in monocyte an granulocyte 

numbers. Both doses of DEP caused a reduction 

of RBC numbers and hemoglobin concentration. 

TEM analysis of RBCs after in vitro incubation (5 

μg/mL) or in vivo administration of DEP, revealed 

the presence of ultrafine-sized aggregates of 

DEP within the RBC. Larger aggregates were 

also taken up by the RBC. The myocardial morphology and capillary bed were not affected by 

DEP exposure. 

Reference: Nemmar et 

al. (2007, 156800) 

Species: Rat 

Gender: Male 

Strain: Wistar Kyoto 

Age: 16 wk 

Weight: 424 ± 8 g 

DEP (SRM 2975) 

Particle Size: NR 

Route: Tail Vein Injection 

Dose/Concentration: 8, 42, or 212 μg DEP/rat 

(150μl of 0.02, 0.1, or 0.5 mg/kg) 

Time to Analysis: 24 h 

Effect of DEP on Blood Pressure: Significant 

decrease on BP in DEP-exposed rats at doses of 

0.02 mg/kg, compared with mean BP observed in 

controls. 

Effect of DEP on HR: Doses of 0.02, 0.1, and 

0.5 mg/kg in rats, resulted in significant reduction 

of HR compared to controls. 

Effect of DEP on Tail Bleeding Time:

Shortening of tail bleeding time in rats exposed to 

0.02, 0.1, and 0.5 mg/kg. The shortening was 

significant at the dose of 0.02 and 0.5 mg/kg 

compared w/ controls. Platelet counts in blood 

did not significantly increased post-DEP 

administration. 

Effect of DEP on WBC and RBC Numbers: No 

significant effect of DEP at doses of 0.02, 0.1 and 

0.5 mg/kg on the numbers of granulocytes, 

monocytes, or lymphocytes compared with 

control. 

Reference: Nemmar et 

al. (2003, 096567) 

Species: Hamster 

Gender: Male and 

Female 

Weight: 100-110 g 

DEP (SRM 1650) 

Particle Size: NR 

Route: IT Instillation 

Dose/Concentration: 120 μl (5, 50, or 500 

μg/animal) 

Time to Analysis: In-vivo: formation and 

embolization of thrombus were continuously 

monitored for 40 min. Ex-vivo: animals were 

ITly instilled w/ DEPs (0 or 50 μg per animal), 

and blood was collected 5, 15, 30, and 60 min 

post-instillation. In-vitro: Saline or salinecontaining DEPs (0.1, 0.5, 1, and 5 μg/mL) was 

added to venous blood from untreated 

hamsters, and closure time was measured in 

the PFA-100 after 5 min/animal. 

Doses of 5-500 μg enhanced experimental 

arterial and venous platelet-rich thrombus 

formation in-vivo. Blood samples taken from 

hamsters 30 and 60 min after instillation of 50 μg 

of DEPs yielded accelerated aperture closure 

(platelet activation) ex-vivo, when analyzed in the 

PFA-100. The direct addition of as little as 0.5 

μg/mL DEPs to untreated hamster blood 

significantly shortened closure time in vitro. 

Reference: Nemmar et 

al. (2004, 087959) 

Species: Hamster 

Gender: Male and 

Female 

Weight: 100-110 g 

DEP (SRM 1650); Positively Charged 

Polystyrene Particles (PCPSP) 

Particle Size: PCPSP: 400 nm; DEP: 

NR 

Route: IT Instillation 

Dose/Concentration: DEP: 50 μg/animal, or 

PCPSP: 500 μg/animal 

Time to Analysis: Pretreatment Phase: 

Hamsters were pretreated w/ Dexametasone IP 

(5 mg/kg) or IT (0.1 or 0.5 mg/kg) or Sodium 

Cromoglycate given IP (40 mg/kg), 1 h before 

DEP or vehicle instillation. Thrombosis: In-vivo 

thrombogenesis assessed 24 h post-instillation 

of DEP or vehicle. 

DEP increased thrombosis without elevating 

plasma vWF. The IT instillation of PCPSP equally 

produced histamine release and enhanced 

thrombosis. Histamine in plasma resulted from 

basophil activation. IP pretreatment with 

Dexametasone abolished the DEP-induced 

histamine increase in BALF and plasma and 

abrogated airway inflammation and 

thrombogenicity. The IT pretreatment with 

Dexametasone showed a partial but parallel 

inhibition of all these parameters. Pretreatment 

with Sodium Cromoglycate strongly inhibited 

thrombogenicity, and histamine release. 

Reference: Nemmar et 

al. (2003, 097487) 

Species: Hamster 

Gender: Male and 

Female 

Weight: 100-110 g 

Ultrafine Particles: Unmodified 

Polystyrene Particles (UPSPs); Negatively Charged Carboxylate-Modified 

Polystyrene Particles (NCC-MPSPs); 

Positively-Charged Amine Modified 

Polystyrene Particles (PCA-MPSPs) 

Particle Size: UPSPs: 60 nm; NCCMPSPs: 60 nm; PCA-MPSPs: 60 or 

400 nm 

Route: IT Instillation 

Dose/Concentration: 5, 50, and 500 μg/animal 

in 120 μl saline 

Time to Analysis: 1 h post-instillation 

Unmodified and negative UFPs did not modify 

thrombosis. Positive UFPs increased thrombosis 

at 500 and 50 μg/animal, but not at 5 μg/animal. 

Positive 400 nm particles (500 μg/animal) did not 

affect thrombosis. PFA-100 analysis showed that 

platelets were activated by the in-vitro addition of 

positive UFPs and 400 nm particles to blood. 

December 2009 D-17 
Study Pollutant Exposure Effects 

Reference: Nemmar et 

al. (2003, 087931) 

Species: Hamster 

Weight: 100-110 g 

DEP (SRM 1650) 

Particle Size: NR 

Route: IT Instillation 

Dose/Concentration: 50 μg/animal in 120 μl 

saline 

Time to Analysis: 1, 3, 6, and 24 h 

At 1, 6, and 24 h after instillation of 50 μg DEPs, 

the mean size of in-vivo induced and quantified 

venous thrombosis was increased by 480, 770, 

and 460%, respectively. Platelets activation in 

blood was confirmed by a shortened closure time 

in the PFA-100 analyzer. In plasma, histamine 

was increased only at 6 and 24 h. Pre-treatment 

with a H1 receptor antagonist (diphenhydramine, 

30 mg/kg intraperitoneally) did not affect DEPinduced thrombosis or platelet activation at 1 h; 

however both were markedly reduced at 6 and 

24 h. 

Reference: Niwa et al. 

(2007, 091309) 

Species: Mouse 

Gender: Male 

Strain: LDLr/KO 

Age,: 6 wk (n = 20) 

Use: IT CB dispersion; 

10-14 wk acute effect 

of CB dispersion on 

circulating CRP 

Carbon Black 

Particle Size: 23-470 nm (mean size 

120.7 nm) 

Route: IT Dispersion 

Dose/Concentration: IT CB Dispersion Study: 

1 mg per animal/wk; Acute Effect of CB 

Dispersion on Circulating CRP Study: 

1mg/animal (single administration) 

Time to Analysis: IT CB Dispersion Study: 

1x/wk for 10 wk 

Acute Effect of CB Dispersion on Circulating 

CRP Study: Single CB administration, blood 

samples collected 24 h post-administration 

IT CB Dispersion Study: Although no difference 

in body weight (bw) between the four groups was 

observed at baseline, and all mice experienced 

an increase in bw with advancing age, the mice 

treated with CB tended to be smaller than those 

treated with vehicle (air). No significant 

differences were observed in cholesterol and TG 

levels among the for groups. Development of 

aortic lipid-rich lesions occurred in mice under a 

0.51% cholesterol diet with or without CB 

infusion, but not in the mice fed a 0% cholesterol 

diet. 

Acute Effect of CB Dispersion on Circulating 

CRP Study: Circulating levels of CRP were 

significantly higher in mice exposed to CB versus 

those exposed to air, indicating an acute 

inflammatory response. Although the presence of 

CB in pulmonary macrophage-like cells in CB 

treated mice under 0.51% cholesterol diet was 

confirmed, CB was not detected in aortas, livers, 

kidneys, or spleens.

Reference: Niwa et al. 

(2007, 091309) 

Species: Mouse 

Cell Types: RAW264.7 

Carbon Black (CB); Water-Soluble 

Fullerene 

(C60(OH)24); Fluoresbrite Carboxylate 

Microspheres; Ox-LDL; Acetylated-LDL

Particle Size: Carbon Black and 

C60(OH)24: 7.1 nm (SD 2.4); 

Fluoresbrite Carboxylate 

Microspheres: 6 nm 

Route: Cell Culture 

Dose/Concentration: CB: 1, 10, 100 μg/mL; 

C60(OH)24: 20, 100ng/mL 

Time to Analysis: RAW264.7+CB 

for 24 h, 13 days, and 50 days; 

RAW264.7+ C60(OH)24 for 24 h or 10 days; 

RAW264.7+ C60(OH)24 for 8 days, then cotreated w/ Ox-LDL for an additional 48 h; 

RAW264.7+Ox-LDL for 5 days, and then cocultured w/ C60(OH)24 for an additional 48 h; 

RAW264.7+ 6 nm beads: 3 days, the Ox-LDL or 

acetylated-LDL added for 24 h 

CB alone had no significant effects on RAW264.7 

cell growth. C60(OH)24 alone or CB and C60(OH)24 

together w/ Ox-LDL induced cytotoxic 

morphological changes, such as Ox-LDL uptakeinduced foam cell-like formation and decreased 

cell growth, in a dose-dependent manner. 

C60(OH)24 induced LOX-1 protein expression, 

pro-matrix metalloprotease-9 protein secretion, 

and tissue factor mRNA expression in lipid-laden 

macrophages. Although CB or C60(OH)24 alone 

did not induce platelet aggregation, C60(OH)24 

facilitated ADP-induced platelet aggregation. 

C60(OH)24 also acted as a competitive inhibitor of 

ADP receptor antagonists in ADP-mediated 

platelet aggregation. 

Reference: Niwa et al. 

(2008, 156812) 

Species: Rat 

Strain: SD 

Age: 6 wk 

CB from Kyoto, Japan 

Particle Size: Mean size (nm) ± SD 

determined at 1, 8, 15, 22, and 29 day 

post-exposure was 118.1 ± 2.4, 119.1± 

2.7, 122.2 ± 2.0, 122.4 ± 2.5 and 121.0 

± 3.6 respectively 

Route: Whole-body Inhalation 

Dose/Concentration: 15.6 ± 3.5 mg/m3

Time to Analysis: 6 h/day, 5 days/wk, for a 

total of 4 wk. BP and HR were measured by tailcuff plethysmography at 1, 14, and 28 day post 

-exposure. Sacrificed At 1, 7, 14, 28, and 30 

day post-exposure 

Although the presence of CB was confirmed in 

pulmonary macrophages, electron microscopic 

survey did not detect CB in other tissues 

including, liver, spleen and aorta. CB exposure 

raised blood pressure levels in a exposure-time 

dependent manner. Levels of circulating 

inflammatory marker proteins, including 

monocyte chemo attractant protein-1, IL-6, and 

CRP, were higher in the CB treated groups than 

in control groups. 

Reference: Nurkiewicz 

et al. (2004, 087968) 

Species: Rat 

Gender: Male 

Strain: SD 

Age: 7-8 wk 

ROFA (from Everett, MA). Major metal 

contaminants are: Fe, Al, V, Ni, Ca, 

and Z. Main soluble metals are: Al, Ni, 

and Ca. 

Particle Size: 2.2 μm (ROFA mean 

count diameter) 

Route: IT Instillation 

Dose/Concentration: ROFA group: 0.1, 0.25, 

1, or 2 mg/rat. Vehicle control group: 300 μl 

saline. Particle control group: TiO2 0.25 mg/rat. 

Time to Analysis: After single IT instillation of a 

particular dose, all rats recovered for 24 h. 

Saline Treated Rats: A23187 dilated arterioles 

up to 72 ± 7% max. 

ROFA and TiO2 Exposed Rats: A23187-induced 

dilation was significantly attenuated. 

Sensitivity of Arteriolar Smooth Muscle to 

NO: Similar in saline treated and ROFA exposed 

rats. 

Other: Significant increase in venular leukocyteadhesion and rolling observed in ROFA exposed 

rats. 

December 2009 D-18 
Study Pollutant Exposure Effects 

Reference: Nurkiewicz 

et al. (2006, 088611) 

Species: Rat 

Gender: Male 

Strain: SD 

Age: 7-8 wk 

ROFA from Everett, MA 

Particle Size: ROFA mean count 

diameter: 2.2 μm; TiO2 mean diameter: 

1.0 μm 

Route: IT Instillation 

Dose/Concentration: ROFA group: 0.1 or 0.25 

mg/rat. Vehicle control group: 300 μl saline. 

Particle control group: TiO2 0.1 or 0.25 mg/rat. 

Time to Analysis: After single IT instillation of a 

particular dose, all rats recovered for 24 h. 

ROFA or TiO2 Exposure and Arteriolar 

Dilation: Exposure caused a dose-dependent 

impairment of endothelium-dependent arteriolar 

dilation. 

ROFA or TiO2 Exposure and Arteriolar 

Constriction: Exposure did not affect 

microvascular constriction in response to PHE. 

ROFA and TiO2 and Leukocyte Rolling and 

Adhesion: Exposure significantly increased 

leukocyte rolling and adhesion in aired venules, 

and these cells were identified as PMN 

leukocytes. 

ROFA and TiO2 and MPO: MPO was found in 

PMN leukocytes, adhering to the systemic microvascular wall. Evidence suggests that some of 

this MPO had been deposited in the 

microvascular wall. There was also evidence of 

oxidative stress in the microvascular wall. 

Reference: Nurkiewicz 

et al. (2008, 156816) 

Species: Rat 

Gender: Male 

Strain: SD 

Age: 6-7wk 

Weight: NR

TiO2 (DeGussa, Sigma-Aldrich) 

Particle Size: Fine- 1 μm, UF- 21 nm 

Route: Whole-body Inhalation 

Dose/Concentration: Concentrations: Fine- 3-

16 mg/m3

; UF- 1.5-12 mg/m3; Dose: Fine- 8, 20, 

36, 67, 90 μg; UF- 4, 6, 10, 19, 30 μg 

Time to Analysis: Acclimated 5 days. Exposed 

4-12 h. Sacrificed 24 h post-exposure. 

Particle accumulation within AMs, anuclear 

macrophages, particle-laden AMs intimately 

associated with the alveolar wall were all present 

in exposed rats. Calcium ionophore impaired 

arteriolar dilation in a dose-dependent manner in 

UF and fine exposed rats. UF produced greater 

systemic microvascular dysfunction. 

Microvascular dysfunction was the same for 

three groups of rats exposed to 30 μg UF TiO2

under different conditions.

Reference: Nurkiewicz 

et al. (2009, 191961) 

Species: Rat 

Gender: Male 

Strain: SD 

Age: 7-8 wk 

Weight: NR

Fine TiO2 (Sigma-Aldrich, St. Louis, 

MO) (~99% rutile) 

TiO2 nanoparticles (DeGussaAeroxide TiO2 P25, Parsippany, NJ) 

(80% anatase, 20% rutile) 

Particle Size: Fine TiO2- MMAD: 402 

nm, Primary size: <5 μm, ,CMD: 710 

nm; Nano-TiO2- MMAD: 138 nm, 

Primary size: 21 nm, , CMD: 100 nm 

Route: Aerosol Inhalation 

Dose/Concentration: 1.5-16mg/m3

Time to Analysis: Acclimated 5 days. Exposed 

240-720 min. Anesthetized 24 h post-exposure. 

Intravital microscopy, NO measurement, 

microvascular oxidative stress measurement, 

nitrotyrosine staining. 

Arteriolar Dilation: Nano-TiO2 significantly 

impaired endothelium-dependent arteriolar 

dilation. Equivalent levels of arteriolar dysfunction 

were found in fine and nano-TiO2. Arteriolar 

dilation in response to abluminal 

microiontophretic application of SNP was not 

different from the controls or between the 

exposure groups. Arteriolar dilation was partially 

restored by radical scavenging with TEMPOL and 

catalase, NADPH oxidase with apocynin, and 

MPO inhibition with ABAH. 

Microcirculation: ROS increased in both 

groups. Nano-TiO2 significantly increased the 

area of tissue containing nitrotyrosine in the lung 

and spinotrapezius microcirculation. 

NO: Fine and nano-TiO2 significantly and dosedependently decreased stimulated NO 

production in isolated microvessels. NO 

production was increased by radical scavenging 

with TEMPOL and catalase or NADPH oxidase 

with apocynin, and was largest in the fine TiO2

group. 

December 2009 D-19 
Study Pollutant Exposure Effects 

Reference: Okayama 

et al. (2006, 156824) 

Species: Rat 

Cell Type: Ventricular 

Cardiac Myocytes from 

Wistar Rats, 

approximately 3 days 

old 

DEP (Tsukuba, Japan) 

DEPE: 5g of DEP in 5 mL PBS 

containing 0.05% Tween 80. 

Others: Catalase, LDH, MPG and 

SOD. 

Particle Size: DEP mass median 

diameter: 0.34 μm. 

Route: In Vitro 

Dose/ Concentration: DEPE: 0-100 μg/mL; 

MPG: 0-1 mM; SOD: 800 U/mL; Catalase: 500 

U/mL

Time to Analysis: cells were incubated for 1, 2, 

4, or 8, 24 or 48 h. 

LDH Activity of Supernatant: 24 h post-DEPE 

exposure. 

SOD, Catalase, MPG on DEPE-induced 

Toxicity: SOD, catalase or MPG was added to 

cells w/ or w/o DEPE & incubated for 4 or 24 h. 

Medium then replaced w/serum-free & cells 

incubated for another 24 h to analysis. 

Cytotoxic Effects of DEPE on Cardiac 

Myocytes: DEPE above 20 μg/mL damaged 

cardiac myocytes in a time and concentrationdependent manner in both long- and short-term 

exposure conditions. However damage was 

greater after long-term exposure. LDH activity 

showed a concentration-dependent increase at 

higher levels of exposure (greater than 20 

μg/mL). 

Effects of ROS Scavenging Enzymes and 

Antioxidant on DEPE-induced Cell Damage:

SOD or catalase attenuated 50 μg/mL DEPEinduced cell damage compared with DEPEtreated groups lacking antioxidant enzymes. Coincubation with SOD and catalase showed more 

protective effects towards DEPE-induced cell 

damage, although these effects were not 

statistically significant from cells treated with 

SOD only. MPG attenuated 50 μg/mL DEPEinduced cell damage in a concentrationdependent manner in both long and short-term 

exposure conditions. Especially in long-term 

exposure MPG showed strong protective effects 

against DEPE-induced cell damage. Cell viability 

was not affected by SOD, catalase, or MPG. 

Reference: Proctor et 

al. (2006, 088480) 

Species: Rat 

Gender: Male 

Age: 12 wk 

Use: Thoracic Aorta 

from cp/cp and +/? 

Male Rats 

cp/cp = homozygous 

for cp gene. Prone to 

obesity and insulin 

resistant. 

+/? = heterozygous for 

either +/cp or +/+. Lean 

and metabolically 

normal. 

ROFA from Birmingham, AL 

Particle Size: 1.95 ± 0.18 μm 

(aerodynamic diameter) 

Route: Protocol 1: Used two aorta rings per 

each experimental treatment group (4 groups 

total). Protocol 2: Used four rings. 

Dose/Concentration: Protocol 1: exposed to 

12.5 μg/mL ROFA-L (at 10 mg/mL). 

Protocol 2: exposed to 1.56, 3.25, 6.26, 12.5 

μg/mL ROFA-L (at 10 mg/mL). 

Time to Analysis: Protocol 1: Cells treated with 

12.5 μg/mL ROFA-L and/or 104mol/L L-NAME 

for 20 min 

Protocol 2: Parameters measured after ROFA-L 

only treatment 

Contractile response to phenylephrine (PE) was 

measured 

ROFA-L (12.5 μg/mL) increased PE-mediated 

contraction in obese, but not in lean rat aortae. 

Effect was exacerbated by L-NAME, and it 

reduced ACh-mediated relaxation in obese and 

lean aortae. Initial exposure of aortae to ROFA-L 

caused a small contractile response, which was 

markedly greater on second exposure in the 

obese aortae but marginal in lean. 

Reference: Radomski 

et al. (2005, 091377) 

Species: Rat 

Strain: Wistar Kyoto 

Carbon Nano Particles (CNPs) 

(purchased from SES Research, 

Houston, TX): Multiplewall Nanotubes 

(MWNT); Single wall Nanotubes 

(SWNT); C60 Fullerenes (C60CS); 

Mixed Carbon Nanoparticles (MCN) 

PM: (SRM1648) (NIST) 

Particle Size: CNPs: NR; PM: 1.4 μm 

average size 

Route: Simultaneous single PM injection into 

femoral vein as FeCl3 injected to induce carotid 

thrombosis 

Dose/Concentration: 0.5 mL suspension of 50 

μg/mL of PM in 0.9% NaCl solution. 

Time to Analysis: Blood flow continuously 

monitored for 900 s. 

Vascular Thrombosis: FeCl3 induced carotid artery thrombosis and MCN had an amplifying 

effect in the development of thrombosis. 

Infusions of MCN, SWNT, and MWNT significantly accelerated the time and rate of 

development of carotid artery thrombosis in rats. 

SRM1648 was less effective than CNPs in 

inducing thrombosis, while C60CS exerted no 

significant effect on the development of vascular 

thrombosis. 

Reference: Radomski 

et al. (2005, 091377) 

Species: Human 

Cell Types: Platelets 

Use: Human platelet 

aggregation

Carbon Nano Particles (CNPs) 

(purchased from SES Research, 

Houston, TX): Multiplewall Nanotubes 

(MWNT); Singlewall Nanotubes 

(SWNT); C60 Fullerenes (C60CS); 

Mixed Carbon Nanoparticles (MCN); 

PM (SRM1648) 

Particle Size: CNPs: NR; PM: 1.4 μm 

average size 

Route: Cell Culture (2.5×108 platelets/mL) 

Dose/Concentration: CNPs: 0.2-300 μg/mL; 

PM: 5-300 μg/mL 

Time to Analysis: Prostacyclin (PGI2), S-nitroso-glutathione (GSNO), aspirin, 2-methylthioAMP, phenanthroline, EDTA and Go6976 were 

pre-incubated w/ platelets for 1 min before 

particle addition. Particles added to platelets 

and platelet aggregation studied for 8min. 

Platelet Aggregation: All CNPs, except C60CS, 

stimulated platelet aggregation (MCN ≥

SWNT>MWNT>SRM1648). All particles resulted 

in upregulation of GPIIb/IIIa in platelets. In 

contrast, particles differentially affected the 

release of platelet granules, as well as the 

activity of thromboxane-, ADP, matrix 

metalloproteinase- and protein kinase C-dependent pathways of aggregation. Particleinduced aggregation was inhibited by 

prostacyclin and GSNO, but not by aspirin.

December 2009 D-20 
Study Pollutant Exposure Effects 

Reference: Reed et al. 

(2006, 156043) 

Species: Rat, Mouse 

Gender: Male and 

Female 

Strain: CDF 

(F344)/CrlBR (rat), SH 

(rat), A/J (mouse), and 

C57BL/6 (mouse) 

Age: 6-12 wk

HWS (burned mix of hardwood in 

noncertified wood stove using a 

Pineridge model 27000, Heating and 

Energy Systems, Inc. Clackamas, OR) 

Measured Components: EC, OM, NO3, 

SO4, NH4, metals 

Particle Size: ~0.25 μm 

Route: Whole-body Inhalation 

Dose/Concentration: Low: 30 μg/m3 

Mid-low: 100 μg/m3

 

Mid-high: 300 μg/m3

 

High: 1000 μg/m3

 

Time to Analysis: 6 h/day, 7days /wk for 1 wk 

or 6 mo. Immediate post-exposure analysis.

Organ Weights: Liver declined in rats of both 

genders at 1 wk and female rats at 6 mo. Lung 

volume increased and lung weight decreased in 

female rats at 6 mo. Spleen weight increased in 

female mice and rats at 1wk. Thymus weight 

decreased in male rats at 1wk. 

Clinical Chemistry: Cholesterol decreased at 

the high dose for male rats at 1wk and 6 mo and 

increased at mid-low and mid-high doses for 

female rats at 6 mo. ALP decreased for rats of 

both genders at 1wk and 6 mo for mid-low, midhigh and high dose levels (14-38%). AST 

decreased by 24% in male rats at 1wk with high 

dose. No effect on females. Creatinine serum 

levels decreased in males at 1wk at mid-high and 

high dose by 13%. No effect observed at 6 mo. 

BUN/Cre ratio decreased in females at 1wk 

(25%) and both genders at 6 mo at mid-high and 

high dose (18-19%). 

Hematology: Hemoglobin and hematocrit 

increased in 6 mo male rats. Bilirubin increased 

in female rats at 6 mo at high dose. Platelets 

increased for male and female rats at 1wk (21%, 

19% respectively). No effect observed at 6m. 

WBC increased in males at 1wk. 

Reference: Reed et al. 

(2004, 055625) 

Species: Rat, Mouse 

Gender: Male and 

Female 

Strain: CDF 

(F344)/CrlBR (rat), A/J 

(mouse) 

Age: 12 wk

DE: generated from two 2000 model 

5.9 L Cummins ISM turbo diesel 

engines 

Co-exposure to 8 gas and 8 solid 

exhaust components measured 

Particle Size: 0.10 - 0.15 μm 

Route: Whole-body Inhalation 

Dose/Concentration: Low: 30 μg/m3 

Mid-low: 100 μg/m3

 

Mid-high: 300 μg/m3

 

High: 1000 μg/m3

 

Time to Analysis: 6 h/day, 7 days/wk for 1wk 

or 6 mo. Analyzed 1 day post-exposure.

Organ Weights: Kidney weight increased after 

6m for both males and female rats at the high 

dose. Kidney and liver weight increased for 

female mice at all dose levels at 6 mo. Lung 

weight increased at high dose at 6mo for female 

mice and male rats. Spleen weight decreased in 

male mice at the low and mid-high levels. 

Clinical Chemistry: There was a massive 

decrease in cholesterol (24%) for rats of both 

genders after 1 wk and a smaller decrease for 

male rats at 6 mo. GGT significantly increased at 

6 mo for male and female rats at the mid-high 

and high dose. ALP increased in male rats at 1 

wk by 10%. AST decreased at mid-high (15%) 

and high dose in female rats at 6 mo. BUN and 

BUN/Creatine declined (19%, 17%) in female rats 

at mid-high and high doses after 6 mo. BUN 

increased by 21% at mid-low, mid-high and high 

doses in male rats at 1wk. 

Hematology: WBC decreased in high females 

after 6 mo. Factor VII (blood clotting) decreased 

in MH and HR males after 1wk and male and 

female HR after 6 mo. Thrombin-antithrombin 

complex declined massively but only in males 

after 1wk. 

Reference: Reed et al. 

(2008, 156903) 

Species: Rat 

Gender: Male, Female 

Strain: CDF 

(F344)/CrlBR, SH

Age: NR 

Weight: NR

GEE (two 1996 General Motors 4.3-L 

V-6 engines; regular, unleaded, nonoxygenated, non-reformulated 

Chevron-Phillips gasoline, U.S. 

average consumption for summer 

2001 and winter 2001-2002) 

Particle Size: 150 nm (MMAD) 

Route: Whole-body Inhalation 

Dose/Concentration: PM: Low- 6.6 ± 3.7 

μg/m3

, Medium- 30.3 ± 11.8 μg/m3, High- 59.1 ± 

28.3 μg/m3

Time to Analysis: 2 wk quarantine period in 

chamber. Exposed 6 h/day, 7 days/wk, 3 day-6 

mo. SH- surgery to implant telemeter in 

peritoneal cavity. 4 wk recovery. ECG data 

obtained every 15 min beginning 3 day preexposure, 7 day exposure, 4 day postexposure. 

Organ Weight: At 6 mo exposure, the heart 

weights of male and female rats increased and 

male rats’ seminal vesicle weight decreased. 

Clinical Chemistry: Serum alanine 

aminotransferase, aspartate aminotransferase, 

and phosphorus decreased in medium and highexposure females. 

Hematology: Hematocrit, red blood cell count, 

and hemoglobin dose-dependently increased for 

both genders at both time points. Plasma 

fibrinogen increased at 1wk in males. 

CV Effects in SH Rat: Lipid peroxides were 

significantly increased in males in the high 

exposure group. TAT complexes decreased in 

females in the high exposure group. 

Removal of Emission PM: The removal of 

emission PM strongly linked PM to increased 

seminal vesicle weight, red blood cell counts, 

LDH, lipid peroxides, and methylation.

December 2009 D-21 
Study Pollutant Exposure Effects 

Reference: Rhoden et 

al. (2005, 087878) 

Species: Rat 

Gender: Male 

Strain: SD 

Age: Adult 

Weight: 300 g 

Urban Ambient Particles (UAPs): SRM1649; CAPs (Boston, MA) 

Particle Size: NR 

Route: UAPs: IT Instillation. CAPs: Inhalation 

Dose/Concentration: UAPs: 750 μg 

suspended in 300 μl saline; CAPs: 700 ± 180 

μg/m3

Time to Analysis: UAPs: 30 min post-instillation. CAPs: immediately after 5 h exposure 

period 

Oxidative Stress and HR Function: UAPs 

instillation led to significant increases in heart 

oxidants. HR increased immediately after 

exposure and returned to basal levels over the 

next 30 min. SDNN was unchanged immediately 

after exposure, but significantly increased during 

the recovery phase. 

Role of ROS in Cardiac Response: Rats were 

treated with 50 mg/kg NAC 1 h prior to UAPs instillation or CAPs inhalation. NAC prevented 

changes in heart rate and SDNN in UAPsexposed rats. 

Role of the Autonomic Nervous System in 

PM-induced Oxidative Stress: Rats were given 

5 mg/kg atenolol, 0.30 mg/kg glycopyrrolate, or 

saline immediately before CAPs exposure. Both 

atenolol and glycopyrrolate effectively prevented 

CAPS-induced cardiac oxidative stress. 

Reference: Rivero et 

al. (2005, 088653) 

Species: Rat 

Gender: Male 

Strain: Wistar Kyoto 

Age: 3 mo 

Weight: ~250 g 

PM2.5, collected from heavy traffic area 

in Sao Paulo, Brazil. PM2.5 Composition (%): S (3.05), As (0.30), Br (0.21), 

Cl (2.09), Co (2.65), Fe (2.67), La 

(5.42), Mn (0.64), Sb (0.21), Sc (3.25), 

Th (8.14) 

Particle Size: PM2.5

Route: IT Instillation 

Dose/Concentration: 100 or 500 μg of PM2.5. 

Time to Analysis: 24 h post-instillation 

Hematology: Total reticulocytes significantly 

increased at both PM2.5 doses, while hematocrit 

levels increased in the 500 μg group. Quantification of segmented neutrophils and fibrinogen 

levels showed a significant decrease, while 

lymphocytes counting increased with 100 μg of 

PM2.5. 

Pulmonary Vasculature: Significant dose-dependent decrease of intra-acinar pulmonary 

arteriole lumen/wall ratio was observed in both 

PM2.5 groups. 

Wet-to Dry Weight Ratio: Significant increase in 

heart wet-to-dry weight ratio was observed in the 

500 μg group. 

Reference: Rivero et 

al. (2005, 088659) 

Species: Rat 

Gender: Male 

Strain: Wistar Kyoto 

Age: 3 mo 

Weight: ~250 g 

PM2.5, collected from heavy traffic area 

in Sao Paulo, Brazil. PM2.5 Composition (%): S (3.05), As (0.30), Br (0.21), 

Cl (2.09), Co (2.65), Fe (2.67), 

La (5.42), Mn (0.64), Sb (0.21), 

Sc (3.25), Th (8.14) 

Particle Size: PM2.5

Route: IT Instillation 

Dose/Concentration: 50 and 100 μg of PM2.5. 

Time to Analysis: HR and SDNN were 

assessed immediately before instillation, 30 and 

60 min post-instillation. 

HR decreased significantly with time, but no 

significant effect of treatment or interaction 

between time and treatment was observed. In 

contrast, there was a significant SDNN 

interaction between time and treatment. The 

SDNN decreased 60 min after instillation with 

PM2.5 concentration of 50 and 100 μg. 

Reference: Seagrave 

et al. (2008, 191990) 

Species: Rat 

Gender: Male 

Strain: SD 

Age: 10-12 wk 

Weight: 250-300 g

GEE (2 1996 General Motors 4.3-L V6 

gasoline engines; conventional 

Chevron Phillips gasoline, U.S. 

average composition) (CO, NO, NO2, 

SO2, THC) (PM2.5 composition- EC, 

OC, SO4, NH4, NO3) 

Simulated downwind coal emission 

atmospheres (SDCAs) (fly ash, gasphase pollutants, sulfate aerosols, NO, 

NO2, SO2) 

Paved Road Dust (RD) (Los Angeles, 

CA; New York City, NY; Atlanta, GA) 

Particle Size: GEE: 150 nm (MMAD), 

RD: 2.6 ± 1.7 μm, SDCA: 0.1-1.0 μm 

Route: Nose-only Inhalation 

Dose/Concentration: GEE: 60 μg/m3, SDCAs: 

317-1072 μg/m3

, RD: 306-954 μg/m3; GEE: CO104 ppm, NO- 16.7 ppm, NO2- 1.1 ppm, SO2- 

1.0 ppm, THC- 12ppm; SDCAs: CO- <1 ppm, 

NO- 0.19-0.62 ppm, NO2- 0.10-0.37 ppm, SO2- 

0.07-0.24 ppm, THC- <1 ppm 

Time to Analysis: 6 h exposure. Cannula 

ligated into trachea and connected to rodent 

ventilator. Thorax and abdomen opened. 

GEE produced CL in the lungs, heart, and liver. 

RD produced a significant effect in the heart at 

the low dose. SDCAs had no effect on CL. RD 

significantly increased the heart’s oxidative 

stress, as demonstrated by the TBARS levels.. 

Reference: 

Simkhovich et al. 

(2007, 096594) 

Species: Rat 

Gender: Female 

Strain: Fischer 344 × 

Brown Norway hybrid 

Age: 4, 26 mo 

Ultra Fine Particles (UFPs) isolated 

from industrial diesel reference PM 

2975 

Particle Size: UFPs ≤ 0.1 μm 

Route: Heart Perfusion (ex-vivo) 

Dose/Concentration: UFPs 12.5, 25, and 37.5 

mg. 

Time to Analysis: Hearts perfused w/ UFPs for 

30 min and analysis conducted every 10 min. 

Young adult and old hearts demonstrated equal 

functional deterioration in response to direct 

infusion of UFPs. Developed pressure in young 

adult UFPs-treated hearts fell from 101 ± 4 to 68 

± 8 mmHg. In the old UFPs-treated hearts 

developed pressure fell by 35%. Positive dP/dt 

was equally affected in the young adult and old 

UFPs-treated hearts and was decreased by 28% 

in both groups. 

December 2009 D-22 
Study Pollutant Exposure Effects 

Reference: Smith et al. 

(2006, 110864) 

Species: Rat 

Gender: Male 

Strain: SD 

Age: 8 wk 

Weight: 260-270 g

CFA: Coal Fly Ash (400 MW, Wasatch 

Plateau, Utah) (aerodynamic 

separation) 

Particle Size: 0.4-2.5 μm 

Route: Nose-only Inhalation 

Dose/Concentration: 1400 μg/m3 PM2.5

including 600 μg/m3 PM1

Time to Analysis: 4 h/day for 3 consecutive 

days. Parameters measured 18 or 36 h postexposure.

Hematology: Plasma protein increased at 18h. 

Lymphocyte and hematocrit percentage 

decreased at 36 h. 

Reference: Stinn et al. 

(2005, 088307) 

Species: Rat 

Gender: Male and 

Female 

Strain: Crl: (WIU BR 

Age: 40 day

DE (generated from 1.6 L VW diesel 

under USFTP 72) 

CO: 10, 37 ppm 

CO2: 2170, 6540 ppm 

NO: 7.0, 22.8 ppm 

NOX: 8.6, 28.3 ppm 

SO2: 0.83, 3.09 ppm 

NH4: ND 

Measured Major Components: NO, 

SO2, 1-nitropyrene, Zi. 50% by DE 

weight is EC. 

Particle Size: 0.19-0.21 μm (MMAD) 

Route: Nose-only Inhalation 

Dose/Concentration: 3 and 10 mg/m3

Time to Analysis: 6 h/day, 7 day/wk for 24 mo; 

6 mo post-exposure

Hematology: Erythrocytes were unaffected (12, 

24, 30) except in high dose females at 24 and 30 

mo. Hemoglobin and hematocrit increased dosedependently with no gender differences. 

Leukocytes increased in a dose- and timedependent manner. 

Reference: Sun et al. 

(2005, 087952) 

Species: Mouse 

Gender: Male 

Strain: ApoE-/-

Age: 16 wk 

CAPs: PM2.5 from Tuxedo, NY. 

HFCD: High Fat Chow Diet 

NCD: Normal Chow Diet 

Particle Size: PM2.5

Route: Whole-body Inhalation 

Dose/Concentration: PM2.5: 85 μg/m3; Daily 

concentration: 10.6 (SD 3.4) μg/m3

 (mean) 

Average exposure over 6 mo period: 15.2 

μg/m3

. 

Time to Analysis: Study diets fed for at least 

10 wk prior to exposure to PM2.5 or FA. Exposed 

for 6 h/day, 5 days/wk for 6 mo. Sacrificed 15-

47 days after exposure. 

Vasomotor Function: Mice fed HFCD and 

exposed to PM2.5 demonstrated an increase in 

the half-maximal dose for dilation to ACh with no 

changes in peak relaxation compared to the mice 

exposed to FA and fed HFCD and NCD. 

Atherosclerosis Burden with PM2.5: In vivo MRI 

imaging of atherosclerosis burden in the 

abdominal aorta revealed significantly increased 

plaque burden in the mice fed HFCD compared 

with the mice fed NCD. Mean (SD) plaque areas 

in the mice exposed to PM2.5 and fed HFCD vs. 

mice exposed to FA and fed HFCD were 33 (10) 

vs. 27 (13) units, respectively. 

PM2.5 and Vascular Inflammation: A 2.6-fold 

higher inducible NOS content was apparent in 

the mice exposed to PM2.5 and fed HFCD compared with the mice exposed to FA and fed HFCD 

chow and a 4-fold increase in the mice exposed 

to PM2.5 and fed NCD compared with the mice 

exposed to FA and fed NCD. 

Reference: Sun et al. 

(2008, 157033) 

Species: Mouse 

Gender: Male 

Strain: ApoE-/-

Age: 6 wk 

CAPs PM2.5 

Collected from Sterling Forest State 

Park, Tuxedo NY (40 miles NW of 

Manhattan) 

Particle Size: PM2.5 

Route: Whole-body Inhalation 

Dose/Concentration: Average Concentration 

of: 85 μg/m3

 CAPs in chamber. 

Average exposure over 6 mo was 15.2 μg/m3

. 

Time to Analysis: 6 h/day, 5 day/wk for 6 mo. 

Mice received two different diets, high-fat chow 

and normal-chow. 

Macrophage and Tissue Factor Expression in 

Aortic Segments: Tissue Factor (TF) expression 

was noted predominantly in the extracellular 

matrix surrounding macrophages, foam cell-rich 

areas and around smooth muscle cells. 

1. High-Fat Diet: Increased TF and increased 

macrophage infiltration was observed in the 

plaques of high-fat chow mice exposed to PM 

compared to mice exposed to air and high fat 

diet. 

2. Normal Diet: PM-exposed mice saw an 

increase in CD68 expression compared to airexposed. However TF expression was not 

significantly different in PM exposed normal diet 

mice compared to control normal diet mice. 

December 2009 D-23 
Study Pollutant Exposure Effects 

Reference: Sun et al. 

(2008, 157033) 

Species: Human 

Cell Lines: BEAS-2B; 

Vascular Smooth 

Muscle Cells (hSMCs); 

and Monocytes (THP1) 

Ambient Particles collected from 

Sterling Forest State Park, Tuxedo, NY 

(24 h/day for 4 wk) 

Particle Size: Particle size ranges: 1. 

<0.18 μm 

2. 1.8 - 2.5 μm or 

3. 2.5 - 10 μm 

Route: In vitro 

Dose/Concentration: 10-300 μg/ml 

Time to Analysis: Doses were tested for 

durations up to 24 h. 

Dose durations tested for up to 24-h did not 

indicate detectable effects on cell viability. 

Effect of PM on TF Expression and Activity in 

hSMCs: In the PM size range of 1-3 μm, 

significant increases in TF expression was 

observed at doses of 100 and 300 μm /mL. In the 

<0.18 μm size range, significant increase in TF 

expression was observed at all doses. The 

particles with sizes 0.18 - 1.0 μm did not induce 

significant change in TF expression. 

Effect of PM on TF Expression and Activity in 

Monocyte Cells: TF protein expression 

increased with <0.18 μm and the 1- 3 μm range 

particles. Expression was increased in the 0.18-

1.0 μm particle range but it was limited compared 

to the other PM size ranges. In general TF 

expression was higher in monocytes than in 

hSMCs cells, but not significantly. 

Effect on TF Expression and Activity in 

Bronchial Epithelial Cells: 100 μg/mL of the 1-3 

μm and <0.18 μm particles significantly increased 

TF expression. 

TF mRNA Expression: TF mRNA was increased 

rapidly within the first hour in response to SRM1694a PM. The lowest dose of SRM PM10 μg/mL 

induced highest levels of mRNA in hSMCs, no 

further increase was observed at higher 

concentrations. 

Reference: Sun et al. 

(2008, 157032) 

Species: Rat 

Gender: Male 

Strain: SD 

Age: 500-650 g 

PM2.5 or UFP 

Particle Size: PM2.5; UFP: <0.1 μm 

Route: Whole-body Inhalation 

Dose/Concentration: Mean PM2.5

concentration: 79.1 ± 7.4 μg/m3. Normalized 

PM2.5 over 10wk period: 14.1 μg/m3

. 

Time to Analysis: 6 h/day, 5 day/wk random 

exposure to PM2.5, UFP, or FA for a total of 10 

wk. At the end of wk 9 exposure, rats were 

infused w/ 0.75 mg/kg/day of AII for 7 days. 

PM2.5, UFP, or FA, continued during AII infusion 

period. 

All = angiotensin II 

Mean Arterial Pressure (MAP): After AII 

infusion, MAP was significantly higher in PM2.5 -

AII vs. FA-AII group. Aortic Vasoconstriction to 

PE was potentiated with exaggerated relaxation 

to the Rho-kinase (ROCK) inhibitor Y-27632 and 

increase in ROCK-1 mRNA levels in the PM2.5 -

AII group. Superoxide production in the aorta was 

increased in the PM2.5. AII group compared to FAAII group, inhabitable by apocynin and L-NAME 

with coordinate upregulation of NAD(P)H oxidase 

subunits p22phox and p47phox and depletion of 

tetrahydrobipterin. 

Reference: Sun et al. 

(2008, 157032) 

Species: Rat 

Gender: Male 

Strain: SD 

Age: 500-650 g 

Cell Line: Primary Rat 

Aortic Smooth Muscle 

Cells (RASMCs)

PM2.5 or UFP 

Particle Size: PM2.5; UFP: <0.1 μm 

Route: Cell Culture 

Dose/Concentration: UFP, PM2.5: 10 or 50 

μg/mL; All: 100 nmol/L 

Time to Analysis: Exposed to UFP or PM2.5

and parameters measured at 0, 1, 3, 6, and 15 

min. 

All = angiotensin II

Exposure to UFPs and PM2.5 was associated with 

an increase in ROCK activity, phosphorylation of 

myosin light chain, and MYPT1. Pretreatment 

with N-Acetylcysteine and the Rho kinase 

inhibitors (Fasudil and Y-27632) prevented MLC 

and MYPT-1 phosphorylation by UFPs suggesting a superoxide-mediated mechanism for 

PM2.5 and UFPs effects.

December 2009 D-24 
Study Pollutant Exposure Effects 

Reference: Sun et al. 

(2009, 190487) 

Species: Mouse 

Gender: Male 

Strain: C57BL/6, cfmsYFP (transgenic, 

yellow fluorescent 

protein under 

monocyte-specific 

promoter) 

Age: 8, 10 wk 

Weight: NR

PM (concentrated- northeastern 

regional background; Tuxedo Park, 

NY) 

Particle Size: 2.5 μm (diameter) 

Route: Whole-body Inhalation. IT Instillation. 

Dose/Concentration: Exposure chamber 

(mean): 72.7 μg/m3, IT: 1.6mg/kg 

Time to Analysis: C57BL/6 mice, fed high-fat 

chow 10wk. Exposed in vivo 6 h/day, 5 day, 128 

days. fmsYFP rendered diabetic or fed normal 

chow 10 wk. IT instilled with PM 2 times/wk for 

10 wk. 

Metabolic Impairment: PM induced insulin, 

homeostasis model assessment indexes, 

elevated glucose, and abnormalities in lipid 

profile consistent with the IR phenotype. 

Vascular Endothelium: PM decreased peak 

relaxation and ED50 to ACH and peak relaxation 

to insulin. Lower levels of NO release were seen. 

Insulin Signaling: PM reduced the 

phosphorylation of Akt in intact aorta. PKC-β11 

was the only PKC isoform to increase. 

Adipose Inflammation, Visceral Adiposity: PM 

significantly increased TNF-α, IL-6, E-selectin, 

ICAM-1, plasminogen activator inhibitor-1, and 

restin. PM increased visceral and mesenteric fat 

mass. F4/80+ macrophages in fat tissue and 

adipocyte size increased. PM downregulated IL10 and glactose-N-acetylgalactosamine-specific 

lectin. 

YFP Cell Adhesion and Infiltration: PM 

increased YFP cells in the adipose tissue, YFP 

cell infiltration in the mesenteric fat, and YFP cell 

adhesion to endothelium. 

Reference: Tamagawa 

et al. (2008, 191988) 

Species: Rabbit 

Gender: Female 

Strain: New Zealand 

White 

Age: 12 wk 

Weight: Acute 

(average)- 2.4 ± 0.2 kg, 

Chronic (average)- 2.7 

± 0.3 kg

PM10 (urban; Ottawa, Canada) 

Particle Size:: 0.8 ± 0.4 μm (mean 

diameter) 

Route: Intrapharyngeal Instillation 

Dose/Concentration: Acute- 2.6mg/kg, 

Chronic- 2mg/kg 

Time to Analysis: Acute animals exposed days 

1, 3, 5. Chronic animals exposed 2 times/wk for 

4 wk. 

Inflammation: PM10 induced more 

macrophages, AMs, positive and activated AMs, 

and fewer tissue macrophages. NO, WBC and 

PMN were only significantly higher in the first two 

wk and IL-6 in the first wk. 

Vascular endothelial function: PM10

significantly reduced Ach-stimulated relaxation 

and did not alter SNP-stimulated relaxation. A 

significant inverse relationship between IL-6 and 

Ach-induced relaxation occurred at wk 1 in the 

acute model and wks 1 and 2 in the chronic 

model. 

AMs: The chronic model had a significant 

correlation between IL-6 and both positive and 

activated AMs at wk 1. A significant inverse 

relationship occurred between Ach and both the 

volume fraction of positive and activated AMs. 

Reference: Tankersley 

et al. (2008, 157043) 

Species: Mouse 

Gender: Male 

Strain: C57BL/6, 

C3H/HeJ, B6C3F1 

Age: 18, 28 mo 

Weight: NR

Carbon black (CB) (Wright dust feed 

particle generator-BGI, Waltham, MA) 

Particle Size: 0.1-1.0 μm 

Route: Whole-body Inhalation 

Dose/Concentration: Average PM2.5

concentration- 401 ± 46 μg/m3, Average PM10

concentration- 553 ± 49 μg/m3

Time to Analysis: 3 h/day, 4 days 

Hemodynamics: CB significantly elevated right 

atrial and ventricular pressures, pulmonary 

arterial pressure and vascular resistance, all of 

which were more pronounced in the 28 mo-old 

mice. RV contractility (specifically, the ejection 

fraction and maximum change in pressure over 

time) reduced in CB-exposed 28 mo-old mice. 

Heart Tissue: CB significantly declined Ca2+-

dependent NOS activity and was more 

pronounced in 28 mo-old mice, who also had 

NOS2 upregulated. CB enhanced ROS 

generation and NOS-uncoupling and was 

greatest in 28 mo-old mice. CB also increased 

MMP-2, MMP-9, ANP, BNP, which were greatest 

in 28 mo-old mice. CB also reduced PKG-1 in 28 

mo-old mice.

December 2009 D-25 
Study Pollutant Exposure Effects 

Reference: Tankersley 

et al. (2007, 097910) 

Species: Mouse 

Gender: Male 

Strain: C3H/HeJ and 

C57BL/6J 

Age: 10 wk

Weight: 22-26 g 

Carbon Black (CB) 

Particle Size: CB: 2.4 μm (MMAD) 

(GSD 2.75 μm). 

Route: CB: Whole-body Inhalation; 

Sympathetic (S) & Parasympathetic (PS) 

blockade: IP Injection 

Dose/Concentration: CB: 159 ± 12 μg/m3; PS 

(atropine): 0.5 mg/kg; S(propanolol): 1 mg/kg 

Time to Analysis: Successive 3 h CB and FA 

Exposures: conducted from 9 a.m. to 1 p.m., or 

at least 3 h after dark-to-light transition 

(exposure period selected based on the nadir in 

circadian pattern in HR responses). 

Subgroups of both strains exposed to PS & S 

blockade. 

FA Exposure with Saline: A significantly greater 

3 h average response occurred in C3 compared 

with B6 mice. 

PS Blockade: No evident strain difference 

between C3 and B6 was observed. 

S Blockade: 3 h average HR responses for C3 

mice were significantly reduced compared with 

saline. 

CB Exposure: HR responses were significantly 

elevated in C3 compared with B6 mice, but these 

HR responses were not different relative to FA 

exposure. 

S Blockade: HR was significantly elevated in B6 

mice during CB relative to FA, but was not 

changed in C3 mice. 

Reference: Tankersley 

et al. (2004, 094378) 

Species: Mice 

Strain: AKR/J 

Age: ~180 days 

Carbon Black (CB) and Filtered Air 

(FA) 

Particle Size: CB: 0.1 to 1 um. 

Route: Whole-body Inhalation 

Dose/Concentration: CB average 

concentration: 160 ± 22 μg/m3

Time to Analysis: FA exposure on day 1, CB 

exposure 3 h/day for 3 consecutive days (days 

2-4) 

On day 1, HR was significantly depressed during 

FA in terminally senescent mice. By day 4, HR 

had significantly slowed due to the effects of 3 

days CB exposure. The combined effects of 

terminal senescence and CB exposure acted to 

depress HR to an average (± SEM) 445 ± 40 

bpm, ~ 80 bpm lower compared to healthy HR 

responses. The change in rMSSD was 

significantly greater on day 1 and day 4 in 

terminally senescent mice, compared to healthy 

mice. LF/HF ratio was significantly depressed in 

terminally senescent mice on day 1. By day 4, 

significant increases in LF/HF were evident in 

healthy mice during CB exposure. Terminally 

senescent mice modulated a lower HR without 

change in the LH/HF ratio during CB exposure. 

Reference: Thomson 

et al. (2005, 087554) 

Species: Rat 

Gender: Male 

Strain: F344 

Weight: 200-250 g 

Urban Ambient Particles (EHC-93) 

from Ottawa, Canada; O3

Particle Size: Respirable Modes 

(aerodynamic diameter): 1.3 and 3.6 

μm. Non-respirable Mode 

(aerodynamic diameter): 15 μm 

Route: Nose-only Inhalation 

Dose/Concentration: EHC-93: 0, 5, 50 mg/m3; 

O3: 0, 0.4, 0.8 ppm 

Time to Analysis: 4 h to particles, O3, or 

combination of particles and O3. 

Both pollutants individually increased preproET-1, 

ET-1 and endothelial NOS mRNA levels in the 

lungs shortly after exposure, consistent w/ the 

concomitant increase in plasma of ET-1[1-21]. 

Prepro-ET1 mRNA remained elevated 24 h postexposure to particles but no after O3. Both 

pollutants transiently increased ET-B receptor 

mRNA expression, while O3 decreased ET-A 

receptor mRNA levels. Coexposure to particles 

plus O3 increased lung preproET-1 mRNA but not 

plasma ET-1[1-21], suggesting alternative processing or degradations of endothelins. This 

coincided w/ an increase of MMP-2 in the lungs 

(this enzyme cleaves bigET-1 to ET-1[1-32]). 

Reference: Thomson 

et al. (2006, 097483) 

Species: Rat 

Gender: Male 

Strain: F344 

Weight: 200-250 g 

Urban Ambient Particles (EHC-93) 

from Ottawa, Canada; O3

Particle Size: NR 

Route: Nose-only Inhalation 

Dose/Concentration: EHC-93: 0, 50 mg/m3; 

O3: 0, 0.8 ppm 

Time to Analysis: 4 h to particles, O3, or 

combination of particles and O3. Sacrificed 

immediately following exposure or following 

24 h recovery. 

Circulating levels of both ET-1[1-21] and ET-3[1-

21] were increased immediately after exposure to 

PM and O3. While expression of preproET-1 

mRNA in the lungs increased, expression of 

preproET-3 mRNA decreased immediately after 

exposure. PreproET-2 mRNA was not detected in 

the lungs, and exposure to either pollutant did not 

affect plasma ET-2 levels. Coexposure to O3 and 

particles, while altering lung preproET-1 and 

preproET-3 mRNA levels in a fashion similar to 

O3 alone, did not cause changes in the circulating 

levels of the two corresponding peptides. 

December 2009 D-26 
Study Pollutant Exposure Effects 

Reference: Totlandsdal 

et al. (2008, 157056) 

Species: Rat 

Gender: Male 

Strain: WKY/NCrl and 

Crl: WI (Han) 

Age: Adult 

Weight: Crl/WI, 250-

300 g 

Use: Isolation of Rat 

Ventricular 

Cardiomyocytes and 

Cardiofibroblasts 

(RVCMs and RVCFBs) 

Pigment Black Printex 90 (Frankfurt, 

Germany); PM: SRM 1648 

Particle Size: Printex 90: 12-17 nm; 

PM: NR 

Route: Cell Culture 

Dose/Concentration: Printex 90: 0, 50, 100, 

200 or 400 μg/mL; PM: 0, 200 μg/mL 

Time to Analysis: 20 h 

Cardiac Cell Cultures: IL-6 release was strongly 

enhanced upon exposure to conditioned media, 

and markedly exceeded the response to direct 

particle exposure. IL-1, but not TNF-α, seemed 

necessary, but not sufficient, for this enhanced 

IL-6 release. The role of IL-1 was demonstrated 

by use the use of an IL-1 receptor antagonist that 

partially reduced the effect of the conditioned 

media, and by a stimulating effect on the cardiac 

cell release of IL-6 by exogenous addition of IL-1 

α and IL-1 β. 

Reference: Tzeng et 

al. (2007, 097883) 

Species: Rat 

Strain: Wistar Kyoto 

Cell Type: Primary 

Vascular Smooth 

Muscle Cell Culture 

(VSMCs): isolated from 

thoracic aortas from 

200-250 g rats. 

Motorcycle Exhaust Particulate Extract 

(MEPE) collected from a Yamaha 

motorcycle with a 50 cm3

 two-stroke 

engine using 95% octane unleaded 

gasoline. 

Particle Size: PM1, PM2.5, PM10

Route: In vitro 

Dose/Concentration: 10-100 μg/mL 

Time to Analysis: 3 days 

Exposure of VSMCs to MEPE (10-100 μg/mL), 

enhanced serum-induced VSMC proliferation. 

The expression of proliferating cell antinuclear 

antigen was also enhanced in the presence of 

MEPE. VSCMs treated with MEPE induced 

increase COX-2 mRNA, protein expression, and 

PGE2 production, whereas the level of COX-1 

protein was unchanged. MEPE increased the 

production of ROS in VSMCs, in a dosedependent manner. MEPE triggered timedependent ERK1/2 phosphorylation in VSMCs 

which was attenuate by antioxidants (NAC, 

PDTC). The level of translocation of NF-κB-p65 

in the nuclei of VSMCs was also increased 

during MEPE exposure. The potentiating effect of 

MEPE in serum-induced VSMC proliferation was 

abolished by COX-2 selective inhibitor NS-398, 

specific ERK inhibitor PD98059, and antioxidants 

(NAC, PTDC). 

Reference: Tzeng et 

al. (2003, 097247) 

Species: Rat 

Strain: Wistar Kyoto 

Cell Type: Primary 

Vascular Smooth 

Muscle Cell Culture 

(VSMCs) 

Motorcycle Exhaust Particulate Extract 

(MEPE) collected from a Yamaha 

motorcycle with a 50 cm3

 two-stroke 

engine using 95% octane unleaded 

gasoline. 

Particle Size: NR 

Route: In vitro 

Dose/Concentration: MEPE: 10 μg/mL; 

Nifedipine: 10 μmol; Manganese Acetate: 100 

μmol; Staurosporine: 1-2 nM; Chelerythrine: 1 

μm 

Time to Analysis: 18 h 

MEPE induced a concentration-dependent 

enhancement of vasoconstriction elicited by 

phenylephrine in the organ cultures of intact and 

endothelium-denuded aortas for 18h. Nifedipine, 

manganese acetate, and staurosporine, but not 

chelerythrine, inhibited the enhancement of 

vasoconstriction by MEPE. ML-9 inhibited the 

enhancement of vasoconstriction by MEPE. 

MEPE enhanced the phosphorylation of 20k-Da 

in rat vascular smooth muscle cells. Nacetylcysteine significantly inhibited the 

enhancement of vasoconstriction by MEPE. A 

time-dependent increase in ROS production by 

MEPE was also detected in primary cultures of 

VSMCs. 

Reference: Upadhay 

et al. (2008, 159345) 

Species: Rat 

Gender: Male 

Strain: SH

Age: 6 mo 

Weight: NR

Ultrafine Carbon Particles (UFCP) 

Particle Size: Size- 31 ± 0.3 nm, 

MMAD- 46 nm, Surface area 

concentration- 0.139 m2 particles/m3

, 

Mass specific surface area- 807m2/g 

Route: Whole-body Inhalation 

Dose/Concentration: 172 μg/m3

Time to Analysis: Acclimatized 2 day. 1 day 

baseline. 24 h exposure. 4 recovery. Sacrificed 

1st or 3rd day of recovery. 

Cardiophysiology: The mean arterial BP and 

HR increased but returned to baseline levels by 

the 4th recovery day. SDNN and HRV decreased. 

RMSSD and LF/HF decreased but were not 

significant. 

Pulmonary Inflammation: UFCP did not cause 

pulmonary inflammation. 

Pulmonary and Cardiac Tissue: HO-1, ET-1, 

ETA, ETB, TF, PAI-1 significantly increased in the 

lung on the 3rd recovery day. HO-1 was 

repressed in the heart, but the other markers had 

slight, nonsignificant increases. 

Systemic Responses: Neutrophil and 

lymphocyte cell differentials significantly 

increased on the 1st recovery day. Other blood 

parameters were unaffected. The plasma renin 

concentration increased on the first 2 recovery 

days. Ang I and II concentrations increased on 

the 1st recovery day but was not significant. 

December 2009 D-27 
Study Pollutant Exposure Effects 

Reference: Wallenborn 

et al. (2008, 191171)

Species: Rat 

Gender: Male 

Strain: Wistar Kyoto 

Age: 13 wk 

Weight: NR

Zinc Sulfate (ZnSO4, aerosolized) 

Particle Size: NR 

Route: Nose-only Inhalation 

Dose/Concentration: 9.0 ± 2.1 μg/m3, 35 ± 8.1 

μg /m3, 123.2 ± 29.6 μg /m3

Time to Analysis: Exposed 5 h/day, 3 days/wk, 

16 wk. Half of the rats used for plasma/serum 

analysis, other half for isolation of cardiac 

mitochondria.

A trend toward increased BALF protein was 

seen. Cardiac mitochondrial ferritin had a small, 

significant increase. Mitochondrial succinate 

dehydrogenase and glutathione peroxidase had 

small, significant decreases. Subchronic 

exposure to 100 μg/m3

 caused expression 

changes of cardiac genes involved with cell 

signaling events, ion channels regulation, and 

coagulation. No pulmonary-related effects were 

seen. 

Reference: Wallenborn 

et al. (2007, 156144) 

Species: Rat 

Gender: Male 

Strain: WKY, SH, and 

stroke-prone SH 

(SHRSP) 

Age: 12-15 wk

PM: precipitator unit power plant 

residual oil combustion 

Particle Size: PM: 3.76 μm (bulk) ± 

2.15 

Route: IT Instillation 

Dose/Concentration: WKY vs SHRSP: 1.11, 

3.33, 8.33 mg/kg 

SH vs SHRSP: 3.33, 8.33 mg/kg

Time to Analysis: Single, 24 h 

Note: 4 h post-exposure study done on WKY vs 

SHRSP but not published.

Oxidative Stress - Cardiac: SOD increased in 

the SHRSP vs WKY experiment only. Only 

SHRSP at 8.33 mg/kg showed a significant 

increase when compared to the control. 

GPx: No action but SHRSP levels were similar to 

SHR and, in the WKY vs SHRSP experiment, 

SHRSP exhibited higher activity level than WKY. 

Ferritin: Equivocal results were observed. Levels 

decreased at the high dose for WKY and SHRSP 

but increased at medium doses for SH and 

SHRSP. 

ICDH: Levels increased for WKY and decreased 

for SHRSP. 

Reference: Wellenius 

et al. (2003, 055691) 

Species: Dog

Gender: Female 

Strain: Mixed mongrel 

Age: NR 

Weight: 14-17 kg

CAPs 

Particle Size: 0.26 ± 0.04 μm 

Route: Permanent Tracheostomy 

Dose/Concentration: Median: 285.7 μg/m3, 

Range: 161.3-957.3 μg/m3

Time to Analysis: Thoracotomy and 

tracheostomy performed. 5-13 wk recovery. 

Pairs of subjects: exposed 6 h/day either 2nd or 

3rd exposure time and filtered air other days. 5 

min preconditioning occlusion. 20 min rest 

interval. 5 min experimental occlusion. Some 

dogs exposed 6 h/d, 4 days (consecutive), 

filtered air on day 4. 

CAPs increased the ST-segment elevation and 

remained elevated 24 h after exposure. This 

increase was seen in precordial leads V4 and V5. 

Multivariate regression analyses showed that the 

mass concentration of Si was significantly 

associated with the peak ST-segment elevation 

and integrated ST-segment change. Univariate 

regression analyses showed Pb to also be 

significantly associated with these measures. 

CAPs had no effect on peak heart rate during 

occlusion or the maximum occlusion-induced 

increase in heart rate. 

Reference: Wellenius 

et al. (2004, 087874) 

Species: Rat 

Gender: Male 

Strain: SD 

Age: Adult 

Weight: ~250 g 

Use: Rat Model for 

Acute Myocardial 

Infarction (AMI): Leftventricular MI induced. 

Animals allowed to 

recover for at least 12 h 

after surgery. 

CAPs (Boston, MA); exposures during 

the period of 07/2000 and 01/2003. 

CO 

Particle Size: PM2.5

Route: Whole-body Inhalation 

Dose/Concentration: CO: 35ppm; CAPs 

(median concentration): 350.5 μg.m3

; 

CAPs+CO: (CAPs median concentration): 

318.2 μg/m3

 

Time to Analysis: 1 h exposure to CAPs or 

CAPs+CO for 1 h. Exposure to pollutants was 

preceded and followed by 1 h exposure to FA. 

CO exposure reduced the ventricular premature 

beat (VPB) frequency by 60.4% during the 

exposure time compared to controls. This effect 

was modified by both infarct type and the number 

of pre-exposure VPBs, and was mediated 

through changes in HR. Overall, CAPs exposure 

increased VPB frequency during the exposure 

period, but this did not reach statistical 

significance. This effect was modified by the 

number of pre-exposure VPBs. In rats with a high 

number of pre-exposure VPB, CAPS exposure 

significantly decreased VPB frequency (67.1%). 

Overall, neither CAPs nor CO had any effect on 

HR, but CAPs increased HR in specific 

subgroups. No significant interactions were 

observed between the effects of CO and CAPs. 

December 2009 D-28 
Study Pollutant Exposure Effects 

Reference: Wellenius 

et al. (2006, 156152) 

Species: Rat 

Gender: Male 

Strain: SD 

Age: Adult 

Weight: ~250 g 

Use: Rat Model for 

Acute Myocardial 

Infarction (AMI): Leftventricular MI induced. 

Animals allowed to 

recover for at least 12 h 

after surgery. 

CAPs: (Boston, MA) 

Particle Size: PM2.5

Route: Whole-body Inhalation 

Dose/Concentration: CO: 35 ppm; CAPs 

(median concentration): 645.7 μg.m3; 

CAPs+CO: 37.9 ppm 

Time to Analysis: CAPs or CAPs+CO 

exposure for 1 h. Exposure to pollutants was 

preceded and followed by 1 h exposure to FA. 

Among rats in the CAPs group, the probability of 

observing supraventricular arrhythmias (SVA) decreased from the baseline to exposure and postexposure periods. The pattern was significantly 

different than that observed for the FA group 

during the exposure period. In the subset with 

one or more SVA during the baseline period, the 

change in SVA rate from baseline to exposure 

period was significantly lower in the CAPs and 

CO groups only, when compared to the FA group. 

No significant effects were observed in the group 

simultaneously exposed to CAPs and CO. 

Reference: Wichers et 

al. (2004, 055636) 

Species: Rat 

Gender: Male 

Strain: SH 

Age: 75 day 

HP-12 (oil-combustion derived PM 

obtained from inside wall of a Boston 

power plant stack burning residual oil 

number 6). 

Water-leachable constituents (μg/mg): 

SO4 (217.3); Zn (11.4); Ni (6.9); Fe 

(0.0); V (1.3); Cu (0.2); Pb (0.0) 

1M HCl-leachable constituents 

(μg/mg): SO4 (220.6); Zn (15.5); Ni 

(14.8); Fe (15.6); V (32.9); Cu (1.1); Pb 

(1.7) 

Particle Size: 3.76 μm (MMAD) (GSD 

2.16) 

Route: IT Instillation 

Dose/Concentration: HP-12 (mg/kg): 0.00 

(saline control), 0.83 (low), 3.33 (mid), 8.33 

(high) 

Time to Analysis: 96 h or 192 h postinstillation. 

Exposures to mid and high-dose HP-12 induced 

large decreases in HR, BP, and body 

temperature. The decreases in HR and BP were 

most pronounced at night and did not return to 

pre-instillation values until 72 h (HR) and 48 h 

(BP) after dosing. ECG abnormalities (rhythm 

disturbances, bundle branch block) were 

observed primarily in the high dose group. 

Reference: Wold et al. 

(2006, 097028) 

Species: Rat 

Gender: Female 

Strain: SD 

Use: Left jugular vein 

and right carotid artery 

were cannulated. 

UFPs from either ambient air (UFAAs) 

or diesel engine exhaust (UFDGs); 

UFIDs from industrial forklift exhaust 

and soluble fraction UFID suspension, 

particle free (SF-UFID) 

Particle Size: UFAAs diameter ≤ 150 

nm; UFDGs diameter ≤ 100 nm 

Route: IV Infusion 

Dose/Concentration: UFDG (50 μg/m) 

Time to Analysis: Infused w/UFAA or UFDG. 

Monitored continuously for 1 h then sacrificed. 

Infusion of UFDGs caused ventricular premature 

beats (VPBs) in 2 out of 3 rats. Ejection fraction 

increased slightly in rats receiving UFAA and was 

unchanged in the UFDG and saline groups. 

Reference: Wold et al. 

(2006, 097028) 

Species: Rat 

Gender: Female 

Strain: SD

UFPs from either ambient air (UFAAs) 

or diesel engine exhaust (UFDGs); 

UFIDs from industrial forklift exhaust 

and soluble fraction UFID suspension, 

particle free (SF-UFID) 

Particle Size: UFAAs diameter ≤150 

nm; UFDGs diameter ≤ 100 nm 

Route: Lagendorff Heart Perfusion 

Dose/Concentration: UFDG (100 μg/2ml); 

UFID (12.5 μg/l in perfusate); SF-UFID (12.5 

μg/l) 

Time to Analysis: Lagendorff 1: Treated 

w/UFDG. Lagendorff 2: Treated with UFID & 

SFUFID. Both experiments were monitored 

continuously for 1 h after injection. 

UFDGs caused a marked increase in leftventricular and end-diastolic pressure (LVEDP) 

after 30 min of exposure. UFIDs caused a 

significant decrease in left-ventricular systolic 

pressure (LVSP) at 30min after the start of 

infusion. This effect was absent when SF-UFID 

was studied.

Reference: Yatera et 

al. (2008, 157162) 

Species: Rabbit 

Gender: Female 

Strain: WHHL 

Age: 42 wk 

Weight: 3.2 ± 0.1 kg 

(avg) 

EHC-93 from Ottawa, Canada 

Particle Size: NR 

Route: IT Instillation 

Dose/Concentration: PM10 suspension: 5 mg 

EHC-93 in 1 ml saline 

Time to Analysis: Exposed 2 times/wk for 4 

wk. Acute effects observed at 0.5, 1, 2, 4, 8, 12, 

and 24 h after initial instillation. Subchronic 

effects observed once/wk for 4 wk. 

Exposure to PM10 caused progression of 

atherosclerotic lesions in thoracic and abdominal 

aorta. It also decreased circulating monocytes 

expressing high levels of CD31 and CD49 day, 

and increased expression of CD54 (ICAM-1) and 

CD106 (VCAM-1) in plaques. Exposure to PM10

increased the number of BrdU-labeled (*) monocytes into plaques and into smooth muscle 

underneath plaques. 

December 2009 D-29 
Study Pollutant Exposure Effects 

Reference: Ying et al. 

(2009, 190111) 

Species: Mice 

Gender: Male 

Strain: ApoE-/-

Age: 16 wk

CAPs:,New York City (Manhattan), NY; 

May-Sept 2007 

Particle Size: PM2.5

Route: Whole-body Inhalation 

Dose/Concentration: 138.4 ± 83.7 μg/m3

Time to Analysis: 6 h/day, 5 day wk, 4 mo 

Vascular Tone: Significant decrease in PEinduced maximum contraction of aortic rings in 

CAPs-exposed mice. No difference in sensitivity 

to PE between groups. Treatment with the 

soluble guanylate cyclase inhibitor ODQ restored 

the response to PE in CAPs aortic rings. No 

significant differences in relaxation induced by 

ACh. CAPs abolished the relaxation induced by 

Ca ionophore A23187. CAPs exposure slightly 

(but significantly) decreased maximum relaxation 

induced by SNP. 

Protein Expression: iNOS mRNA expression 

was increased in the aortas of CAPs-exposed 

mice. eNOS and GTPCH levels were unchanged. 

Distribution of inOS protein expression was 

limited to plaque in air-exposed mice and was 

found in the plaque and media for CAPs-exposed 

mice. 

Superoxide Production: Superoxide levels in 

CAPs-exposed mice were increased in the aorta 

compared to air-exposed mice. The addition of LNAME significantly increased superoxide 

production. Extensive protein nitration in aortas of 

CAPs mice. NADPH subunits Rac1 and p47 phox 

mRNA expression was increased in aortas of 

mice exposed to CAPs. 

Atherosclerosis: Significant increase in plaque 

area of CAPs-exposed mice. Higher levels of 

macrophage infiltration, collagen deposition, and 

lipid composition of plaques from CAPs-exposed 

mice. 

Reference: Yokota et 

al. (2004, 096516) 

Species: Rat 

Gender: Male 

Strain: SD 

Weight: 345-498.2 g 

DEP (obtained from the Japan 

Automobile Research Institute) 

Particle Size: NR 

Route: IT Instillation 

Dose/Concentration: Group 1: DEP: 1 mg/0.1 

ml; Group 2: DEP: 0.2 ml (10, 12.5 or 25 

mg/ml); Group 3: DEP 2.5 or 5 mg/0.2 ml 

Time to Analysis: DEP pre-treatment 24-72 h 

before ischemia/reperfusion. 

DEP Effects on Mmyocardial 

Ischemia/Reperfusion-induced Arrhythmia: 

An increased mortality was observed in the DEP 

group compared to the vehicle-treated group. 

46% of the animals in DEP died during the first 3 

min reperfusion period. The animals of other 

groups were intratracheally instilled with DEP at 

the beginning of ischemia/reperfusion experiment, or were pretreated with polyethylene 

glycol-conjugated SOD (1000 IU/kg, iv). In these 

animals, incidences of both arrhythmia and 

mortality were similar to those in the animals 

treated with the vehicle. 

DEP Rffects on the Biochemical and 

Hematological Parameters: Neutrophil count 

was elevated by a higher dose (5 mg) of DEP at 

24 h after the IT instillation, and oxygen radical 

production, which was induced by 12-Otetradecanoylphorbol 13-acetate, was enhanced 

at 72 h. 

Reference: Yokota et 

al. (2005, 096003) 

Species: Rat 

Gender: Male 

Strain: SD 

Weight: 303-472.2 g 

DEP from Japan 

Particle Size: NR 

Route: IT Instillation 

Dose/Concentration: DEP: 5 mg/animal 

Time to Analysis: Single exposure 0.5, 1, 2, 3, 

6, 12, 24, 48 h. 

At 12 and 24 h post-instillation, circulatory 

neutrophil counts in the 5 mg DEP group were 

significantly elevated, and were 2.1-fold (12 h) 

and 2.3 fold (24 h) in vehicle treated animals. 1 

mg DEP caused an increase of approximately 

0.4-fold in CNC at 6 h. 12-Otetradecanoylphorbol 13-acetate induced 

oxyradical production (ORP) in the isolated 

neutrophil was enhanced at 12 and 24 h after 

instillation with 5 mg DEP. In Serum, a marked 

elevation of CINC-1 and a slight elevation of MIP2 were also observed, while TNF-α was not 

detected. GM-CSF was not detected in serum 24 

h post-instillation. 

December 2009 D-30 
Study Pollutant Exposure Effects 

Reference: Yokota et 

al. (2008, 190109) 

Species: Mouse 

Gender: Male 

Strain: ddy 

Age: NR 

Weight: 39.6-46.0 g

DEP (DMSC (dichloromethane 

soluble-component), RPC (residual 

particle-component)) 

Particle Size: NR 

Route: IT Instillation 

Dose/Concentration: 5 mg/kg, 10 mg/kg 

Time to Analysis: DMSC and RPC extracted 

from DEP. Mice acclimatized 7 day 

. DEP, DMSC, or RPC instilled. BALF and blood 

obtained and G-CSF, GM-CSF, IL-6 measured 

2, 4, 12, 24 h post-instillation. 

Inflammation: At 5 mg/kg DEP increased the 

total cell and macrophage count. DEP or RPC 

increased neutrophils at 5 and 10 mg/kg. 10 

mg/kg DEP or RPC increased macrophages at 

4 h and decreased at 12 h. 

Hematology: Compared to 5 mg/kg DEP, RPC 

increased RBC, WBC, and neutrophils. 10 mg/kg 

RPC or DEP caused sustained increases in RBC, 

WBC, and neutrophils. 

Cytokines: 5 mg/kg RPC markedly increased GCSF and IL-6. Other cytokine increases at this 

dose were transient. 10 mg/kg DEP increased IL6 at 4 h, and DEP or RPC increased G-CSF and 

IL-6 at 12 h. DEP or RPC also increased IL-1β. 

Myocardium: Myocardial MPO activity 

significantly increased in 5 mg/kg RPC at 12 and 

24 h. Myocardial MIP-2 increased the most in 5 

mg/kg RPC, while LIX tended to be lowered by 

RPC. 

Table D-2. Respiratory effects: in vitro studies. 

Study Pollutant Exposure Effects 

Reference: Aam and 

Fonnum (2007, 

155123) 

Species: Human, Rat 

Tissues/Cell Types: 

Human-Neutrophil 

Granulocytes (NG); 

Rat- AM 

DEP: SRM 1975 

Particle Size: NR 

Route: Cell Culture 

Dose/Concentration: NG: 8.8 - 280 μg/mL 

AM:140, 280 μg/mL 

Vitamin E = 5 μM 

Time to Analysis: 1 h 

ROS of NG: Formation of ROS in NG decreased 

with increased doses of DEP. Lucigenin 

chemiluminescence of ROS formation diminished 

25% at 8.8 μg/mL DEP and luminol 

chemiluminescence 32% with 17.5 μg/mL DEP. DCF 

fluorescence required much higher doses of DEP. 

Controls without PMA stimulation had highly reduced 

lucigenin and luminol with DEP dose of 140 μg/mL 

while DCF increased 116%. 

ROS of AM: 280 μg/mL of DEP decreased ROS 

level by 19% with DCF. DEP with PMA-unstimulated 

cells increased 24% with DCF. 

Necrosis: NG cell death was DEP dose-dependent. 

At 280 μg/mL, cell death increased 5.4% as 

compared to control. LDH concentration increased 

1.6% with 70 μg/mL DEP and 3.9% with 280 μg/mL 

after 1 h. 

Reference: Agopyan 

et al. (2003, 056065) 

Species: Human 

Tissues/Cell Types: 

BEAS-2B, NHBE, 

SAEC 

PC: synthetic carboxylate-modified 

particles 

Particle Size: 2, 10 μm 

Route: Cell Culture 

Dose/Concentration: 

PC2 = 0.83 g/mL or 3.4x109 particles/mL 

PC10 = 0.8 g/mL or 3x106

 particles/mL 

Time to Analysis: 

PC2 = 12, 24, 8 h 

PC10 = 2, 6, 12, 24 h 

Calcium Imaging: PC10 induced increase of Ca2+

concentration in all capsaicin-sensitive cells 100%. 

Similar reaction observed in cells exposed to PC2. 

However, more than 3-PC2s were required to induce 

a Ca increase unlike PC10. CPZ (10um) and 

amiloride could fully block PC-induced response. 

cAMP: Post 6 h, a dose-dependent increase in 

cAMP was observed. Again, CPZ blocked increase 

by 70-90% depending on cell type: SAEC >NHBE ~ 

BEAS-2B. 

Apoptosis: PC10 and PC2 induced apoptosis timedependently. PC2 was slower in induction than 

PC10. Post 48 h, 80-95% cells were apoptotic in all 

cell types. Noncapaisin-sensitive cells (which did not 

bind to particles) did not exhibit apoptosis. CPZ 

reduced apoptosis by 97% BEAS-2B, 96% NHBE 

and 98% SAEC. Amiloride did not block apoptosis. 

Necrosis: Induction of necrosis by PC2 and PC 10 

was negligible. A slight increase from 1% to 2% was 

observed at 24-48 h in NHBE and SAEC. BEAS-2B 

showed slight decrease from 3% to 4% in same time 

period. 

December 2009 D-31 
Study Pollutant Exposure Effects 

Reference: Agopyan 

et al. (2004, 156198) 

Species: Human, 

Mouse 

Tissues/Cell Types: 

Human-NHBE, SAEC; 

Mouse-Wildtype and 

TRPV1(-/-) Terminal 

Ganglion Neurons 

(TG) 

ROFA 

MSHA: Mt St Helen Ash 

Particle Size: NR 

Route: Cell Culture 

Dose/Concentration: 100 μg/mL ROFA or 

MSHA 

Time to Analysis: ROFA/MSHA in NHBE 

and SAEC = 2, 6, 24, 48 h 

ROFA/ MSHA in TG = 24 h 

cAMP measurements with NHBE and SAEC 

exposed to ROFA/MSHA = 6 h 

Calcium Imaging in NHBE and SAEC: In 100% of 

reactive cells, ROFA/MSHA induced an increase in 

Ca2+. Levels remained elevated as long as PM 

bound to plasma membrane. Washing and disjoining 

PM from membrane caused Ca2+ to slowly decline to 

baseline. CPZ (or CPZ and amiloride) reversibly 

inhibited PM-induced rises in Ca2+. 

Calcium Imaging in TRPV1(+/+) and (-/-) mice 

sensory neurons: All sensitive neurons in 

TRPV1(+/+) increased Ca2+ in response to ROFA. 

No effect of ROFA in TRPV1(-/-). 

cAMP: ROFA and MSHA induced increases in Ca2+

in NHBE and SAEC cells, which was completely 

blocked by cAMP. 

Apoptosis: ROFA or MSHA induced timedependent apoptosis, peaking at 24 h. CPZ again 

inhibited this response. Neurons bound to PM 

(<25um) induced apoptosis in TRPV1(+/+). Cells 

without bound PM or bound with PM (>25 μm) 

showed no effect. No apoptosis occurred in the 

absence of Ca2+. 

Necrosis: Necrosis for any of the cell types was 

negligible. 

PKA: Inhibition of PKA resulted in 90+% apoptosis 

in NHBE and SAEC. Again, no apoptosis was 

observed in a Ca2+ free environment. 

Reference: Ahn et al. 

(2008, 156199) 

Species: Human 

Tissues/Cell Types: 

A549 

DEP: (6 cyl, 11L, turbo-charged, heavyduty diesel engine, South Korea) 

Dex: anti-inflammatory (Sigma, St. 

Louis, MO) 

Particle Size: NR 

Route: Cell Culture 

Dose/Concentrations: 0, 1, 5, 10, 50 and 

100 μg/mL of DEP 

Some cells pre-treated with 10, 20, 40, 50 

pg/mL of Dex. 

Time to Analysis: 24 h 

COX-2 Expression: Cells expressed dosedependent increases in COX-2 expression after 

treatment with 10-100 μg/mL of DEP. Treatment of 

50 μg/mL for 24 h induced statistically significant 

COX-2 expression in both mRNA and protein levels. 

Pre-treatment with Dex significantly reduced 

expression of COX-2 mRNA and protein. Dex 

treatment induced dose-dependent suppression of 

DEP-induced protein levels. 

PGE2 Levels: Levels of the inflammatory mediator, 

PGE2, increased when were cells exposed to 50 

μg/mL of DEP. Pre-treatment with 50 μg /mL Dex 

completely inhibited DEP-induced release of PGE2. 

Reference: Ahsan 

(2005, 156200) 

Species: Human 

Tissues/Cell Types: 

Trx-1-transfected 

Clone of Murine L-929 

cells; Control Clone (L929-Neo1); A549 

DEP: provided by Dr. Masaru Sagai, 

University of Health and Welfare, 

Aomori, Japan 

Particle Size: NR 

Route: Cell Culture 

Dose/Concentration: 

DEP: 50 μg/mL 

hTrx-1- or L-929-Neo1: 40 μg/mL 

Pretreatment: rhTrx-1 (10 μg/mL) or DMrhTrx-1 (NR)

Time to Analysis: Pretreatment for 1 h. 

Parameters measured 3 h post exposure. 

ROS: DEP induced significant increases of ROS in 

L929-Neo1 cells. hTRx-1 cells showed no affect. RTPCR revealed hTrx-1 mRNA expression in 

transfected cells but not control L929-Neo1 cells. 

Endogenous murine Trx-1 mRNA expression 

increased in control cells, but not in hTrx-1 cells. 

A549 cells had increased ROS levels but these 

levels were suppressed with rhTrx-1 pretreatment. 

Pre-treatment with DM-rhTrx-1 increased ROS 

levels more. 

Akt (antiapoptotic molecule): Phosphorylated Akt 

prevents apoptosis. DEP induced phosphorylation of 

Akt in control cells after 3 h and dephosphorylation 

after 5 h. In hTrx-1 cells, Akt remained 

phosphorylated after 5 h. In A549 cells, Akt 

phosphorylated at 3 h and slowly turned off at 12-

24 h. Pre-treatment with rhTrx-1 blocked 

dephosphorylation. This suggests that Trx-1 

preserves active form of Akt and thereby protects 

against cytotoxicity from DEP. 

December 2009 D-32 
Study Pollutant Exposure Effects 

Reference: AlfaroMoreno et al. (2002, 

156204) 

Species: Human, 

Mouse, Rat 

Strain: Human-A549; 

Mouse-J7 74A.1, 

BALB-c 

Tissues/Cell Types: 

HUVEC, Mouse 

Fibroblasts, Rat Lung 

Fibroblasts (RLF)

PM10: Collected from 3 zones in Mexico 

City: North (industrial), Center 

(business) and South (residential) 

Particle Size: PM10

Route: Cell Culture 

15000 cells/cm2

 except: 

Cytotoxicity: Confluent Cultures 180,000 

cells/cm2

. 

DNA Breakage: 20,000 cells/well. 

Cytokine Assays: 180,000 cells/cm2

Dose/Concentration: Cytotoxicity: 10, 20, 

40, 80, 160 μg/cm2

Apoptosis: 160 μg/cm2 

DNA Breakage: 2.5, 5, 10, 20, 40 μg/cm2

 

Cytokine Assays: 10, 20, 40, 80 μg/cm2

 

E-Selectin Expression: 40 μg/cm2

Time to Analysis: Cytotoxicity: 24, 48, 72 h; 

Apoptosis: 24 h; DNA Breakage: 72 h; 

Cytokine Assays: 24 h 

Cytotoxicity: Cytotoxic effect exhibited dosedependency after 72 h in proliferating cells of 

J774A.1, BALB-c and RLF cell lines. 

Proliferating Cells: Northern particles induced a 

statistically larger effect than central or southern 

particles. J774A.1 was more susceptible while 

BALB-c was less susceptible. A549 was most 

resistant to decreased viability during exposure. No 

significant variation in viability was observed when 

compared to the control. Particles were not cytotoxic 

among confluent cell growth for any cell lines when 

exposed to 20-160 μg/cm2

. 

Apoptosis: Overall, particles induced low rates of 

cell death via apoptosis. J774A.1 depicted similar 

levels of apoptosis when exposed to three PM 

zones, ~15% apoptotic cells measured. BALB-c was 

not reported. Results for the A549 measured 

apoptotic cells were: South- 4%, Central- 11% and 

North- 15%. HUVEC cells indicated an increase in 

apoptosis with northern particles. 

DNA Breakage: PM10 from all zones induced DNA 

breakage. A dose-dependent relationship was 

established with PM2.5 particles at concentrations of 

10 μg/cm2. The Southern zone required a higher 

dose of PM (10 μg/cm2

) to produce the same effect 

as other zones (2.5 μg/cm2

). 

Cytokines: Particles induced TNF-α and IL-6 

secretion in J774A.1 cells dose-dependently. IL-6 

increased significantly with central particles. PGE2 

secretion in RLF cells induced by exposure to PM 

showed dose-dependent responses. PM from the 

central zone induced the most PGE2 secretion. Max 

secretion was observed at doses of 40 μg/cm2

 from 

all three PM zones. 

E-Selectin Expression: HUVEC cells showed a 

25% increase in E-selectin expression after 

exposure to 40 μg/cm2 of PM. 

Reference: Amakawa 

et al. (2003, 156211) 

Species: Mouse, 

Human 

Strain: Mouse-ICR 

Tissues/Cell Types: 

AMs 

Gender: Male 

Age: Mouse 6-7 wk; 

Human 20-24 yr 

DEP (obtained from a 4JB1, Isuzu, 

1500 rpm, 4cyl diesel engine) 

DEPE = DEP Extract (methanol) 

CB = Charcoal (Sigma) 

Particle Size: DEP- 0.4 μm, CB- 0.7 

μm 

Route: Cell Culture 

Mouse: 5×105

 cells/mL; Human: 3×105

cells/mL 

Dose/Concentration: DEP = 1 or 10 μg/mL; 

DEPE = 1 or 10 μg/mL; CB = 1, 10, 100 

μg/mL 

Time to Analysis: Human cells pre-treated 

with LPS 1 μg/mL. Murine cells pre-treated 

with SOD 300 IU/mL. Parameters measured 

24 h post exposure. 

Cells: For mice, more than 90% of the cells were 

macrophages and over 90% were viable. For 

humans, 96% of the cells were macrophages, 3% 

lymphocytes and 1% neutrophils; over 95% of the 

human cells were viable. 

DEP Cytotoxicity: None observed 

Cytokines: DEP (10 μg/mL) suppressed release of 

TNF-α and IL-6 for both mice and humans in a dosedependent manner. Murine cells pre-treated with 

LPS or IFN-y released even less TNF-α and IL-6. IL10 was unaffected. Human macrophages pretreated with LPS also released lower levels of TNFα, IL-6 and IL-8. 

ROS: Pre-treatment of SOD on murine cells partially 

attenuated the suppressive effect of DEP as well as 

decreased the production of ROS generated by DEP 

(10 μg/mL). 

Carbon: Carbon particles did not suppress TNF-α or 

IL-6 release from murine AMs; however,100 μg/mL 

of CB stimulated TNF-α production. 

Methanol: No cytotoxicity nor cytokine release 

effects were observed. 

DEPE: DEPE suppressed TNF-α and IL-6 release in 

a similar way as DEP. 

December 2009 D-33 
Study Pollutant Exposure Effects 

Reference: Amara et 

al. (2007, 156212) 

Species: Human 

Cell Lines: A549, NClH292 

DEP = SRM 2975 

CSC = cigarette smoke condensates 

(collected from Kentucky standard 

cigarettes, 2R4F; University of 

Kentucky) 

DC = DEP + CSC 

CB (Degussa, Frankfurt, Germany) 

Particle Size: CB: 95 nm; DEP: NR 

Route: Cell Culture 

Dose/Concentration: DEP = 5-10 μg/cm2

CB = 10 μg/cm2

 

CSC = 10 μg/cm2

Time to Analysis: 6 or 24 h 

Inflammatory Markers: LDH of A549 was 

unaffected at either time point with DEP or CB. LDH 

increased with CSC at concentrations high than 10 

μg/mL at both time points. DC had no effect. 

Proteases: MMP-1 mRNA expression showed a 

dose dependent increase with DEP in A549 cells. 

DEP also increased MMP-1 in NCI-H292 cells. CB 

and CSC had no effect. MMP-1 mRNA expressions 

were inhibited by N-acetylcysteine antioxidant. 

Similar inhibition was observed with NOX4 oxidase. 

DC induced a similar effect to DEP. MMP-1 protein 

expression increased post 24 h with DEP. MMP-2, 

TIMP-1, TIMP-2 mRNA expression was unaffected. 

TGF: TGF-β mRNA expression was unaffected. 

ROS: DEP and DC increased ROS formation after 1 

h. DEP effect was inhibited by N-acetylcysteine 

antioxidant pre-treatment. 

MAP-Kinase: DEP induced MMP-1 expression 

increased ERK1/2 phosphorylation after 10 min, 

peaking at 30 min, and returning to normal levels at 

60 min. Treatment with CBPs did not increase 

ERK1/2 phosphorylation whereas treatment with 

CSC resulted in phosphorylation. Only inhibitors of 

ERK1/2 reduced DEP induced MMP-1 activity. P38 

and JNK inhibitors had no effect. 

Reference: Anseth et 

al. (2005, 088646) 

Species: Human 

Cell Lines: A549; 

A549-p0 (lacking 

mitochondria) 

s-ROFA: soluble portion 

Particle Size: 1.95 ± 018 μm 

Route: Cell Culture (3X105 cells/mL) 

Dose/Concentration: 100 μg/mL 

Time to Analysis: Experiments conducted 

by spreading monolayer of Infasurf (calf lung 

surfactant extract on PBS, PBS+ROFA or 

conditioned media from A549 AEC. 

Parameters measured after one 6-h 

incubation period. 

Lung Surfactant Gelation: ROFA alone and A549 

conditioned media alone did not significantly alter 

Infasurf rheology. However, conditioned media from 

A549 AEC at 16 h induced a significant increase in 

elastic storage and viscous loss moduli. Inhibiting 

ROS production lowered effect, indicating s-ROFA 

gelation mediated through ROS. 

ROS: ROS mediated through mitochondria as 

evidenced by the effect of ROFA-AEC on surfactant 

gelation in the presence of mitochondria ROS 

inhibitors as well as A549-p0 cells. 

Reference: Auger et 

al. (2006, 156235) 

Species: Human 

Tissue/Cell Type: 

Nasal Epithelial Cells 

DEP: SRM1650 

PM2.5: obtained from a highway in 

Paris, France 

Particle Size: DEP: 400 nm (mean 

diameter); PM2.5

Route: Cell Culture (2-3.5x104 cells/cm2) 

Dose/Concentration: 10-80 μg/cm2 

Time to Analysis: Cells treated on apical 

side. Parameters measured 24 h following 

treatment. 

Cytotoxicity (LDH): No cytotoxicity for DEP or 

PM2.5 (80 μg/cm2

). 

Cytokines: In non-stimulated ALI cultures, IL-8 was 

the most abundantly secreted cytokine, followed by 

GM-CSF, TNF-α, and IL-6 in decreasing levels of 

production. Amphiregulin was moderately, but 

consistently, secreted. After treatment, both DEP 

and PM2.5 induced IL-8 and amphiregulin release in 

a dose-dependent manner through the basolateral 

surface. PM2.5 stimulated IL-6 and GM-CSF release 

through the apical surface. 

ICAM-1 expression: No effect from DEP or PM2.5. 

ROS: DEP and PM2.5 both increased ROS 

production in a dose-dependent manner. 

December 2009 D-34 
Study Pollutant Exposure Effects 

Reference: Bachoual 

et al. (2007, 155667) 

Species: Mouse 

Cell Type: RAW 264.7 

PM10 from two Paris, France subway 

sites: RER and Metro 

CB (Frankfurt, Germany) 

TiO2 (Calais, France) 

DEP: SRM1650 (NIST) 

Particle Size: CB: 95 nm; TiO2:150 nm; 

DEP: NR 

RER PM10: 79% <0.5 μm, 20% 0.5-1 

μm; 

Metro PM10: 88% <0.5 μm, 11% 0.5-1 

μm. 

Route: Cell Culture (40,000 cells/mL) 

Dose/Concentration: All particles: 0.01, 

0.1, 1, 10 μg/cm2

Time to Analysis: 3, 8, 24 h 

Cell Viability: No effects from any particulate at 

concentrations up to 10 μg/cm2

 for 24 h. 

Inflammatory Effect: Exposure of cells to 10 

μg/cm2

 of RER or Metro induced time-dependent 

increase in TNF-α and MIP-2 protein release. This 

effect was similar to both locations. No effect was 

observed at low concentrations of PM10. No effect of 

CB, TiO2 or DEP was observed. 

GM-CSF or KC production: RER and Metro PM10

did not induce any effect at any concentration. 

Effect on Protease mRNA Expression: Exposure 

of cells to 10 μg/cm2

 RER or Metro PM10 did not 

modify mRNA expression of MMP-2 or -9 or their 

inhibitors TIMP-1 and -2. MMP-12 expression 

significantly increased after exposure to RER or 

Metro PM10 for 8 h. 

Effects on HO-1 Protein Expression: Exposure to 

10 μg/cm2

 of RER or Metro PM10 for 24 h induced 

positive cytoplasmic staining for HO-1. 

Reference: Baulig et 

al. (2007, 151733) 

Species: Human 

Cell Line: 16-HBE14oWUB: Winter Urban Background 

Particles (obtained from Vitry-surSeine, suburb of Paris, France) 

SUB: summer Urban Background 

Particles Vitry-sur-Seine) 

WC: Winter Curbside Particles, 

SRM1648 (obtained from Ported’Auteuil, ring road of Paris, France) 

SC: Summer Curbside Particles, SRM 

1648 (Porte-d’Auteuil) 

DEP: SRM 1650a (NIST) 

DPL (control) 

Particle Size: WUB, SUB: PM2.5; WC, 

SC, DEP: NR 

Route: Cell Culture (20,000 cells/cm2) 

Dose/Concentration: 10 μg/cm2

Time to Analysis: 18 or 24 h 

EGF: All native PM2.5 induced similar AR secretion 

by bronchial epithelial cells (in decreasing order WC, 

WUB, SC, SUB), but this release was significantly 

greater than the release induced by DEP. β-cellulin 

increased with SC, WUB and WC. No data was 

available for SUB or DEP. 

Interleukins: IL-1α increased significantly with 

WUB, WC,SC, DEP, DPL (in decreasing order). No 

data was available for SUB. Exposure to WUB 

caused IL-1β to increase to induction factor of over 

2. IL-11 R α decreased significantly with SUB. 

Cytokines: Exposure to WUB caused G-CSF to 

increase with an induction factor of over 2.Though 

not statistically significant, TNF-R1 also increased. 

Proteases: TIMP-2 decreased with WUB but 

significantly increased with SUB. Overall, SUB 

downregulated integrins and interleukins seen with 

other particles while upregulating neurotrophic 

factors, chemokine receptors and adhesion 

molecules. MMPs were not measured. 

Chemokines: CCR-3 significantly increased with 

SUB. GRO-γ and GRO-α increased with WC at both 

18 and 24 h. DEP had no effect with GRO-α. 

Removal of metal from particles lowered response of 

GRO-α. 

December 2009 D-35 
Study Pollutant Exposure Effects 

Reference: Bayram et 

al. (2006, 088439) 

Species: Human 

Cell Type: A549 

DEP: (obtained from a 4JB1-type, lightduty, 4 cyl, 2.74-L Isuzu diesel engine) 

DEP-FCS: DEP + FCS 

DEP-NAC: DEP + N-acetylcystine, 

antioxidant 

DEP-A: DEP + AEOL10113, catalytic 

antioxidant 

DEP-S: DEP + SP600125, inhibitor of 

JNK 

DEP-N: DEP + SN50, inhibitor of NFkB 

Particle Size: DEP: 0.4 μm (mean 

diameter) 

Route: Cell Culture 

Dose/Concentration: DEP: 0, 5, 10, 50, 

100, 200 μg/mL 

Time to Analysis: 24, 48, 72 h 

Cell Growth: With 10% FCS (as a positive control), 

A549 cells exhibited time dependent growth. A 

mixture of FCS and DEP did not affect cell growth 

for up to 48 h. With DEP alone, cell growth was 

prevented from cell number reduction due to 

removal of serum at 48 and 72 h. A dose of 10 

μg/mL induced a maximum proliferation effect. 

Cell Cycle: DEP increased the percentage of 

serum-starved cells in S phase at 48 h. DEP 

decreased the percentage in G0/1 phase and G2/M 

phase. 

Apoptosis: DEP prevented the increase in 

apoptotic, serum-starved cells.

Protein Expression: p21CIP1/WAF1 expression 

increased at 48 h. DEP dose-dependently 

decreased this expression. 

NAC: NAC alone, at 33 mM, induced an increase in 

cell numbers. DEP-NAC inhibited cell numbers at 48 

h. DEP-NAC inhibited cell numbers in S phase; thus, 

cells in G0/1 phase increased. DEP-NAC induced a 

further decrease of cells in G2/M phase. 

AEOL10113: DEP-A caused a dose-dependent 

decrease in cell numbers. 

SP600125: Alone, SP600125 increased cell 

numbers at 33 mM. DEP-S decreased cell numbers. 

Reference: Becher et 

al. (2007, 097125) 

Species: Rat 

Strain: Crl/WKY 

Cell Type: AM, 

Alveolar Type II 

Gender: Male 

Weight: 200 g 

SPM = suspended PM SRM-1648 

Particle Size: 6-8 μm 

Route: Cell Culture (1.5×106 cells/well AM; 

6×106

 cells/well Type II) 

Dose: 200 μg/mL = 20 μg/cm2

Time to Analysis: 20 h 

Cytokines in Macrophages: SPM increased TNF-α

and MIP-2. NADPH inhibitor DPI reduced MIP-2 

response, whereas iNOS inhibitor 1400W did not 

affect either. 

Cytokines in Type 2 Cells: SPM increased IL-6 and 

MIP-2 significantly. This SPM effect was inhibited by 

DPI, whereas1400W reduced the IL-6 response 

significantly. 

ROS in Type 2 Cells: SPM significantly increased 

ROS formation. DPI largely blocked this SPM effect. 

ROS in Macrophages: No significant increases 

were observed. 

Reference: Becker et 

al. (2005, 088590) 

Species: Human 

Gender: Male and 

Female 

Age: 18-35 yr 

Cell Types: Alveolar 

Macrophages, NHBE 

PM (Coarse, Fine, Ultrafine): Chapel 

Hill, NC 

Particle Size: PM-C: PM2.5; PMF: PM0.1; PM UF: <0.1μm 

Route: Cell Culture (0.5-1×105 cells/well 

NHBE; 2-3×105/mL AM) 

Dose/Concentration: NH BE: 25, 50, 100, 

250 μg/mL of PM; AMs: 50 μg/mL of DEP or 

10 ng/mL of LPS 

Time to Analysis: 18h for NHBE; overnight 

for AMs 

Cytokines: All 3 fractions induced dose-dependent 

increases in IL-8 secretion with PM-c, PM-F, PM-UF 

(in order of decreasing effects). TLR-2 antibody 

blocked these particle induced IL-8 effects. 

Inhibitors of Endotoxin effects and TLR-4 

activation: No effects were observed in NHBE, but 

all 3 fractions repressed the IL-6 release in AMs. 

TLR mRNA Expression: PM did not affect TLR-2 

mRNA in NHBEs. PM-C and PM-F induced a slight 

increase in TLR-4 mRNA in NHBEs while PM-UF 

induced a substantial increase. PM-C increased 

TLR-2 mRNA in AMs and decreased TLR-4 mRNA 

in AMs. 

Induction of Hsp70: PM-C and PM-F induced 

Hsp70 in NHBE dose-dependently. Hsp70 was not 

induced in AM following particle stimulator. 

December 2009 D-36 
Study Pollutant Exposure Effects 

Reference: Becker et 

al. (2005, 088592) 

Species: Human 

Gender: Male 

Age: 18-35 yr 

Cell Types: AM, 

NHBE 

PM (Coarse, Fine, Ultrafine): Chapel 

Hill, NC 

ROFA 

Fe, Si, Cr Components 

Oct 2001, Jan 2002, April 2002, July 

2002 

Particle Size: PM-C: 2.5-10 μm; PM-F: 

<0.1 μm; PM-UF: <0.1 μm 

Route: Cell Culture (3-5×105 cells/well 

NHBE; 2-3×105

 cells/mL AM) 

Dose/Concentration: NHBE: 11 μg/mL of 

PM; AM: 50 μg/mL of PM 

Time to Analysis: 18-24 h NHBE; 18 h AM 

IL-8 Release in NHBE: PM-C and PM-UF induced 

effects. No effects from PM-F (all 4 dates). 

IL-6 Release in AM: All 3 fractions induced increase 

with later dates having generally lower effects. 

ROS (DCF): NHBE, at lower exposures, were 

observed to be more responsive to PM than AMs. 

AM exhibited highly variable results over time. 

ROS (DHR): NHBE cells were observed to be more 

responsive to PM than AMs. AM responsiveness to 

PM increased over 4 time periods; this was not 

observed in NHBE. 

Seasonal Variability: Coarse particles were more 

potent than F and UF regardless of the month, and 

the potency for PM to induce IL-6/IL-8 production 

varied significantly. Coarse particles induced a 5-25 

fold change in IL-6 release for AMs and a 3-6 fold 

change in IL-8 release for NHBEs. 

Metal Correlation to IL-6/8 induction: Fe and Si 

were positively associated with IL-6 release in AMs 

incubated with the coarse fraction. Cr was positively 

associated with IL-8 release in NHBE cells 

incubated with F or UF. 

Reference: BeckSpeier et al. (2005, 

156262) 

Species: Human, 

Canine (Beagle) 

Cell Types: Human 

AMs, Canine AM 

(CAM) 

DEP = SRM 1650a (NIST) 

EC = Ultrafine EC (spark discharge) 

P90 = Printex 90 (Carbon Black, 

Degussa) 

PG = Printex G (Carbon Black, 

Degussa) 

Particle Size: DEP: 20-40 nm; EC: 5-

10 nm; P90: 14 nm; PG: 51 nm 

Route: Cell Culture (1×106 cells/mL AM) 

Dose/Concentration: All particles: 1 (EC 

only), 3.2, 10, 32, 100 μg/mL 

Time to Analysis: 60 min 

Phagocytosis: All particles were phagocytosed by 

CAM within 60 min. 

Oxidative Potential: EC showed a very high effect. 

DEP, P90 and PG had no effect 

Formation of Lipid Mediators: DEP, EC P90 and 

PG increased arachidonic acid and PGE2/TXB2 in 

CAM in a dose-dependent manner. Only EC 

increased LTB4 and 8-isoprostane. 

ROS Activation: All particles increased activity in 

canine macrophages with EC, P90 and PG 

increasing activity in a dose-dependent manner. 

DEP increased activity in canine macrophages. 

Similar results were observed human alveolar 

macrophages but only EC and P90 were tested. 

Particle Mass vs Particle Surface Area:

PGE2/TXB2 effects were highly correlated with 

particle surface area. 

Reference: Bitterle et 

al. (2006, 156276) 

Species: Human 

Cell Type: A549 

C-UFP = ultrafine carbonaceous 

particles (obtained from a spark 

discharge aerosol generator GFG 

1000, Palas, Karlsruhe, Germany) 

Particle Size: 90 nm (count median 

mobility diameter) 

Route: Cell Culture (3×107 cells) 

Dose/Concentration: 44 ± 4 ng/cm2; 87 ± 

23 ng/cm2

; 230 ± 70 ng/cm2 

Time to Analysis: 6 h 

Cell Viability: Exposure to clean air resulted in a 

93.7 ± 9.1% viability. Exposure to low, mid and high 

doses of C-UFP resulted in a 94.9 ± 9.5% viability. 

Thus C-UFP had no effect on cell viability. 

Interleukins: Clean air controls induced a 2-3 fold 

increase in IL-6 and IL-8 production vs submersed 

control. U-CFP exposures induced a similar effect 

on IL-8 and IL-6 levels. 

Antioxidant enzyme HO-1: The mid dose 

increased transcription of HO-1 by 2.7 fold. There 

was no observed effect at the high dose level which 

indicates possible cytotoxicity. 

December 2009 D-37 
Study Pollutant Exposure Effects 

Reference: Blanchet 

et al. (2004, 087982) 

Species: Human 

Cell Type: 16HBE 

PM2.5 

(Vitry-sur-Seine, Paris, France) 

DEP = SRM 1650a 

CB = Carbon Black (Degussa) 

TiO2 (Huntsman) 

Particle Size: CB: 95 nm; TiO2: 150 nm

Route: Cell Culture (45,000 cells/cm2) 

Dose/Concentration: All particles: 0.1, 1, 

10, 30 μg/cm2

Time to Analysis: 6, 18, 24, 30 h 

Amphiregulin Expression: DEP and PM2.5 both 

increased AR mRNA expression from 6 to 30 h, with 

PM2.5 inducing higher expression levels than DEP. 

Both DEP and PM2.5 increased AR protein secretion. 

No observed effect for CB and TiO2. PM2.5 induced 

protein secretion dose-dependently. 

Signal Pathways in AR Secretion: MAP kinase 

and tyrosine kinase inhibitors reduced effects of 

DEP and PM2.5 but p38MAP kinase inhibitor did not. 

Role of Oxidative Stress: N-Acetylcysteine blocked 

AR secretion following PM2.5. Antioxidant enzyme 

catalase had no effect. 

Cytokines: DEP induced a significantly high release 

of GM-CSF, higher than PM2.5. EGFR antibody 

reduced GM-CSF release at 0.25 μg/mL dose. 

Reference: Bonvallot 

et al. (2001, 156283), 

Species: Human 

Cell Type: 16HBE14oDEP: SRM 1650 

OE-DEP: dichloromethane extract (2x) 

of DEP 

nDEP: native DEP 

sDEP: nDEP - OE-DEP 

CB: Carbon Black FR103 (Degussa) 

BaP: Benzo[a]pyrene 

CB: 95 nm 

NR 

Particle Size: CB: 95 nm; DEP: NR 

Route: Cell Culture (3×106 cells) 

Dose/Concentration: DEP, sDEP, nDEP 

and CB = 10 μg/cm2

OE-DEP = 15 μg/mL 

BaP = 0.25, 50 and 250 μg/mL 

Time to Analysis: 24 h 

Proinflammatory Response: At 10 μg/cm2, nDEP 

induced GM-CSF release by 4.7 fold. OE-DEP 

increased GM-CSF by 3.7 fold. BaP and sDEP also 

induced increases of CN-CSF but had smaller effect. 

CB had no effect. 

NF-κB Activation: nDEP and OE-DEP induced 

enhanced degradation of IκB at 2-4 h and 1 h 

respectively. NF-κB DNA binding was enhanced by 

OE-DEP (15 μg/mL, peak <1 h) and nDEP (10 

μg/cm2

, peak at 2-h with plateau till 4 h). Both OEand nDEP enhanced NF-kB DNA binding levels 

were higher than BaP enhanced binding levels. 

CYP1A1 mRNA: The CYP1A1 mRNA level was 

markedly increased in nDEP and OE-DEP treated 

cells in comparison with their respective controls. 

Radical Scavengers (decreased ROS in situ):

Increases of GM-CSF and NF-κB DNA binding by 

nDEP and OE-DEP was attenuated by radical 

scavengers. 

MAPK Activation: Increases by nDEP and OE-DEP 

of GM-CSF was inhibited by Erk1/2 inhibitor but not 

by p38 inhibitors. Both nDEP and OE-DEP triggered 

Erk1/2 and p38 phosphorylation. sDEP affected p38 

phosphorylation only. 

Reference: Brown et 

al. (2007, 156300) 

Species: Human, 

Mouse 

Cell Type: PBMC, 

A549 (Human); 

J774A.1 (Mouse) 

PM10 (London, England) 

CM from PM10-treated human 

monocytes 

Particle Size: PM10

Route: Cell Culture (1×106 cells/mL 

J774A.1; 5×106

 cells/mL PBMC; 5×105

cells/well A549) 

Dose/Concentration: PM10: 75 μl (10 

μg/mL); CM: 250 μl; tBHP: 12.5 μm (in 

J774); TNF: 0, 500 pg, 1 ng, 10 ng 

Time to Analysis: tBHP:1, 2, 4 h; PM: 4 h; 

TNF: 18 h 

Cytokines: PM10 induced release TNF-α protein 

from PBMCs at 10 μg/mL for 4 h. Further inhibited 

by verapamil and BAPTA-AM. Calmodulin inhibitor 

W-7 had no effect. CM increased IL-8 from A549 

cells 3 fold. Verapamil, BAPTA-AM and W-7 

significantly inhibited IL-8 release induced by CM. 

ICAM-1: A549 cells treated with TNF-α showed 

dose-dependently effect of TNF-α on ICAM-1 

upregulation at 18 h. CM also induced upregulation. 

Verapamil, BAPTA-AM and W-7 fully inhibited CMinduced upregulation. 

December 2009 D-38 
Study Pollutant Exposure Effects 

Reference: Calcabrini 

et al. (2004, 096865) 

Species: Human 

Cell Type: A549 

PM2.5 (Rome, Italy) 

Particle Size: PM2.5 

Route: Cell Culture (5×104 cells/well) 

Dose/Concentration: 30, 60 μg/cm2 (aliquot 

of 0.1 μg/μl) 

Time to Analysis: 5, 24, 48, 72 h 

Particle Characterization: Components measured 

include C-rich particles, Ca sulfates, silica, silicates, 

Fe-rich particles, metals. Carbonaceous particles 

made up majority of PM. 

Cell Surface Changes: PM deposited on the cell 

surface showed dose and time-dependent increases 

in microvilli rearrangement and cell shape alterations 

without affecting apoptotic markers for up to 72 h. 

PM internalization: At 24 h with the low dose, 

aggregates of PM in cytoplasm or surrounded by 

membrane was observed. With the high dose, large 

particle aggregates often close to nuclear envelopes 

were observed. 

Cytoskeleton: At 72 h PM induced dose-dependent 

alterations from rearrangement/interweaving of 

microtubules to bundling of microtubules with some 

shortening/disruption. 

Cell Growth: PM decreased cell growth in a dose 

and time-dependent manner 

ROS: PM increased ROS at the high dose for 5 h 

but not at 24 h or with the low dose. 

Cytokines: PM induced TNF-α peaked at 5 h at 

high dose and 48 h at low dose, both ND at 72 h. 

PM induced IL-6 starting at 24 h thru 72 h in time 

and dose dependent manner. 

Reference: Cao et al. 

(2007, 156322) 

Species: Human 

Cell Type: HAEC 

NIST-DEP: collected using a diesel 

forklift and hot bag filter system. (NIST, 

Minneapolis, MN) 

C-DEP: obtained from a 30-kw (40 hp) 

four-cylinder Deutz BF4M1008 diesel 

engine (U.S. EPA) 

Organic extract fraction of particles 

NIST- DEP 2% 

C-DEP 20 % 

Particle Size: NR 

Route: Cell Culture (5×105 cells) 

Dose/Concentration: NIST-DEP, C-DEP: 0, 

12.5, 25, 50, 100, 200 μg/mL 

Time to Analysis: 1-4 h 

Cell Viability: DEP had no effect. 

Stat3: Both DEPs induced time-dependent 

phosphorylization of Stat3 in cytoplasm. NIST-DEP 

induced phosphorylization dose-dependently from 

12.5 to 50 μg/mL but stayed level at 100 and 200 

μg/mL. p-Stat3 induction was inhibited by antioxidant 

BHA though it was reactivated with exposure to 

H2O2. Reaction induced by H2O2 was similar to that 

of DEP. 

pStat3 Nuclear Transport: NIST-DEP induced 

cytoplasmic pStat3 to move from cytoplasm into 

nucleus. 

pEGFR Dephosphorylation: After 4 h of NIST-DEP 

exposure, dephosphorylation was inhibited for up to 

90 min. 

Reference: Chang et 

al. (2005, 097776) 

Species: Human 

Cell Type: A540, THP1 

UfCB (Printex 90, Degussa) 

Particle Size: 14 nm 

Route: Cell Culture (7×105 cells) 

Dose/Concentration: 100 μg/mL 

Time to Analysis: 4 h 

ROS in THP-1 and A549: UFCB increased ROS. 

NAC pretreatment blocked most of the UFCBinduced ROS production. 

VEGF in THP-1: UFCB increased VEG. NAC 

decreased the UFCB effects below those of the 

control. 

VEGF in A549: Produced similar, but less marked, 

results as with THP-1. 

December 2009 D-39 
Study Pollutant Exposure Effects 

Reference: Chauhan 

et al. (2004, 096682) 

Species: Mouse 

Strain: BALB/c 

Cell Type: RAW 

264.7; J774A.1; 

WR19M.1 

EHC-T: total EHC-93 (Env Health Ctr, 

Ottawa, Canada) 

EHC-I: insoluble EHC 

EHC-S: soluble EHC 

SRM1648: urban particulate St. Louis 

(NIST) 

SRM1649: urban dust/organics 

Washington (NIST) 

VERP: fine PM2.5 (Vermillion, Ohio) 

Cristobalite: SRM 1879 (NIST) 

TiO2: SRM 154b (NIST) 

Particle Size: EHC-93: 0.5 μm (median 

diameter); Cristobalite, SRM 1648, 

SRM 1649, TiO2,: NR; VERP: PM2.5 

Route: Cell Culture (15000 cells/well) 

Dose/Concentration: Particle suspensions: 

20, 50, 100 μg/well 

LPS: 0-5 μg/mL 

IFN-y: 0-1000 U/mL 

Time to Analysis: Particles added to culture 

at 0h, LPS and IFN-γ added at 2 h. 

Parameters measured after 22 h incubation 

period. 

Stimulation with LPS/IFN-y: LPS and IFN-y each 

induced NO release. Combination of LPS and IFN-y 

produced larger effect in all cell lines. L-NMMA, 

NOS inhibitor, suppressed most of the NO 

production with 100 nmol/L. 

Cellular Viability and Cytotoxicity: Exposure of 

cells to particulates did not result in overt cytotoxicity 

or excessive loss of cellular material. There was no 

correlation between the cytotoxicity of the particles 

in the surviving cells and the loss of protein mass in 

monolayers. 

Nitrite Production: EHC-T, EH-93-I, SRM1648 and 

SRM 1649 produced dose-dependent decreases. 

Cristobalite only decreased at higher doses. No 

effect from EHC-S, VERP or TiO2. 

iNOS: EHC-I, EHC-T, Crisobalite and SRM1648 

inhibited iNOS expression. TiO2 had no effect. EHC 

sol, SRM 1649 and VERP were not tested. 

Reference: Chauhan 

et al. (2005, 155722) 

Species: Human 

Cell Type: A549 

EHC-T: total EHC-93 

EHC-I: insoluble EHC 

EHC-S: soluble EHC 

Cristobalite (SiO2): SRM-1879 

TiO2: SRM-154b 

Particle Size: EHC-93: 0.4 μm (median 

physical diameter); TiO2, SiO2: 0.3-0.6 

μm 

Route: Cell Culture (150000 cells/flask) 

Dose/Concentration: All particles: 0, 1, 4, 8 

mg/5ml 

Time to Analysis: 24 h 

Cellular Viability: Decreased after exposure to 

EHC-T, EHC-I and cristobalite. Rate of reduction 

was not consistent across doses. EHC-S and TiO2

had no effect on viability. 

ET-1: Release of ET-1 peptide decreased dosedependently for EHC-T, -S and -I. Fractions of EHCS and EHC-I were more potent than EHC-T. TiO2

and Cristobalite also reduced ET-1 secretion 

although this was not consistent across the dose 

range. 

Cytokines: Results showed no detectable amounts 

of GM-CSF, IL-1β or TNF-α in cell culture 

supernatants. IL-8 increased dose-dependently with 

EHC-T, EHC-I and cristobalite. 

VEGF: VEGF significantly increased dosedependently with EHC-T, EHC-S and cristobalite. 

EHC-S induced a significant decrease in VEGF. 

Gene Expression: mRNA levels for preproET-1 

reduced at 24 h for all particle types. EHC-S induced 

a significant decrease in ET-1 expression at this high 

dose. ECE-1 mRNA expression increased with EHCT and EHC-I. Other particles had no effect. ETaR 

mRNA increased with EHC-T, EHC-S, and TiO2 in 

biphasic manner where the highest expression of 

mRNA was seen at the middle dose levels. EHC-S 

had no effect. ETbR mRNA increased with a low 

dose of EHC-T and decreased with a high dose of 

EHC-T. EHC-S, EHC-I and cristobalite induced an 

increase of ETbR. TiO2 induced a significant 

decrease. 

Proteases: mRNA levels for MMP-2 reacted 

similarly to preproET-1. mRNA levels for TIMP-2 was 

significantly induced with EHC-I. EHC-T and EHC-S 

induced small effects. 

Reference: Cheng et 

al. (2003, 156337) 

Species: Human 

Cell Type: A549 

DEP-h: DEP with high sulfur 

DEP-LS: DEP with low sulfur 

GEP: gasoline engine exhaust particles

Primed cells pretreated with TNF-α

Particle Size: DEP-h: 15.9 nm; DEPLS: 17.7 nm; GEP: 8.3 nm 

Route: In Vitro Cellular Exposure (Exhaust 

flow-through cell culture with air-cellinterface, exhaust diluted 10-15x with 8×105

cells/mL) 

Dose/Concentration: DEP (total): 1.5-

3.5×106

 particles/cm3; GEP (total): 1-2×106

particles/cm3

; TNF-γ: 5ml (25 ng/ml) 

Time to Analysis: 60-360 min 

IL-8: DEP-h induced a 3 fold increase in IL-8 than 

that of the control. DEP-LS also induced increases. 

Primed cell cases had higher levels (10x) than 

unprimed when exposed to DEP-LS. DEP-h induced 

higher levels of IL-8 than DEP-LS. This response 

lasted for up to 6 h. GEP induced a statistically 

insignificant increase of IL-8 in unprimed cells. With 

primed cells, GEP induced levels of IL-8 that 

exceeded those of DEP-h and DEP-LS. This 

response lasted for 1-2 h. 

December 2009 D-40 
Study Pollutant Exposure Effects 

Reference: Chin et al. 

(2003, 156340) 

Species: Rat, Human 

Cell Line/Type: RAW 

264.7, MHS (Alveolar 

Macrophage Cell 

Line), A549 

CB: (N339, with benzo[a]pyrene 

absorbed on surface. Manufactured in 

Cabot, Boston, MA) 

BaP 

Benzo [a] pyrene 1, 6-quinone: BP-1,6-

Q (obtained from NCI, Kansas City, 

MO) 

Particle Size: CB 0.1 μm (mean 

diameter) 

Route: Cell Culture 

Dose/Concentration: 

CB: 1, 2, 4 μg/mL 

BaP: 2 μg/mL 

BP-1,6-Q: 1 μM 

Time to Analysis: 1-24 h 

HO-1 mRNA Expression: In RAW264.7, HO-1 

mRNA levels increased with 2 and 4 μg/mL at 2 h. 

Increases continued to 8 h and declined by 24 h. 

BaP had no effect. BP-1,6-Q increased HO-1 mRNA 

after 1 h and was maintained until 8 h. In A549 and 

MHS, HO-1 mRNA increased after 1 h, peaking at 8 

h in A549 and 4 h in MHS. 

HO-1 Protein Expression: An increase of protein 

was observed from 4-8 h in RAW264.7. 

AP-1: Increases in binding activity were observed in 

RAW 264.7 cells at 2 h. 

Reference: Churg et 

al. (2005, 088281) 

Species: Rat 

Strain: SD 

Weight: 250 g 

Cell Type: Epithelial 

Cells of Tracheal 

Explants

EHC93 (Ottawa Urban Air Particles) 

TiFe = Iron-loaded fine TiO2 (obtained 

from Aldrich Chemicals, Milwaukee, 

WI) 

Particle Size: EHC-93: 3-4 μm 

(MMAD); TiFe: 0.12 ± 1.4 μm 

(geometric mean diameter) 

Route: Cell Culture 

Dose/Concentration: EHC-93, TiFe: 500 

μg/cm2

Time to Analysis: 1, 24 h. Some 

experiments (referred to as 2 h) explants 

transferred to different dish and incubated 

for additional hour. Pre-treated with 

Inhibitors/Chelators for 2 h. 

Activation of NF-κB: Both particle types increased 

nuclear translocation of NF-κB. TiFe and EHC-93 

increased NF-κB 1.5 fold at 1 h. TiFe increased NFκB 3.5 fold at 2 h. EHC-93 increased NF-κB more 

than 2 fold. TiO2 by itself did not increase NF-κB at 

any exposure duration. 

Morphological changes in tracheal epithelial 

cells: No evidence of dust particles was observed 

(EHC-93 or TiO2) in the epithelial cell cytoplasm at 2 

h. No evidence of morphologic cell damage from 

particles was observed. 

Colchicine: Treatment with colchicine did not 

prevent NF-κB activation. 

Inhibitors/Activators: Tetramethylthiourea (TMTU) 

(membrane-permeable active oxygen scavenger), 

Deferoxamine (redox-inactive metal chelator), PPS 

(Src inhibitor) AG1478 (epidermal growth factor 

receptor inhibitor) prevented NF-κB activation in 

both EHC93 and TiFe exposed-cells. Iron-containing 

citrate extract of both dusts increased NF-κB 

activation in both EHC93 and TiFe exposed-cells. 

Reference: Courtois et 

al. (2008, 156369) 

Species: Rat 

Strain: Wistar 

Cell Line: Dissected 

intrapulmonary arteries 

from rats used in 

corresponding in vivo 

experiments 

PM (SRM 1648) 

(63% in, 4-7% , mass fraction >1%: Si, 

S, Al, Fe, K, Na) 

UF carbon black (FW2, P60) 

Particle Size: SRM 1648 mean 

diameter 0.4 μm; ultrafine carbon black: 

FW2- 13 nm, P60- 21 nm 

Route: Cell Culture 

Dose/Concentration: 100, 200 μg/mL 

Time to Analysis: 24 h incubation 

NO: Generally, Ach-induced relaxation in 

intrapulmonary arteries decreased, Ach-induced 

cGMP accumulation decreased, and relaxation by 

SNP or DEA-NO also decreased. UF carbon black 

did not affect NO responsiveness. 

Oxidative Stress, Inflammatory: Dexamethasone 

prevented SRM 1648-induced impairment of the Ach 

relaxation response but antioxidants did not. TNF-α, 

MIP2, IL-8 increased. ROS was not affected.

Reference: Dagher et 

al., (2007, 097566) 

Species: Human 

Cell Type: L132 

(Normal Lung 

Epithelial Cells) 

LC10, LC50 = PM2.5 

(collected Jan-Sept in Dunkerque, 

France) 

Particle Size: cumulative frequency: 

0.5 μm: 34%; 1 μm: 64%; 1.5 μm: 79%; 

2 μm: 87%; 2.5 μm: 92%; 5 μm: 98%; 

10 μm: 100% 

Route: Cell Culture (3×106, 1.5×106, 

0.75×106

 cells/20mL) 

Dose/Concentration: LC10: 19 μg/mL; 

LC50: 75 μg/mL 

Time to Analysis: 24, 48 or 72 h 

p65 Protein: Phosphorylation of p65 increased in 

PM-exposed L132 cells in dose-dependent manner. 

IκBα Protein: Phosphorylated IκBα protein 

concentrations increased in cytoplasm with both 

particle types at all time points. 

p65 and p50 DNA: p65 DNA binding increased at 

24 h with LC10 and LC50, at 48 h with LC10, and at 

72 h with LC10 and LC50. p50 DNA binding 

increased at all time points with LC10 and LC50. 

December 2009 D-41 
Study Pollutant Exposure Effects 

Reference: Dai et al. 

(2003, 087944) 

Species: Rat 

Strain: SD 

Weight: 250 g 

Cell Type: Tracheal 

Explants 

EHC-93 (Environmental Health Center, 

Ottawa) 

DEP: SRM 1650a (NIST) 

Particle Size: EHC-93: 3-4 μm 

(MMAD); DEP 1.55 ± 0.04 μm (CMD) 

Route: Cell Culture 

Dose/Concentration: ECH, DEP: 500 

μg/cm2

Time to Analysis: Exposed for 1 h. 

Parameters measured following a 7 day 

incubation period. 

Hydroxyproline: EHC93 induced an almost 3 fold 

increase in explant hydroxyproline. DEP increased 

tissue hydroxyproline 2.5 fold. 

Procollagen: EHC-93 doubled gene expression of 

procollagen. Procollagen gene expression could be 

fully inhibited by SN50, TMTU or treatment of the 

PM with DFX. Treatment of explants with p38 or 

ERK (inhibitors) had no effect on procollagen 

expression. DEP induced an increase in procollagen 

gene expression but this increase was completely 

prevented by SN50 and MAP kinase inhibitors 

(SB203580 and PD98059). Neither TMTU or DFX 

has any effect. 

TGFβ1: Treatment of explant with EHC93 

approximately doubled gene expression for TGFβ1. 

Treatment with SN50, TMTU and fetuin (TGFβ

antagonist) blocked increase. DFX, MAP kinase 

inhibitors (SB203580 and PD98059) had no effect. 

DEP roughly doubled TGFβ1 expression.SN50 and 

MAP kinase inhibitors (SB203580 and PD98059) 

fully blocked this effect. TMTU and DFX had no 

effect. 

Reference: Doherty et 

al. (2007, 096532) 

Species: Rat 

Strain: NR8383 

Cell Types: AMs 

Ratios of: V: Fe; Al: Fe; Mn: Fe 

V = sodium vanadate (NaVO3) 

Al = aluminum chloride hexahydrate 

(AlCl3) 

Mn = manganese chloride tetrahydrate 

(MnCl2) 

Fe = ferric chloride hexahydrate (FeCl3)

Ratios based on PM2.5 measurements 

from NYC, LA and Seattle 

Particle Size: Metals from PM2.5

samples 

Route: Cell Culture (2×105 cells/mL) 

Dose/Concentration: Fe = 16 μmol 

(equivalent to urban NYC 500 μg PM2.5); V 

and Mn tested in molar rations of 0.02 to 0.4 

relative to Fe; Al tested in molar ratios of 

0.125 to 8 relative to Fe. 

Time to Analysis: 20 h 

IRP: Addition of V increased IRP activity 5 to 9 fold. 

Though there was no seeming dose responsivity, 

IRP activity remained strongly elevated over the 

range of V:Fe ratios tested. Addition of Mn only 

resulted in an effect at 0.1 molar ratio (two-fold), not 

at higher or lower ratios. Al resulted in peak 

increases of 5 fold at molar ratios 2 while declining 

to 2 fold at molar ratios 4 and 8. 

Cytotoxicity: Al was cytotoxic at molar ratios of 4 

and 8. All other Al, V, Mn ratios had no effect. 

Mixtures: The combination of metals tested at NYC 

PM ratios and V drove all the Fe transport activity. 

Combinations of V+Mn and V+Al increased activity 

more than V:Fe alone. 

Reference: Doornaert, 

et al. (2003, 156410) 

Species: Human 

Cell Line/Type: 

16HBE14o-; P-HBE 

DEP: SRM 1650 (NIST) 

CB: (Sigma, France) 

DPC: Dipalmitoyl phosphatidylcholine 

(positive control) 

0.5 um 

Particle Size: NR 

Route: Cell Culture 

Dose/Concentration: DEP and CB: 1-100 

μg/mL 

Time to Analysis: Parameters measured 

24, 48, 72 h post exposure. 1-HBE Cell 

Deadhesion Capacity: 24 h, evaluation of 

detachment performed every 5min for 40 

min after. Cell Wound Repair Capacity: 24 h, 

repair evaluated 3.5, 7, 24 h after. 

Cytotoxicity: DEP was cytotoxic at 100 μg/mL at all 

time points in a time-dependent manner. CB and 

DPC cytotoxicity was substantially lower but 

significant at 72 h. 

Phagocytosis: 1-HBE cell levels that were in 

contact with DEP or CB or have phagocytized those 

particles increased in a dose-dependent manner. 

DEP induced greater levels of cell contact and 

phagocytosis than CB. 

F-actin: Only DEPs were engulfed by F-actin 

stained cell fragments. 

Actin CSK Stiffness: DEP (5, 20, 100 μg/mL) 

induced net dose-dependent decrease in 

cytoskeleton stiffness and a dose-dependent 

decrease in actin cytoskeleton stiffness. CB 

produced no significant decrease. 

Adhesion Molecules: DEP induced a concomitant 

reduction of both CD49 (α3) and CD29 (β1) integrin 

subunits and a decrease in level of CD44 (HBE cellcell and cell-matrix adhesion molecule) at both 20 

and 100 μg/mL. 

Proteases: DEP also induced an isolated decrease 

in MMP-1 expression without change in tissue 

inhibitor of TIMP-1 or TIMP-2 at 100 μg/mL. CB 

produced no change or insignificant results. 

1-HBE Cell Deadhesion Capacity: DEP exposure 

induced a dose-dependent amplification of cell 

detachment at 5 min of incubation and onward. 

Cell Wound Repair Capacity: DEP inhibited wound 

repair/wound closure in a dose-dependent manner. 

December 2009 D-42 
Study Pollutant Exposure Effects 

Reference: Dostert et 

al. (2008, 155753) 

Species: Human 

Cell Line/Type: THP1, 

monocyte-derived 

macrophages (MM) 

Asbestos 

Silica 

DEP 

CSE: cigarette smoke extract 

MSU: monosodium urate crystals 

Particle Size: NR 

Route: Cell Culture 

Dose/Concentration: Asbestos: 0.1, 0.2 

mg/mL; Silica: 0.1, 0.2, 0.25, 0.5 mg/mL; 

DEP: 0.2, 0.25, 0.5 mg/mL; CSE: 5%, 10% 

in solution mg/mL; MSU: 0.1, 0.2 mg/mL 

Time to Analysis: 1, 3, 6 h 

IL-1β: Increased levels of IL-1β with asbestos and 

silica were observed in THP1 at 6 h. CSE and DEP 

had no effect. MM also had increased levels with 

asbestos, silica and MSU at high dose levels only. 

Caspase-1: Asbestos increased caspase-1 activity. 

ROS: Asbestos doses in THP1 exhibited an 

increase in ROS formation. 

Reference: Doyle, et 

al. (2004, 088404) 

Species: Human 

Cell Type: A549 from 

non-smoking adults 

BD: 1,3-butadiene, known carcinogen 

Acrolein: photochemical and NO 

product of BD in atmosphere 

Acetaldehyde: photochemical and NO 

product of BD in atmosphere 

Formaldehyde: photochemical and NO 

product of BD and ISO in atmosphere 

ISO: isoprene, 2-methyl analog of BD 

Methacrolein: photochemical and NO 

product of ISO in atmosphere 

Methyl vinyl ketone: photochemical and 

NO product of ISO in atmosphere 

Particle Size: NR 

Route: Environmental Irradiation (smog) 

Chambers 

Dose/Concentration: 50 ppb NO; 200 ppbV 

ISO, BD 

Time to Analysis: Exposed to gases for 5 h. 

Analysis 9 h post exposure. 

Cytotoxicity: ISO+NO and BD+NO induced small 

increases of LDH in A549. However, ISO+NO+light 

and BD+NO+light increased LDH levels 4-6 fold 

indicating photochemical products of ISO and BD 

are highly cytotoxic. LDH levels of each combination 

were equivocal. 

IL-8 Protein: Methacrolein, methyl vinyl ketone and 

formaldehyde (products of ISO) increased IL-8 

protein levels significantly. ISO+NO had no effect. 

BD photochemical products (acrolein, acetaldehyde 

and formaldehyde) also increased IL-8 protein, more 

than doubling the photochemical products induced 

by ISO. BD+NO had no effect. 

IL-8 mRNA: IL-8 mRNA expression also increased 

with photochemical products of ISO and BD but did 

not reach a statistically significant level. 

Reference: Duvall et 

al. (2008, 097969) 

Species: Human 

Cell Type: Airway 

Epithelial Cells 

PM-F, -C, -UF 

Particles collected from: Seattle, WA 

(PM-S); Salt Lake City, UT (PM-SL); 

Phoenix, AZ (PM-P); South Bronx, NY 

(PM-SB); Hunter College, NY (PMSterling Forest, NY (PM-SF) 

Particle Size: Coarse: >2.5 μm; Fine: 

<2.5 μm; UFP: <0.1 μm 

Route: Cell Culture (100,000 cells/cm2) 

Dose/Concentration: 5 mg/ml 

Time to Analysis: 1, 24 h post exposure 

Particle Characterization: PM-HR, PM-SL and PMS contained the highest UF, F, and C concentrations. 

PM-SB and PM-HR had similar F and C 

concentrations. Sulfate was highest in PM-F for all 

sites except in PM-SB and PM-HR. Wood 

combustion was highest in PM-SL, PM-S, PM-P. Soil 

dust was highest in PM-SL and PM-S. 

IL-8: PM-UF induced a greater increase in IL-8 than 

other types of PM except PM-P. PM-UF is 

associated with vanadium, lead, copper, sulfate. PMF-HR caused the greatest increase followed by PMSB. PM-F-SF and PM-F-P was least effective. PM-C 

also caused an increase in IL-8 levels and was 

associated with vanadium and EC. 

COX-2: PM-F-S induced the greatest increase in 

COX-2 expression. Other PM-F sites induced similar 

increases. UF PM had no effect. PM-C, associated 

with EC, induced increases. 

HO-1: PM-F-SF induced the greatest increase in 

HO-1. PM-F-SL was the least effective. UF PM had 

no effect. PM-C, associated with copper, barium and 

EC, caused an increase. 

Reference: Dybdahl et 

al. (2004, 089013) 

Species: Human 

Cell Type: A549 

DEP: SRM 1650 (NIST) 

Particle Size: 90 nm (MMAD) 

Route: Cell Culture (105 cells/mL) 

Dose/Concentration: 0, 10, 50, 100, 500 

μg/mL 

Time to Analysis: 2, 5, or 24 h 

Cytokines: DEP induced dose-dependent increases 

of IL-1α, IL-6, IL-8 and TNF-α at 24 h. Cytokines 

increased between 4 and 18 fold at the highest DEP 

dose as compared to controlled cells. DEP also 

increased IL-6 mRNA expression levels in a dose 

and time-dependent manner.IL-6 mRNA levels 

increased 14 fold at 24 h, 8 fold at 5 h, and 2 fold at 

2 h. 

Cell Viability: DEP exposure did not decrease cell 

viability at any dose tested. 

December 2009 D-43 
Study Pollutant Exposure Effects 

Reference: Fritsch et 

al. (2006, 156452) 

Species: Mouse 

Cell Type: RAW 264.7 

MAFO2: incinerator fly ash (collected by 

electrostatic precipitation in commercial 

municipal waste incinerator facility) 

composition representing 12% of total 

mass (mg/g): 

Fe (9.1); Pb (23.3); Zn (75.7); C (7.5) 

Particle Size: 165 nm (modal value) 

Route: Cell Culture (1×106 cells/well) 

Dose/Concentration: 6.3-188 μg/cm2 for 

Toxicity; 2.6, 6.5, 13.2 μg/cm2 for 

Arachidonic Acid; 13.2 μg/cm2

 for MAPK 

Pathway; Other doses noted in Effect of 

Particles 

Time to Analysis: 1, 2.5, 5, 24 h 

Toxicity: Viability decreased from 99% to 18% at 

62.5-188 μg/cm2

. Lower doses had no effect. 

Arachidonic Acid: At 2.5 h, AA level increased 2 

fold for 6.5 μg/cm2

 and 6 fold for 13.2 μg/cm2. No 

increase was observed after 5 h. 

MAPKs: Cells pretreated with PD98059, an inhibitor 

of MEK-1, inhibited AA liberation due to MAFO2

treatment of 13.2 μg/cm2

COX-2: A time-dependent increase of COX-2 protein 

expression was exhibited at 2.5 and 5 h. 

ROS: A dose-dependent increase in ROS formation 

was observed at concentrations greater than 31.3 

μg/cm2

 after 3 h. 

GSH: There was an observed increase of production 

at 20 h. Doses greater than 60 μg/cm2

 reduced total 

glutathione. 

HO-1: There was an observed dose-dependent 

increase in expression at 4 h. 

Reference: Fujii et al. 

(2002, 036478) 

Species: Human 

Cell Type: HBEC 

(from current 

smokers), AMs, CoCulture: AMs+HBEC 

Age: HBEC: 48-70 yr 

PM10: EHC-93 (Ottawa, Canada) 

Particle Size: PM10

Route: Cell Culture (HBEC: 2.5-3× 106

cells/well); (AMs: 1.0×107

 total) 

Dose/Concentration: 100, 500 μg/mL 

Time to Analysis: 2, 8, 24 h 

Viability: Over 90% of HBEC were viable after a 24 

h exposure of up to 500 μg/mL of PM. AMs 

incubated with and without 100 μg/mL saw no 

significant difference in viability. 

Cytokine mRNA: TNF-α, GM-CSF, IL-1β, IL-6, LIF, 

OSM and IL-8 mRNA expression increased in coculture with 100 μg/mL at 2 and 8 h. In AMs, TNF-α, 

IL-1β, IL-6 mRNA expression increased with 100 

μg/mL at 2 h. IN HBECs, IL-1β and LIF increased 

with 100 μg/mL at 2 h. HBECs added to AMs 

exposed to PM10, further increase in mRNA of IL-1β, 

LIF and IL-8. 

Cytokine Protein: In co-culture and AMs, significant 

increase in protein production of GM-CSF, IL-8, IL1β, IL-6 and TNF-α in dose-dependent manner. GMCSF and IL-6 production significantly higher in coculture then AM or HBEC alone. 

Bone Marrow: Co-culture instillation of 

supernatants increased circulating band cell counts 

at 6 and 24 h with 100 μg/mL. 

Reference: Fujii et al.. 

(2001, 156455) 

Species: Human 

Cell Type: HBEC from 

current smokers 

Age: 48-70 yr

PM10:EHC93 (Ottawa, Canada)99% 

<3.0um 

Particle Size: PM10( 99% < 3.0 μm) 

Route: Cell Culture (2.5-3×106 cells/dish) 

Dose/Concentration: 10, 100, 500 μg/mL 

Time to Analysis: 2, 8, 24 h 

Phagocytosis: 18.6% of cells engulfed particles 

when exposed to 100 μg/mL. Over 90% remained 

viable. 

Cytokine mRNA: LIF mRNA increased dosedependently at 2 h but declined at 8 and 24 h. GMCSF increased dose-dependently at 8h and peaked 

at 24 h. IL-1α increased at 2 h, increased dosedependently at 8 h and peaked at 24 h. M-CSF, 

MCP-1, IL-8 were unaffected. 

Cytokine Protein: LIF, GM-CSF, IL-1β and IL-8 

increased dose-dependently. Soluble fraction of 100 

μg/mL PM10 did not affect cytokine production. 

December 2009 D-44 
Study Pollutant Exposure Effects 

Reference: Garcon et 

al. (2006, 096633) 

Species: Human 

Cell Type: L132 

PM2.5 (collected in Dunkerque, France 

for 9mo, Jan-Sept) 

Particle Size: PM2.5- 0-0.5 μm 

(33.63%), 0.5-1.0 μm (30.61%), 1.0-1.5 

μm (14.33%), 1.5-2.0 μm (8.69%), 2.0-

2.5 μm (4.89%), >2.5 μm (7.87%) 

Route: Cell Culture: 3×106 cells/20ml (24 h); 

1.5×106

 cells/20ml (48h); 0.75×106

cells/20ml (72 h) 

Dose/Concentration: 18.84, 37.68, 56.52, 

75.36, 150.72 μg/mL; LC10- 18.84 μg/mL; 

LC50- 75.36 μg/mL 

Time to Analysis: 24, 48 or 72 h 

Cytotoxicity: PM induced dose-dependent 

(R2 =.9907) cytotoxic effect in proliferating L132 

cells. 

LDH: Increase at 72 h with 56.52 and 75.36 μg/mL. 

Oxidative Stress: A decrease in MDF activity was 

observed at all exposure levels at 24, 48, and 72 h 

(72-h <5 % of control). MDA levels showed increase 

concentration after 72 h, both LC10 and LC50. LC10 

and LC50 saw an increase in SOD activity at 24 h; 

LC50 saw a decrease in activity after 48 and 72 h. 8-

OHdG and PARP exhibited increases at all time 

points with LC10 and LC50. 

Inflammatory Response: Increases of TNF-α

concentration was exhibited at 24 h at LC50, and at 

48 h and 72 h at LC10 and LC50. iNOS activity 

increase at all time points at LC10 and LC50.NO 

concentration exhibited increases at all time points 

after exposure to LC10 and LC50. 

Reference: Geng et 

al. (2005, 096689) 

Species: Rat 

Strain: Wistar Kyoto 

Tissue/Cell Type:

Lung macrophages 

BPM: Blowing PM2.5; PM collected from 

Wuwei City, Gansu Province, China 

(Blowing days correspond to desert 

storm days) 

NPM: Non-blowing (normal) PM2.5 

Particle Size: PM2.5

Route: Cell Culture 

Dose/Concentration: 0, 33, 100, 300 μg/mL

Time to Analysis: 4 h 

Cytotoxicity: Dosages greater than 150 μg/mL 

decreased cell viability. 

Plasma Membrane Fluidity: Dose-dependent 

decrease had no effect on membrane lipid 

hydrophilic region. 

Plasma Membrane Permeability: LDH enzyme 

activity and extracellular AP activity increased dosedependently, indicating increased membrane 

permeability, but this was only statistically significant 

at 300 μg/mL dose. NPM may affect some 

parameters at 100 μg/mL. Overall, NPM induced a 

slightly higher increase than BPM. 

Intracellular Ca2+: A dose-dependent increase was 

observed. 

Lipid Peroxidation (TBA): An increase was 

observed only at 300 μg/mL. 

Antioxidant (GSH): A decrease was observed only 

at 300 μg/mL. 

Reference: Geng et 

al. (2006, 097026) 

Species: Rat 

Strain: Wistar Kyoto 

Tissue/Cell Type: 

Lung macrophages 

DPM: dust storm samples; PM 

collected from Baotou City, Inner 

Mongolia, China in March 2004 

NPM: normal PM 

Particle Size: PM2.5 

Route: Cell Culture 

Dose/Concentration: 0, 33, 100, 300 μg/mL

Time to Analysis: 4 h 

Cytotoxicity: MTT reduction assay revealed a 

significant decrease in cell viability at 150 μg/mL and 

300 μg/mL. LDH enzyme activity significantly 

increased at 150 and 300 μg/mL. 

GSH levels: Significant decreases were seen in 

cellular GSH levels and increases in TBARS levels 

in both groups with a 300 μg/mL dose. 

Plasma Membrane Activity: In the plasma 

membrane, Na, K-ATPase were significantly 

inhibited. Ca2+Mg2+-ATPase were unaffected. 

Plasma Membrane Lipid Fluidity: Results indicate 

that DPM could increase the surface fluidity of 

membrane lipid. 

Intracellular Ca2+: A dose-dependent increase in 

free intracellular Ca2+ levels was observed. 

Reference: Ghio et al. 

(2005, 088272) 

Species: Human 

Cell/Tissue Type: 

BEAS-2B 

FAC: ferric ammonium citrate 

(component of ROFA) 

VOSO4: vanadyl sulfate 

(component of ROFA) 

Particle Size: NR 

Route: Cell Culture 

Dose/Concentration: 100 μM FAC - 

preexposed before metal compounds or oil 

fly ash 

50 μM VOSO4 - preexposed before metal 

compounds or oil fly ash 

100 μg/mL ROFA 

Time to Analysis: 0-1 h, 4 h 

IRE DMT1: FAC increased mRNA and protein 

expression for -IRE DMT1. VOSO4 decreased 

mRNA and protein expression for -IRE DMT1. +IRE 

DMT1 unaffected by any treatment. 

Metal transport: Uptake of iron increased after preexposure to FAC and decreased after pre-exposure 

to VOSO4. Pre-exposure to FAC again increase the 

uptake of both iron and vandium. VOSO4 induced 

opposite effect, decreasing Fe uptake. 

ROS: Increased acetaldehyde, indicating increased 

oxidative stress. ROS decreased with FAC 

pretreatment. ROS increased with VOSO4

pretreatment. 

December 2009 D-45 
Study Pollutant Exposure Effects 

Reference: Gilmour et 

al. (2004, 057420) 

Species: Rat 

Strain: SD 

Cell/Tissue Type: AM 

Coal Fly Ash 

MU = Montana Ultrafine 

MF = Montana Fine 

MC = Montana Coarse 

KF = West Kentucky Fine 

KC = West Kentucky Coarse 

Coal combustion using a laboratoryscale down-fired furnace rated at 

50kW. Montana subbituminous coal 

and western Kentucky bituminous coal 

Particle Size: Coarse: >2.5 μm; Fine: 

<2.5 μm; UFP: <0.2 μm 

Route: Cell Culture (2×105 cells/mL) 

Dose/Concentration: 125 μg/mL or 250 

μg/mL 

Time to Analysis: 4 or 24 h 

LDH: Mid and high doses of Montana ultrafine 

particles showed significant increase after 4 h 

exposure vs control. Other particle types had no 

effect. After 24 h, LDH level was not statistically 

significant between particles tested and control. 

Cytokines: Treatment with Montana ultrafine 

particles resulted in a significant production increase 

of TNF-α. MIP-2 showed increases in all the fine and 

ultrafine treatments, with Montana ultrafine and W. 

Kentucky fine PM showing the highest increases. IL6 increased with Montana ultrafine particles although 

there was some variability and the increases were 

not statistically significant. 

Reference: Gilmour et 

al. (2005, 087410) 

Species: Human 

Cell/Tissue Types:

monocyte derived 

macrophages, 

HUVECs, A549, 

16HBE 

PM10: Collected from the Marylebone 

and Bloomsbury monitoring sites in 

London, UK 

Particle Size: PM10

Route: Cell Culture 

Dose/Concentration: 50 μg/mL 

Time to Analysis: 4 h, 6 h, 20 h 

IL-8: PM10 at 50 μg/mL induced a significant 

increase in IL-8mRNA and protein expression in 

PMM and 16HBE at 6 and 20h. A less substantial 

increase was also observed in A549. 

Procoagulant Activity: PM10 induced a significant 

decrease in macrophage mediated clotting time in 

16HBE. Other cell types were unaffected. 

Annexin V Binding: At 100 μg/mL, PM10 induced a 

significant increase in binding macrophages at 4 and 

20 h. There was no effect at 50 μg/mL. 

Tissue Factor mRNA Expression: Expression was 

increased in macrophages at 6 h only. 

tPA Expression: mRNA expression decreased at 

6 h. Protein expression decreased at 4 h and 20 h in 

a dose-dependent manner. 

TF Expression: TF mRNA expression increased in 

a dose-dependent manner at 6 h in HUVECs. 

Protein levels also increased at 4 h but declined to 

basal levels by 20 h. 

Reference: Gilmour et 

al. (2003, 096959) 

Species: Human 

Cell/Tissue Type: 

A549 

PM10: Collected from the Marylebone 

and Bloomsbury monitoring sites in 

London, UK 

TSA 

H2O2

NAC 

Mannitol 

Provided by Sigma Chemical, Poole, 

UK or GIBCO-BRL, Paisley, UK 

Particle Size: PM10

Route: Cell Culture 

Dose/Concentration: PM10: 100 μg/mL; 

TSA: 100 ng/mL; H2O2: 200 μM; NAC and 

Mannitol: 5 mM 

Time to Analysis: 24 h 

IL-8: PM10, TSA and H2O2 treatment induced an 

increase of IL-8. Concomitant exposure of TSA with 

PM10 or H2O2 significantly increased IL-8 release 

when compared to PM10 or H2O2 alone. IL-8 mRNA 

expression with PM10 or H2O2 exposure and TSA 

coincubation caused significant increases. Silver 

staining of PCR products indicated that the IL-8 

gene promoter was associated with acetylated H4 

following TSA, PM10 and TNF treatment. 

H4: PM10 exposure significantly increased 

acetylation levels of H4 over controls. Increased 

acetylated H4 was mediated by PM10 in a dosedependent manner. Treatment with PM10 and H2O2

increased HAT activity associated with H4 by 245% 

and 166% respectively. Significant increases in 

acetylation of H4 following treatment of cells with 

TSA, PM10 and H2O2 for 24 h was observed. 

PM10induced HAT activity was significantly 

decreased in the presence of NAC and mannitol. 

Nuclear presence of HDAC2 protein was 

significantly reduced by exposure to both HDAC 

inhibitor and PM10. There was a decreasing trend in 

HDAC2 gene expression following TSA and PM10

treatment. 

NF-κB: The activation of the transcription factor NFκB was enhanced following the inhibition of HDAC 

with TSA and by treatment with 

December 2009 D-46 
Study Pollutant Exposure Effects 

Reference: Graff et al. 

(2007, 156488) 

Species: Human 

Cell/Tissue Type: 

HAEC 

PM 

-UF: ultrafine 

-F: fine 

-C: coarse 

Particles collected from Seattle, WA (-

S), Salt Lake City, UT (-SL), Phoenix, 

AZ (-P), South Bronx, NY (-SB), Hunter 

College, NY (-H), Sterling Forest, NY (-

SF) 

Particle Size: UF: <0.1 μm; F: 0.1- 2.5 

μm; C: 2.5-10 μm 

Route: Cell Culture 

Dose/Concentration: 250 μg/mL 

Time to Analysis: 6 h, 24 h 

Gene Expression: PM-UF, PM-F, and PM-C both 

upregulated and downregulated genes in the HAECs 

though downregulation was far more common for all 

the three PM fractions. PM-F affected the greatest 

number of transcripts, followed by the UF and C 

fractions. 

IL-8: mRNA expression increased, with PM-F-S 

having the greatest impact. Aluminum, strontium, 

manganese and potassium were highly associated 

with expression. Wood combustion was moderately 

associated. 

HOX-1: mRNA expression increased, with PM-F-SF 

having the greatest impact. Potassium, manganese, 

strontium and wood combustion were highly 

associated with expression. Aluminum and 

vanadium were moderately associated. 

Reference: Gualtieri et 

al. (2005, 097841) 

Species: Human 

Cell Type: A549 

TD: Tire debris extracted in methanol, 

constituent of PM10

(generated by spinning a new 

automotive tire against abrasive 

surface) 

Particle Size: 10-80 μm 

Route: Cell Culture 

Dose/Concentration: 10, 50, 60, 75 μg/mL 

Time to Analysis: 24, 48, 72 h 

Cytotoxic Effect: Treated cells presented inhibitory 

effect on reduction of MTT which appeared to be 

dose and time-dependent. A statistically significant 

reduction was observed at 48 and 72 h. Trypan blue 

showed a significant PM lethality as well as a dosedependent increase in mortality. 

DNA Damage: At 24 and 72 h, DNA damage 

increased dose dependently in damaged and ghost 

cells. 

Cell Cycle Analysis: At 24 h, TD extract-treated 

cells presented a significant increase in the 

percentage of cells in G1 phase when compared 

with control. This increase was associated with a 

decrease in the percentage of cells in S phase. At 48 

and 72 h, the increase in percentage of cells in G1 

was associated with a decrease in the percentage of 

cells in both S and G2/M phases. Cells exposed to 

TD extracts presented changed morphology. 

Modifications most obvious at 72 h. The highest dost 

produced increased vacuolization in cytoplasm and 

apoptotic nuclear images. 

Reference: Hetland et 

al. (2005, 087887) 

Species: Rat 

Gender: Male 

Strain: Crl/Wky 

Cell Type: AMs

PMC = Coarse 

PMF = Fine 

-A = Amsterdam 

-L = Lodz 

-R = Rome 

-O = Oslo 

Coexposures PAH, Fe, Al, Zn, Cu, V 

Particle Size: PMC: 2.5-10 μm; PMF: 

0.2-2.5 μm 

Route: Cell Culture (1.5×106 cells/well) 

Dose/Concentration: 50, 100 μg/mL PM 

Time to Analysis: 20 h 

IL-6: PMC from all cities exhibited increases in IL-6 

release with spring and summer roughly equal and 

both inducing higher levels than the winter PMC. For 

the Spring and Summer samples, PMC-L exhibited 

the highest IL-6 releases (440% and 460% 

respectively) followed by Rome, A’dam/Oslo, and 

Oslo/A’dam. For the winter samples, Rome and 

Amsterdam induced higher IL-6 levels (340% and 

300% respectively) than Lodz and Oslo (165% and 

160%). The fine fractions did not induce any 

significant cytokine release. 

TNF-α: PMC from all cities increased TNF-α release 

with 50 μg/mL generally inducing a slightly higher 

increase than 100 μg/mL. 

Constituent Correlation: Levels of Fe, Al, Zn, Cu 

and V as well as PAH (total and fractions) showed 

no correlation with IL-6 release. 

Endotoxin Correlation with IL-6 release: A 

confirmatory test revealed no correlation. 

December 2009 D-47 
Study Pollutant Exposure Effects 

Reference: Hetland et 

al. (2004, 097535) 

Species: Rat, Human 

Cell Type: Alveolar 

Macrophages (Rat), 

A549 

Strain: Wky/NHsd 

Gender: Male 

Weight: 180-230 g 

AMC = Ambient Coarse 

AMF = Ambient Fine 

AMUF = Ambient Ultrafine 

(AM samples taken at a suburban site, 

without a dominating PM source, near 

Utrecht, Netherlands) 

Road PM: PM10, (collected in a road 

tunnel with predominating road 

abrasion due to use of studded tires in 

Trondheim, Norway) 

Particle Size: AMC: 2.5-10 μm; AMUF: 

<0.1 μm 

Route: Cell Culture (1×106 cells/well) 

Dose/Concentration: 0, 100, 200, 400, 600, 

800, 1000 μg/mL 

Time to Analysis: 20h (Type 2 cells); 40h 

(A549 cells) 

IL-8: All 3 AM fractions showed dose-dependent 

increases in A549 cells until 600 μg/mL; at that 

concentration, levels declined. AMC showed the 

most pronounced decline which correlates with 

decreased viability. Road PM showed a near linear 

response until 1000 μg/mL, whereas DEP plateaued 

at 600 μg/mL in A549. 

MIP-2: AMC and AMUF had no effect on Type 2 

cells. DEP induced increases at 200 μg/mL, 

whereas Road PM induced the strongest increase, 

peaking at 600 μg/mL in Type 2 cells. 

IL-6: AMC induced increases at 100 μg/mL in Type 

2, but levels declined below normal at 200 μg/mL. 

AMUF induced a decline of IL-6 levels. Road PM 

induced significant increases in Type 2. DEP had a 

slight effect. AM fractions induced increases in A549 

cells, peaking at 600 μg/mL with AMF. DEP and 

Road PM induced a dose-dependent increase. 

Cell Survival: AMC showed major effects at 200 

μg/mL in Type 2. AMUF showed effects at 400 

μg/mL. Road PM and DEP showed a gradual 

decline from 75% to 50% at 800 μg/mL in Type 2. All 

AM fractions induced a decrease in viability after 

600 μg/mL in A549 with AMC inducing a larger 

decrease than AMUF and AMF; AMUF and AMF 

induced similar levels. Road PM and DEP had no 

effect on A549. 

Apoptosis: AMC elicited a marked induction of 

apoptosis 200 μg/mL in Type 2 cells. AMF showed a 

dose-dependent increase in A549. Other AM 

fractions showed some slight increases in both cell 

types. Statistical significance was reached for all 

particles except for Road PM. 

Reference: Holder et 

al. (2008, 093322) 

Species: Human 

Cell Type: 16HBE14o 

DEP: generated from a single cylinder 

diesel engine using , commercial 

certified #2 diesel fuel 

Copollutants: NOX 7 ppm, CO2 0.1% 

Particle Size: Suspension: 223 nm 

(mean diameter); ALI: 122 nm (mean 

diameter) 

Route: Suspension (1×105 cells/cm2), Air 

Liquid Interface (ALI, 1×105

 cells/cm2) 

Dose/Concentration: Suspension: 0.13, 

0.24, 1.88, 2.5, and 12.5 μg/cm2; ALI: 1 

g/cm3

 (total number of particles: 2.3×107

particles/cm2

 ) 

Time to Analysis: Exposure for 6 h. 

Parameters measured 20 h post-exposure. 

ALI vs Tracheal Bronchial (TB) Deposition: The 

TB region deposition is 1.5 nominally x ALI, but 

particle diameter deposited in the TB was 62 nm 

(geometric mean diameter) as compared to the 

particle deposition in the ALI, measuring 260 nm. 

Inflammatory Response: Suspended DEP 

decreased viability at concentrations of 2.5 μg/cm2 or 

higher. IL-8 release (corrected for viability) increased 

at concentrations of 1.88 μg/cm2

 or higher in a dosedependent manner. IL-8 exhibited intermediate 

levels of secretion between in vitro levels of 0.25 

and 1.88 μg/cm2

. No statistically significant results 

were observed in ALI. Viability for ALI was near 

100% (75% uncorrected). 

Reference: Huang et 

al. (2003, 087376) 

Species: Human, 

Mouse 

Cell Type: BEAS-2B, 

RAW 264.7 

PMC: PM coarse 

PMF: PM fine 

PMSM: PM submicron 

Collected between SeptemberDecember 2000 from 4 ambient 

monitoring stations in Taiwan that 

represented background, urban, traffic, 

and industrial sites 

Particle Size: PMC: 2.5-10 μm; PMF: 

1-2.5 μm; PMSM: <1 μm 

Route: Cell Culture (5×105 cells/mL) 

Dose/Concentration: All PM: 50, 70, 100 

μg/mL 

Time to Analysis: BEAS-2B: 8h; RAW 

264.7: 16 h 

Viability: None of the PM fractions affected cell 

viability. 

IL-8: Only PMSM induced a significant IL-8 increase 

in BEAS-2B. IL-8 response was associated with a 

combination of Mn and Cr (R2 = 0.28). Response 

was also correlated with nitrate, although 

significance disappeared when 1 extreme nitrate 

value was removed. 

Lipid Peroxidation: Only PMSM enhanced lipid 

peroxidation in BEAS-2B, correlating with both 

elemental and . 

TNF-α: In RAW264.7, PMSM increased TNF-α

production. Polymixin pretreatment significantly 

reduced TNF-α levels for all 3 PMs which indicates 

an endotoxin role in macrophage response. TNF-α

production (after polymixin pretreatment only) was 

associated with Cr and Fe content. 

December 2009 D-48 
Study Pollutant Exposure Effects 

Reference: Hutchison 

et al. (2005, 097750) 

Species: Mouse 

Cell Line: J774.1A

PM10: Samples collected for 7 day 

during closure (-C) and reopening of 

steel plant (-R) 

PMT: PM total (aqueous sonicate) 

PMS: PM soluble aqueous 

PMI: PM insoluble aqueous 

Particle Size: PM10 

Route: Suspension 

Dose/Concentration: 500 μl (estimated 

concentrations of 112, 143, 156, 180, 233, 

255 μg/1ml water) 

Time to Analysis: 4 h 

Particle Characterization: Reopening of the plant 

showed a significant increase in the total and acid 

extractable metal content of PM. Aqueous 

extractable metal content did not change. Soluble 

zinc, copper and manganese also increased 

significantly post reopening. Iron was the most 

abundant in acid extractable metals and increased 

greatly at the reopening. 

TNF-α: PMT-R and PMT-C induced a statistically 

significant increase. Treatment with chelation agent 

reduced effect to control levels. 

Reference: Imrich et 

al. (2007, 155859) 

Species: Rat 

Gender: Female 

Age: 12-14 wk 

Cell Type: AM 

UAP: SRM 1649 (positive control) 

TiO2: Particle control 

CAPs (Boston, MA) 

All cells primed with LPS 

Coexposure with NAC, 

dimethylthiourea (DMTU), H2O2 or 

catalase 

Particle Size: CAPs: ≤2.5 μm; UAP: 

PM2.5; TiO2: ~1 μm 

Route: Cell Culture (2×105 cells/well) 

Dose/Concentration: Caps 100 μg/mL; 

UAP: 50 or 100 μg/mL; LPS 250 ng/mL; 

NAC, DMTU: 2, 10, 20 mM; Catalase: 1, 5, 

10 mM; H2O2 0-50 μm/hr 

Time to Analysis: 18-20h 

TNF-α: DMTU at 20 mM reduced TNF in LPSprimed cells in control and UAP-treated groups. 

NAC at 20 mM reduced TNF release but this was 

not statistically significant. Catalase significantly 

inhibited TNF in control and UAP-treated groups. 

CAPs (especially the insoluble portion) significantly 

increased TNF unless co-exposed with NAC, DMTU 

or catalase. All three reduced levels back to around 

basal levels. DMTU was particularly effective at 

diminishing TNF release. H2O2 increased TNF 

release in CAPs-exposed cells. TiO2 had no 

increased ability to induced cytokine release when 

mixed with H2O2. 

Cell Death: Viability decreased substantially when 

exposed to H2O2 + CAPs. The soluble fraction of 

CAPs showed to be more effective with H2O2 than 

the insoluble portion. TiO2 had no significant effect. 

NO: Some CAPs induced slight increases when 

mixed with H2O2. No difference was observed 

between soluble and insoluble portions of CAPs. 

DFO: DFO at 0.05 mM completely inhibited 

oxidation induced with soluble CAPs + H2O2. 

Insoluble CAPs + H2O2 was also DFO-sensitive. 

DFO was ineffective against the insoluble CAPs 

induction of TNF and MIP-2. 

Reference: Ishii et al. 

(2004, 088103) 

Species: Human 

Cell Type: A549 

(collected from 6 

lobectomy or 

pneumonectomy 

smokers), HBEC 

EHC-93:PM10 (obtained from 

Environmental Health Directorate, 

Ottawa, Ontario, Canada) 

Particle Size: PM10 

Route: Cell Culture (1×107 cells) 

Dose/Concentration: 100 μg/mL 

Time to Analysis: 3, 6, 24 h 

Cytokines: TNF-α, IL-1β, GM-CSF, IL-6, and IL-8 

levels were significantly increased in A549 cells. 

mRNA Expression: MCP-1, ICAM-1 and IL-8 

mRNA expression increased in untreated AM 

supernatants at 3 h. Only the MCP-1 levels were 

statistically significant at 3 h. Levels declined by 6 h. 

When A549 cells were exposed to PM10 exposed 

AM, levels of RANTES, TNF-α, ICAM-1, IL-1β, and 

LIF increased. Except for RANTES mRNA, these 

differences were less in the 6 h samples. VEGF 

increased as well, but this increase was not 

statistically significant. 

TNF-α and IL-1β-neutralizing Antibodies: IL-1β

antibody alone or in combination with TNF-α

significantly reduced expression of all eight mRNAs. 

Combinations for some mRNAs reduced expression 

by up to 1/2. This effect was not observed when 

A549 was treated with the control AM. 

Transcription Factor Binding Activity: Binding of 

AP-1 and Sp1 increased when A549 treated with 

supernatants from PM10-exposed AM, but not from 

control AM. 

December 2009 D-49 
Study Pollutant Exposure Effects 

Reference: Ishii et al. 

(2005, 096138) 

Species: Human 

Cell Type: AMs 

(obtained from 10 

smokers who stopped 

smoking 6 wk prior), 

HBEC 

EHC-93: PM10 (obtained from 

Environmental Health Directorate, 

Ontario, Canada) 

Particle Size: PM10 

Route: Cell Culture (HBEC: 2.5-3.0×106

cells; AM: 1×107

 cells; co-culture of 

AM/HBEC: 5×106 cells) 

Dose/Concentration: 100 μg/mL 

Time to Analysis: 2, 24 h 

 mRNA Expression After 2 h Exposure: AM or 

HBEC exhibited no effect. In contrast, co-culture 

increased expression of MIP-1β, GM-CSF, M-CSF, 

IL-6, MCP-1 and ICAM-1-mRNA. 

mRNA Expression After 24 h Exposure: AMs 

exhibited no effect. HBEC increased levels of GMCSf, LIF and ICAM-1. Co-culture, on the other hand, 

increased expression of MIP-1β, GM-CSF, M-CSF 

and ICAM-1 mRNA. 

Protein Levels: AM and HBEC both increased GMCSF, IL-6 and MIP-1β release into the supernatant. 

Co-culture effect was not additive but synergistic 

(i.e., higher than expected). MCP-1 levels did not 

increase significantly. Co-culture appeared to 

decrease protein levels for both the control and PM 

values. M-CSF levels increased for co-culture only. 

Surface Expression of ICAM-1: Upon 24 h 

exposure to PM, HBEC exhibited an increase in 

expression. Expression in AMs were not affected by 

2 h PM stimulation. 

ICAM-1 Inhibitors: IgG or anti-CD11b antibody was 

unaffected in co-culture. 

Reference: Jalava et 

al. (2005, 088648) 

Species: Mouse 

Cell Type: RAW 264.7 

UPM: SRM1649a (Washington, DC) 

DEP: SRM1650 (NIST) 

EHC-93: Ottawa dust (Environmental 

Health Center, Ottawa, Canada) 

HFP-00: Pooled ambient air PM2.5

sample from Helsinki, Finland 

M-UPM: methanol extract of UPM 

Particle Size: SRM 1649a, SRM 1650, 

EHC-93: NR; HFP-00: PM2.5 

Route: Cell Culture (5×105 cells/mL) 

Dose/Concentration: 150 μg/mL 

Time to Analysis: Methanol treatment of 

PM samples: 24 h; Exposure to ambient PM 

samples: 2, 4, 8, 16, or 24 h. 

TNF-α: All the PM samples increased TNF-α. 

Cell Viability: SRM1649a exhibited the most 

cytotoxicity, followed by HFP-00 and EHC-93. 

Methanol significantly affected cytotoxicity of the 

EHC-93 sample only. 

Cytokines: TNF-α concentrations in the cell culture 

medium significantly increased at all time points 

between 2 and 24 h. The highest increase was seen 

in EHC-93. IL-6 production also increased at 

different levels with the highest increase observed in 

EHC-93. No response was observed for IL-10. 

Cell Viability: Duration of exposures had no 

significant effect on any of the samples. A 2 h 

exposure time was sufficient to induce the typical 

reductions in cell viability. 

Reference: Jalava et 

al. (2006, 155872) 

Species: Mouse 

Cell Type: RAW 264.7 

PM: Collected east of Helsinki, Finland 

between Aug 23 and Sept 23, 2002 

Divided in 12 groups (4 sizes by 3 

exposure types): 

-S: seasonal average 

-W: wildfire 

-M: mixed 

-B: blank 

Particle Size: PM10-2.5; PM2.5-1; PM1-0.2; 

PM0.2

Route: Cell Culture (5×106 cells/mL) 

Dose/Concentration: 15, 50, 150 and 300 

μg/mL 

Time to Analysis: 24 h 

Particulate Mass Concentrations in HVCL Size 

Ranges: The largest increase of PM concentrations 

was observed in PM1-0.2. 

NO: All 12 samples increased NO production when 

compared to corresponding unexposed controls. 

Peaks were observed at 150 μg/mL, except in PM1-

0.2. 

Cytokines: All 12 samples increased TNF-α and IL6 production. PM10-2.5 and PM2.5-1 produced a much 

larger response than PM1-0.2 and PM0.2. IL-6 

production for PM0.2 was not measured. MIP-2 

production also increased with similar trends. 

Cytotoxicity: All 12 samples induced dosedependent decreases in cell viability. PM10-2.5 were 

the least active inducers of apoptosis while PM0.2 

showed the highest activity (4-17% of apoptotic 

cells). 

December 2009 D-50 
Study Pollutant Exposure Effects 

Reference: Jalava et 

al. (2007, 096950) 

Species: Mouse 

Cell Type: RAW 264.7 

Urban background PM 

PM10, PM2.5, and PM0.2 collected from 

6 European cities during different times 

of the year from October 2002 to July 

2003: 

-D: Duisburg (Fall) 

-P: Prague (Winter) 

-A: Amsterdam (Winter) 

-HR: Helsinki (spring), 

-B: Barcelona (spring) 

-AT: Athens (summer) 

Particle Size: PM10: 2.5-10 μm; PM2.5: 

0.2-2.5 μm; PM0.2: <0.2 μm 

Route: Cell Culture (5×105 cells/mL) 

Dose/Concentration: 15, 50, 150, 300 

μg/mL 

Time to Analysis: 24 h 

PM Characterizations: The highest mass 

concentrations of PM10 and PM0.2 were measured in 

Athens. Prague had the highest PM2.5

concentrations. 

NO: All PM fractions induced statistically significant 

NO production in macrophages. PM2.5 -P and PM2.5 - AT produced significantly larger responses, though 

all samples at 150 and 200 μg/mL induced 

statistically significant production. When compared 

to the other PM0.2 samples, -P and -HR produced 

significantly larger responses. 

Cytokines: PM10 showed average cytokine 

production to be 7.8 fold and 83 fold for TNF-α, and 

4.4 fold and 530 fold for MIP-2 when compared to 

PM2.5 and PM0.2 respectively. PM10 induced 

statistically significant increases in production of 

TNF-α, MIP-2 and IL-6. PM2.5, with exception of 

Prague, caused significant increases in cytokines. 

PM0.2-A and -AT showed small yet statistically 

significant increases in TNF-α. An increase in MIP-2 

was observed with -P and -HR. IL-6 increased 

significantly with PM10 and slightly with PM2.5. In the 

PM0.2 range, only the -A and -AT samples caused a 

small, statistically significant TNF- α production. 

MIP-2 production was only detected from the -P and 

-HR samples. PM0.2 effects on IL-6 response were 

negligible. 

Cytotoxicity: The average cytotoxicity of PM10 and 

PM2.5 were roughly equal, but PM0.2 were less 

cytotoxic with the exception of -P. The doseresponse trends for most of the samples were 

linearly declining, with PM10 and PM2.5 exhibiting 

statistically significant declines in viability. 

Reference: Jimenez et 

al. (2002, 156610) 

Species: Human 

Cell Type/Line: A549, 

THP-1, Mono Mac 6 

(DSMZ) 

PM10: Collected from London and 

Edinburgh air particulate monitoring 

stations. 

TiO2: Tioxide Europe (London, UK) and 

Degussa-Huls (Cheshire, UK) 

UFTiO2: Tioxide Europe (London, UK) 

and Degussa-Huls (Cheshire, UK) 

Particle Size: PM10, TiO2: 200 nm; 

UFTiO2: 20 nm 

Route: Cell Culture (110,625 cells/well) 

Dose/Concentration: PM10, TiO2, UFTiO2: 

100 μg/mL; TNF-α: 10 ng/mL 

Time to Analysis: 4 h 

NF-κB and AP-1 DNA Binding: NF-κB DNA binding 

increased in PM10 and TNF-α exposed macrophages 

by 9.5 and 12 fold. NF-κB activity remained 

unaltered in TiO2 and UFTiO2 exposed 

macrophages. 

IL-8: Cells treated with PM10 conditioned media 

increased transcription binding of NF-kB to IL-8 

promoter sites. Increases were observed in gene 

expression after exposure to TNF-α and PM10. TiO2

or UFTiO2 had no effect. Increases observed in IL-8 

production with PM10. 

IL-8 Promoter CAT Activity: PM10 media increased 

CAT expression by 65% over control. No differences 

observed with TiO2 or UFTiO2 media. 

Neutrophil Chemotaxis: PM10 conditioned media 

induced a 2.3 fold increase compared to control. 

TNF-α and IL-1β Production: PM10 media 

increased TNF-α and IL-1β production. No increases 

were observed in TiO2 and UFTiO2 media. 

December 2009 D-51 
Study Pollutant Exposure Effects 

Reference: Jung et al. 

(2006, 132421) 

Species: N/A 

Type: Surrogate Lung 

Fluid 

Soot Particles: Generated using a coflow, laminar, diffusion flame system 

CB (Degussa) 

PM2.5: Collected using IMPROVE air 

pollution samplers 

Particle Size: Soot: 185 nm; CB: 25 

nm, PM2.5 

Route: Surrogate Lung Fluid 

Dose/Concentration: Soot: 0-30 mg; CB: 5-

10 mg; PM2.5: 50 or 100 μg/mL 

Time to Analysis: Parameters measured 

continuously over 2 h. 

OH Radical Formation: Formation occurred with 

linear dependence on soot mass. Average response 

was 0.89 nmol OH produced per mg of soot. 

Formation also occurred with soot + hydrogen 

peroxide. Hydrogen peroxide alone did not form OH 

radicals. 

Fe: Average Fe concentration in soot particles was 

305 ± 172 nM. Observed negative correlation 

between amount of Fe and amount of OH radical 

formation. DSF inhibited iron-induced increase in 

OH radical formation. 

Carbon Black: OH radical generation by carbon 

black was significantly less than soot. OH generation 

by CB was observed to be linearly proportional to 

PM mass, but CB was much less efficient at 

generating the OH radical. 

PM2.5: A high variability in the increase of OH 

radicals was observed with PM2.5. Pretreatment with 

DSF partially blocked OH radical production, but a 

significant level remained. This may be due to PM2.5

containing high levels of Fe and Cu. 

Reference: Kafoury 

and Madden (2005, 

156617) 

Species: Mouse 

Cell Type: RAW 264.7 

DEP: SRM 1975 (purchased from 

NIST, Rockville, MD) 

BAY11-7082, NF-κB inhibitor 

(coexposure) 

IL-1ß: obtained from Santa Cruz 

Biotechnology (Santa Cruz, CA) 

Particle Size: DEP: 0.3 μm (mean 

diameter) 

Route: Cell Culture (3-4×105 cells) 

Dose/Concentration: DEP 25, 100, or 250 

μg/mL; IL-1ß: 100 ng/mL 

Time to Analysis: DEP: 4 h pre-treated with 

BAY11-7082 for 1.5 h; IL-1β: 4 h 

TNF-α: DEP induced a significant release of TNF-a 

at 100 and 250 μg/mL dose-dependently. Exposure 

at 25 μg/mL had no effect. IL-1β containing PM 

samples at 100 μg/mL also resulted in a significant 

release of TNF-α. 

NF-κB Binding Activity: Treatment of RAW 264.7 

with BAY11-7082 significantly inhibited IL-1βinduced TNF-α release. Similar effects observed 

with DEP-induced TNF-α release. 

Apoptosis: Inhibition of NF-kB binding activity by 

BAY11-7082 resulted in DEP-induced apoptotic 

response. Without BAY11-7082, apoptosis was not 

induced even at the DEP dose of 250/μg/mL for 4 h. 

The control, U937 cells with campothecin, induced 

apoptosis. 

Reference: Karlsson 

et al. (2006, 156625) 

Species: Human 

Cell Type: A549, 

Monocytes (isolated 

from heparinized 

whole blood) 

PM 

(W1: wood burning in old-type boiler; 

W2: wood burning in modern boiler; 

P: wood pellets burning in pellets 

burner; T1: PM10 tire debris with 

studded tires and ABT pavement; 

T2a: PM10 tire debris with studded tires 

and ABS pavement; T2b: PM2.5 tire 

debris with studded tires and ABS 

pavement; T3: PM10 tire debris with 

friction tires and ABS pavement; 

St: PM10 from busy street in Stockholm, 

Sweden; Su: PM10 from platform of 

subway station in Stockholm) 

Particle Size: W: NR, T1, T2a, T3, St, 

Su: PM10, T2b: PM2.5 

Route: Cell Culture 

Dose/Concentration: Suspension: 40 

μg/cm2; Culture: 100 μg/cm2 (1 ml/well) 

Time to Analysis: Suspension: 4 h; Culture: 

18 h 

PM Characterization: Boiler emitting PM-W1 led to 

4 times higher emission of particles when compared 

to PM-W2 and 8 times higher emissions when 

compared to PM-P. Total concentration and CO was 

substantially higher in the old-type wood boiler. 

Effects with Filter Fibers: No increase of DNA 

damage was observed compared to the water 

control. Filter fibers led to the induction of cytokines 

in human macrophages. 

Genotoxicity: All particulate samples induced DNA 

damage in A549 cells. PM-Su exhibited the most 

genotoxicity and induced 4-5 times more DNA 

damage than others. 

Cytokines on Glass Fiber Filters: PM-W2 induced 

a significant increase in IL-8. PM-St induced the 

highest increases of IL-6, IL-8, and TNF-α. 

Cytokines on Teflon Filters: PM-2a and PM-2b 

samples caused significant increases of IL-6, IL-8, 

and TNF-α. 

December 2009 D-52 
Study Pollutant Exposure Effects 

Reference: Katterman 

et al. (2007, 096358) 

Species: Rat 

Cell Type/Line: RLE6TN (Alveolar 

Epithelial Cell Line) 

PM: Oils: OAAF, Oil Q, OII l II, NF2 

PM: Coal Germany and Ohio 

Diesel particulates: ZODDA (doped with 

Zn), ZSDDA (doped with Zn and S): S: 

PMs washed in solution; F: Fresh 

samples; L: Leached 

Al2O3, Fe2O3, SiO2, TiO2, ZnO also 

tested 

Particle Size: NR 

Route: Cell Culture (cytotoxicity: 50,000 

cells/well; SEM: 25,000 cells) 

Dose/Concentration: Oils 0.2 mg/mL; 

Coals 0.7 mg/mL; Diesel 0.01 mg/mL; Al2O3

0.5 mg/mL; Fe2O3 0.7 mg/mL; SiO2 0.7 

mg/mL; TiO2 0.7 mg/mL; ZnO 0.05 mg/mL 

Time to Analysis: 24 h 

Metabolic Activity: For oils comprised of 3/4 fresh 

and 1/4 leached, metabolism decreased. Coals 

(fresh and leached) had no effect. ZODDA-F and 

ZSDDA-F both induced decreases in activity. 

ZSDDA-L had no effect. 

Cellular Morphology: PM-S had a minimal effect. 

PM-F induced widespread cell damage. 

Constituent Differences between PM-F and PML: In oil samples Cu, Ti and Ca salts were removed 

upon washing. Fe, Al, Si remained constant. 

Grinding Effects: Coal toxicity increased upon 

grinding, whereas diesel PM toxicity decreased upon 

grinding. 

Metal Oxide Effects: Only SiO2, and ZnO (much 

higher at lower concentrations than other metal 

oxides) decreased metabolic activity. Fresh, washed 

and sonicated samples exhibited similar results. 

Grinding only affected TiO2 (increase) and ZnO 

(decrease). 

Reference: Kendall et 

al. (2004, 156634) 

Species: Human 

Tissue Type: BALF 

(obtained by 

bronchoscope from 6 

nonsmokers and 3 

smokers) 

PM2.5 sample sites; 2 schools in Bronx, 

NY, 6 background urban, 6 urban 

roadside. Sampling occurred 24 h/day 

for 12 days. 

Particle Surface Chemistry: 79-87% 

carbonaceous material (Ch, COO, C-

(O,N)), 10-17% O (O1s), 1.5-4% N 

(NH4

+

, N-C, NO3

2-), 0.6-1% S, and 0.3-2 

% Si. 

Only NO3 - higher in roadside samples. 

NH4 and NO3 - correlated with NO and 

NOX in air but not NO2. 

Particle Size: PM2.5 

Route: BALF interaction 

Dose/Concentration: 5-10 ml of 0.5 M NaCl 

or BALF 

Time to Analysis: Filters treated with BALF 

for 4 h 

Saline Washing: Removed particles and decreased 

NH4, NO3, O and S relative to C1. 

BALF treatment (XPS): PM2.5 surfaces interacted 

strongly with BALF within hours of contact. Specific 

surface components of PM2.5 immersed in BALF 

were desorbed while biomolecules from BALF were 

adsorbed to particles. N-C on the PM surface 

increased 3 fold for smokers and 4 fold for 

nonsmokers (range 1.4-7.4). This is most likely 

related to protein-like adsorption on PM. Treatment 

also induced a slight increase in COO and 

decreases in NH4, NO3, O and S. 

ToF-SIMS - Organics: Particle loading and surface 

hydrocarbons showed a linear correlation. Loss of 

hydrocarbons from PM2.5 surface averaged 55% (10-

75) after undergoing saline and BALF washes. In 

only 3/12 samples BALF removed less hydrocarbon. 

BALF treatment increased the amino acid and 

phospholipid content of the PM2.5 surface. 

ToF-SIMS - Inorganic: Saline washing appeared to 

increase Al and Si but with extreme variability; this 

increase was not statistically significant. Both saline 

and BALF washing decreased NH4 and Na levels to 

a similar extent. BALF washing did not affect Al or 

Si. 

Reference: Kim et al. 

(2005, 088454) 

Species: Human 

Cell Type/Line: 

BEAS-2B 

Zn2+

Particle Size: NA 

Route: Cell Culture 

Dose/Concentration: 15, 50, 100 μmol 

Time to Analysis: 1-20 h 

Cell Viability: At 50 μM for 20 h, no apoptosis was 

induced. 

IL-8: At 12 h, IL-8 increased in dose-dependent 

manner. At 15 or 50 μM, Zn2+ increased protein 1.6 

and 4.6 fold respectively. IL-8 mRNA expression 

increased dose-dependently, reaching statistical 

significance at 2 h and continuing until 4 h. 

EGFP (adenoviral IL-8 promoter): Levels 

increased 2.4 fold with 50 μM Zn2+. 

Proteases: With 50 μM Zn2+, phosphorylation of 

MAPKs ERK, JNK and p38 increased by 15 min and 

continued increasing up to 2 h. Pre-exposure of 

inhibitors of MEK, JNK, before Zn2+ exposure 

caused inhibition of Zn-induced IL-8 mRNA and 

protein production. Inhibitor of p38 had no effect. 

Dephosphorylation of ERK and JNK was partially 

inhibited with exposure to Zn2+. 

December 2009 D-53 
Study Pollutant Exposure Effects 

Reference: Kleinman 

et al. (2003, 087938) 

Species: Rat 

Strain: Wistar Kyoto, 

F344 

Age: 22-24 mo, 10 wk 

Cell Type/Line: AM

UF1: Utrecht 1 Fine (urban freeway) 

UC1: Utrecht 1 Coarse 

UF2: Utrecht 2 Fine (urban, freeway, 

light industrial) 

UC2: Utrecht 2 Coarse 

SRM 1650 

SRM 1648 

Particle Size: UF1: 0.2-2.5 μm; UC1: 

2.5-10 μm; UF2: 0.2-2.5 μm; UC2: 2.5-

10 μm 

Route: Cell Culture (105 cells/well at 106

cells/mL) 

Dose/Concentration: 1.2 to 1200 ng/106

cells 

Time to Analysis: 4, 18 h 

Macrophage PMA-stimulated respiratory burst 

activity: SRM 1648 and 1650 induced dosedependent decreases approaching 0 at 50 -100 

μg/105 cells. Large dose-dependent decreases from 

old rat AMs exposed to fine PM exposure were 

followed by young rat AMs exposed to fine PM. 

However, no age-related effects were statistically 

significant. 

Free radical production: All coarse particles 

depressed free radical production in a semi-dosedependent manner, with UC2 exhibiting more 

potency than UC1. Both fine particles also showed 

dose-dependent responses but UF1 and UF2 

responses were greater than the control at 3 μg/106

cells. 

PM Characterization: Ratios between coarse and 

fine PM were similar for metals tested (Al, Fe, Mn, 

Zn). Al was higher in coarse samples and Zn higher 

in fine PM, although large variability was observed. 

Fe and Mn results were roughly equivalent for all 

samples. 

Reference: Kocbach 

et al. (2008, 198874) 

Species: Human 

Cell Type/Line: THP-1 

PMW: Wood smoke particles 

Collected from conventional Norwegian 

wood stove burning birch 

PMT+: Traffic-derived particles; 

collected from road tunnel in winter 

when studded tires were used 

PMT-: Traffic-derived particles; 

collected from road tunnel in summer 

without studded tires 

DEP: SRM2975 

Porphyr: fine grain syenite porphyry 

(prepared by SINTEF, Trondheim, 

Norway) 

Polymyxin B Sulphate (endotoxin 

inhibitor) 

Particle Size: PMW, PMT, DEP: NR; 

Porphyr 8 μm (mean) 

Route: Cell Culture (1×106 cells/mL) 

Dose/Concentration: 30-280 μg/mL 

Time to Analysis: 2, 5, 12 h 

Particle Characterization: PMT+ contained a high 

mineral particle content. PMT- contained carbon 

aggregates, and polycyclic aromatic hydrocarbons 

(PAH). PMW and DEP contained carbon 

aggregates. PAH content of PMW was greater than 

DEP. Porphyr was not included in the analysis. 

Cytokines: PMT± induced releases of TNF-α, IL-1β, 

and IL-8 with 30 or 70 μg/mL. PMW similarly 

induced TNF-α and IL-8. DEP induced IL-1β and IL8. Porphyr induced IL-8 increases. IL-4, IL-6 and IL10 were unaffected. Overall, the order of effective 

cytokine induction from most to least effective was 

PMT±, PMW, DEP, and Porphyr. mRNA expression 

of TNF-α, IL-1β, IL-8, and IL-10 increased with 140 

μg/mL of PMT± and slightly for PMW. 

LDH: PMT ± induced small but statistically 

significant increases at low doses. DEP increased 

LDH at 280 μg/mL only. 

Polymyxin B Sulphate: The endotoxin inhibitor 

significantly inhibited LPS-induced cytokine release 

by 80-90% and reduced PMT± induction by 50-60%. 

Organic Extraction: PMT+ washed and native 

particles showed equivocal induction of cytokine 

release. PMT+ organic extract had no effect. PMTand PMW organic extracts significantly increased 

TNF-α and IL-8. Washed particles induced less 

significant increases of IL-8. DEP organic extract 

had no effect. 

December 2009 D-54 
Study Pollutant Exposure Effects 

Reference: Kristovich 

et al. (2004, 087963) 

Species: Human 

Cell Type/Line: 

HUVEC, HPAEC, 

HPMVEC, HPBMC 

CP: carbon particle (carbonaceous 

negative image of zeolite) 

CFE: C/Fe particulate (synthesized) 

CFE+: C-Fe/F-Al-Si particulate 

(synthesized) 

CFA: Coal Fly Ash (Coal-fired power 

plant, NOS) 

DEP: (exhaust pipe of diesel powered 

truck) 

CP, CFE, CFE+ approx 1 μm 

(resembling zeolite) 

Particle Characterization (Surface 

chemistry): CP = 88% C, 1% Si, 10% 

O, 1% N. CFE = 80% C, 2% Fe, 2% Si, 

16% O. CFE+ = 20% C, 6% Al, 3% Si, 

50% F, 6% O, 11% N, 4% Na. 

CFA = 25% C, 3% Fe, 13% Al, 17% Si, 

41% O, 1 % N. DEP = 70% C, 3% Fe, 

24% O, 1% N, 2% S. 

Particle Size: CP, CFE, CFE+: 

approximately 1 μm (resembling 

zeolite); CFA: <2 μm; DEP: 150 nm 

Route: Cell Culture (4×106 cells/well) 

Dose/Concentration: CP: 5-50 μg/cm2; 

CFE: 2.5-25 μg/cm2; CFE+: 2.5-25 μg/cm2; 

CFA: 10-100 μg/cm2

; DEP: 2.5-25 μg/cm2

Time to Analysis: 4, 8, or 24 h 

Cytotoxicity: CP exhibited no effects. DEP and 

CFE exhibited intermediate toxicities in the range of 

50-70 μg/cm2

. No toxicity was apparent when 

treated with CFA (up to 200 μg/ cm2

) or synthesized 

C particulates. 

Endothelial Activation: ICAM-1, VCAM-1, and Eselectin were activated dose-dependently by DEP, 

CFE, and CFE+. No effects observed for CFA or CP. 

These effects were not the result of endotoxin 

release. 

Individual Variability: Donors (humans) showed 

variability in responses especially for CFA. 3/9 had a 

medium response negated by ND responses in 6/9. 

Reference: Kubatova 

et al. (2006, 198835) 

Species: Rat, Human 

Cell Type/Line: RAW 

264.7, BEAS-2B 

PMW: Wood Smoke 

Collected from airtight wood stove 

burning hardwoods 

-P: Polar (fraction extracted from 25-50 

C) 

-MP: Mid Polar (fraction extracted from 

100-150 C) 

-NP: Nonpolar (fraction extracted from 

200-300 C) 

-C: P + MP + NP 

Particle Size: NR 

Route: Cell Culture (RAW 264.7: 106

cells/mL; BEAS-2B: 105

 cells/mL) 

Dose/Concentration: 50, 100, 200 μg/mL 

Time to Analysis: 12 h 

GSH: PMW-MP and PMW-NP induced GSH 

depletion substantially in a dose dependent manner 

starting at 50 μg/mL in both cell types. DMSO had 

no effect. 

Cytotoxicity: PMW-MP and PMW-NP increased 

cytotoxicity at 200 μg/mL in RAW 264.7. BEAS-2B 

was unaffected. 

Particle Characterization: PMW-MP contained 

higher concentrations of oxy-PAHs, disyringyls, 

syringylguaiacyls and PAHs. oxy-PAHs include 9-

fluorenone, 1-phenalenone, 9,10-anthraquinone and 

hydroxycadalene. PAHs included phenanthrene, 

fluoranthene and pyrene. 

Effects of Individual Components of PMW-MP on 

GSH: 1,8-dihydroxy-9-10anthraquinone and 9,10-

phenanthraquinone depleted GSH. 9,10-

anthraquione, anthrone, 1-hydroxypyrene increased 

GSH. Phenanthrene, 1-methylpyrene, 9-fluorenone 

and xanthone had no effect. 

Reference: Kubatova 

et al. (2004, 087986) 

Species: Monkey 

Cell Type/Line: 

African green monkey 

kidney cells 

designated COS-1 

(CV-1 cells with origin -

defective mutants of 

SV40), E coli PQ 37 

(SOS Chromotest) 

DEP: Obtained from diesel bus 

PMW: Wood smoke particulates 

obtained from airtight wood stove 

burning hardwood 

HSF: Hot pressure fractionation 

-C: P + MP + NP 

-P: Polar 

-MP: Mid Polar 

-NP: Nonpolar 

OE: Organic Extraction 

-HNP: n-hexane nonpolar 

-MEP: methanol polar 

Particle Size: NR 

Route: Cell Culture (10,000 cells/180 μl) 

Dose/Concentration: 0, 50, 100, 150, 200, 

250, 300 μg/mL 

Time to Analysis: Cytotoxicity: 24 h; 

Chomotest: 2 h SOS 

Cytotoxicity: PMW induced cytotoxicity in a dosedependent manner. PMW-HNP induced low 

cytotoxicity, followed by PMW-C (intermediate) and 

PMW-MEP (highest). Levels above 25 μg/mL were 

cytotoxic. DEP-HNP induced cytotoxicity but was not 

dose-dependent. Results similar for all 3 fractions 

(highly variable). All fractions with concentrations 

higher than 100 μg/mL were cytotoxic. 

Extraction Water Temperature Effect: PMW was 

cytotoxic at temperatures over 50 C. DEP was 

cytotoxic at temperatures higher than 200 ̊ C. At 

250 ̊, cytotoxicity between DEP and PMW was 

similar. At 300 ̊ C, PMW cytotoxicity declined and 

DEP stayed high, resulting in DEP inducing higher 

cytotoxicity than PMW. 

SOS Chromotest: β-Galactosidase formation 

increased, peaked at 200 ̊ C with DEP and declined 

to control at 300 ̊ C. Individual fractions showed 

linear dose response from 25-200 μg/mL with 150 ̊ C 

and 200 ̊ C extracts significantly higher. 

December 2009 D-55 
Study Pollutant Exposure Effects 

Reference: Lee et al. 

(2005, 156682) 

Species: Human 

Cell Type/Line: A549 

MEP: Motorcycle Exhaust Particles 

(Yamaha Cabin engine, 95 octane 

unleaded gasoline,150 rpm) 

MEPE: MEP Particle Free 

Particle Size: MEP 0.5 μm; MEPE<0.2 

μm 

Route: Cell Culture (1×105 cells/well) 

Dose/Concentration: MEP 0.02, 0.2, 0.2, 2, 

20 μg/mL; MEPE 20 μg/mL 

Time to Analysis: 24 h 

IL-8: MEP induced IL-8 at concentrations greater 

than 0.2 μg/mL. Levels increased 2fold at 24 h with 

20 μg/mL. MEPE induced similar responses at 20 

μg/mL. Induction of IL-8 mRNA expression was 

dose-dependent with MEP and MEPE. 

Cytotoxicity: Exposure to particles did not affect 

cytotoxicity. 

NF-κB: MEP (20 μg/l) induced time-dependent 

activation for 2 h and continued at same level for up 

to 6 h. Pretreatment of PDTC (1mM) fully inhibited 

MEP induction. 

MAP Kinase: MEP induced time-dependent 

activation up to 30 min and stayed elevated for at 

least 60 min. 

ROI: MEP treatment induced a time-dependent 

increase in ROI for up to 1 h and then continued the 

at same level for up to 6 h. 

Reference: Lee and 

Kang (2002, 198864) 

Species: Mouse 

Cell Type/Line: 

Peritnoeal 

Macrophages, RAW 

264.7 

MEP Yamaha 2-stroke engine using 

unleaded gas) 

MEPE(particle-free MEP) 

Particle Size: 0.5 μm 

Route: Cell Culture (5×105 cells/mL 

(Cytotoxicity), 3×105

 cells/mL (Apoptosis), 

2×106

 cells (MMP and ROI), 1×107 cells 

(GSH) 

Dose/Concentration: 5, 10, 50, 100, 300, 

1000 μg/mL 

Time to Analysis: 6, 12, 18, 24 h 

Cytotoxicity: Viability decreased dose and timedependently in all cell types at 24 h. 

Apoptosis: subG1 significantly and dosedependently increased at the 300 MEP μg/mL dose 

in all cell types, indicating increased apoptosis. 

MEPE induced similar results. Inhibition was 

successful against MEP-induced apoptosis by 

calcium chelators EGTA, BAPTA-AM, cyclosporin A 

and antioxidants NAC, GSH, catalase and SOD. 

Ca2+: MEP and MEPE increased Ca2+ at 300 

μg/mL. BAPTA-AM completely inhibited induction. 

ROI: MEP increased ROI in a time-dependent 

manner. Calcium chelators and antioxidants 

substantially attenuated induction. 

GSH: MEP significantly decreased GSH. 

MMP: Mitochondria membrane potential decreased 

dose-dependently with MEP 100 μg/mL and 300 

μg/mL. Calcium chelators and antioxidants partially 

inhibited reduction. 

Reference: Li et al. 

(2002, 042080) 

Species: Mouse 

Cell Line: RAW 264.7, 

THP-1 

VACES (Biosampler PM10 in Downey, 

CA -DEP concentrate in water) 

DEPM (DEP methanol extract) 

DEPME (DEP methylene chloride 

extracts) 

DEPAL (DEPME aliphatic (hexane)) 

DEPAR (DEPME aromatic 

(hexane/methlene chloride)) 

DEPPO (DEPME polar (methylene 

chloride/methanol)) 

Particle Size: NR 

Route: Cell Culture (2×106 cells/well Mouse 

RAW 264.7 and THP-1; 0.67×106

 cells/well 

Murine RAW 264.7) 

Dose/Concentration: 10-200 μg/mL 

JNK Activation and IL-8 Production: THP-1 

cells- 0, 10, 25, 50, 100 μg/mL DEPM; THP1 cells- 0, 10, 25, 50, 100 μg/mL of DEP; 

RAW264.7 cells- 10 -100 DEP μg/mL 

Cytotoxicity: 1, 10, 25 (THP-1 cells only), 50, 

100, 200 μg/mL 

GHS/GSSG: 0, 10, 25, 50, 100 μg/mL 

HO-1 Expression: 0, 25, 50, 100, 200 μg/mL 

Time to Analysis: GHS/GSSG: DEPM, 

whole DEP (RAW 264.7 only) 8 h. 

HO-1, MnSOD Expression: RAW 264.7, 

THP-1 7h. RAW 264.7 cells exposed to 

whole DEP 16 h. 

JNK Activation, IL-8 Production: THP-1 cells 

30 min, 16 h. RAW 264.7 cells 90 min. 

Cytotoxicity: RAW264.7, THP-1 18 h. 

GSH/GSSG Ratio: DEPM induced dose-dependent 

decrease in GSH/GSSG ratios in both cell lines. 

DEP induced decreases at comparable doses to 

DEPM. 

HO-1 Expression: Cells exhibited dose-dependent 

increases in HO-1 expression. 

HO-1 Expression in Murine RAW 264.7: VACES-F 

consistently induced HO-1 expression over a 9m 

period, whereas VACES-C was effective in inducing 

HO-1 during fall and winter. HO-1 induction 

positively correlated to higher OC and PAHs that 

were represented in VACES-F, but also seen with a 

rise in PAHs in VACES-C during winter months. 

MnSOD: At doses of 2.5 μg/mL, DEPM increased 

MnSOD in THP-1 cells. 

JNK Activation: DEPM dose-dependently 

increased JNK phosphorylation but did so without a 

change in the JNK expression level. DEP-exposed 

mouse RAW264.7 cells exhibited similar increases 

in JNK phosphorylation but without increasing JNK 

expression. 

IL-8: Exposure to DEPM elicited dose-dependent 

increase in IL-8 levels of THP-1 cells. 

December 2009 D-56 
Study Pollutant Exposure Effects 

Reference: Li et al. 

(2002, 087451) 

Species: Human 

Cell Line: BEAS-2B, 

NHBE, THP-1 

macrophages 

DEPM (DEP methanol extract) 

DEPME (DEP methylene chloride 

extracts) 

DEPAL (DEPME aliphatic (hexane)) 

DEPAR (DEPME aromatic 

(hexane/methlene chloride)) 

DEPPO (DEPME polar (methylene 

chloride/methanol)) 

Particle Size: 0.05-1 μm 

Route: Cell Culture (106 cells/mL) 

Dose/Concentration: 0, 10, 25, 50, 100 

μg/mL 

Time to Analysis: 30, 60, 120 min 

ROS: BEAS-2B cells demonstrated increased HE 

fluorescence, indicating increased ROS formation. 

THP-1 cells were unaffected. 

GSH/GSSG Ratio: DEPM dose-dependently 

decreased GSH/GSSG in THP-1 and BEAS-2B 

cells. Similar changes occurred with NHBE cells. 

THP-1 cells maintained a higher ratio of GSH/GSSG 

than BEAS-2B and NHBE cells. 

NAC on GSH/GSSG Ratio: Exposure to DEPM in 

the presence of NAC did not affect the GSH/GSSG 

ratio in BEAS-2B and NHBE cells. In THP-1 cells, 

NAC prevented a decline in the GSH/GSSG ratio. 

MnSOD and HO-1: THP-1, BEAS-2B and NHBE 

cells showed constitutive MnSOD expression and 

dose-dependent expression of HO-1 protein and 

mRNA. No change occurred in the expression of βactin. 

DEPAL, DEPAR, DEPPO, CoPP on HO-1 

Expression: DEPPO was more potent than DEPAR. 

DEPAL lacked activity for THP-1 and BEAS-2B cells. 

The potency of DEPPO was sufficient to affect 

cellular viability and HO-1. CoPP induction of HO-1 

failed in THP-1 cells, but succeeded in BEAS-2B 

cells. However, it did not protect against the 

oxidizing effects of DEPM. 

JNK: JNK activation increased in DEP-exposed 

THP-1 and BEAS-2B cells. JNK isoforms were 

observed at doses of ≥ 25 μg/mL. In BEAS-2B cells 

a high rate of cell death diminished this response at 

100 μg/mL. NHBE also showed increased JNK 

phosphorylation at doses 50 - 100 μg/mL. 

NAC on JNK: NAC led to inhibition of JNK 

activation. 

IL-8: THP-1 cells showed dose-dependent increases 

of IL-8. NHBE cells showed incremental increases 

followed by rapid decline at 100 μg/mL attributed to 

apoptosis. BEAS-2B cells responded to 10 μg/mL 

with increased IL-8, but cellular toxicity and cell 

death led to a drop in IL-8 production at higher 

doses. 

Cytotoxicity: Comparing cytotoxicity at 25 μg/mL 

DEP, BEAS-2B cells had a higher rate of cell death 

than THP-1 cells. BEAS-2B cells showed a 

significant rise in cell death at doses larger than 10 

μg/mL. In THP-1 cells, it took doses of 25 μg/mL or 

more before significant increases occurred. 

In BEAS-2B, cell death began at 2 h. In THP-1, 

increases in cell death prolonged for 8h or longer. 

NHBE cells also showed increase rates of 

cytotoxicity compared to macrophages. NAC in 

THP-1 interfered with a generation of cytotoxicity, 

but NAC did not have any decreasing effect on cell 

death in BEAS-2B or NHBE cells. 

December 2009 D-57 
Study Pollutant Exposure Effects 

Reference: Lindbom 

et al. (2007, 155934) 

Species: Mouse 

Cell Line/Type: RAW 

264.7 

PM10: 

-ST: Street 

-S: Subway 

-G: Granite 

-Q: Quartzite 

(-G and -Q generated by road simulator 

at Swedish National Road and 

Transport Research Institute) 

Particle Size: PM10; Bimodal with 

peaks around 4-5 um and 7-8 um. 

Route: Cell Culture (130,000 cells/cm2) 

Dose/Concentration: 1, 10 or 100 μg/mL 

Time to Analysis: 18, 24 h 

Analysis of Arachidonic Release (AA): Cells 

pre-incubated w/ 1 μCI tritium marked for AA 

and washed exposed to 10, 50, 100 and 250 

μg/mL 

Cellular Viability: Viability was not influenced by 

any particle types and in all cases exhibited 90% or 

higher viability, except for the combination of subway 

particles and NAC where viability dropped to 20%. 

Cytokines: All particles induced TNF-α secretion in 

a dose-dependent fashion. PM-S was most potent at 

1 μg/mL. PM-G and PM-ST induced effects at 10 

μg/mL. PM-Q induced increase of TNF-α at 100 

μg/mL.PM-ST induced IL-6 release at 10 μg/mL. 

PM-G, PM-Q, PM-S induced IL-6 secretion at 100 

μg/mL. DFX inhibited TNF-α in cells exposed to PMS and PM-ST. DFX induced increase of TNF-α with 

PM-Q. For all PM types (except PM-ST) DFX 

inhibited induced IL-6 secretion. 

NO: PM-ST and PM-G induced a significant release 

of NO, with PM-ST inducing a higher NO release 

than PM-G. 

NAC: NAC treatment significantly inhibited both 

TNF-α and IL-6 secretion with all PM particles. 

L-NAME: L-NAME caused a decrease in NO 

secretion at 100 μg/mL of PM-ST. L-NAME did not 

have an effect on granite-induced NO secretion at 

100 μg/mL. 

Cytokine Gene Expression: TNF-α mRNA showed 

a trend to increase for -ST, but this did not reach 

significance. IL-6 gene expression increased for PMQ, PM-ST, PM-S but not for PM-Q. 

AA Release: PM-S exposure at 100 and 250 μg/mL 

was the only PM to induce AA release. 

Lipid Peroxidation: All particle types induced lipid 

peroxidation. PM-S and PM-ST induced significantly 

higher lipid peroxidation as compared to PM-Q and 

PM-G. 

ROS: All particle types induced ROS formation. PMS and PM-ST induced significantly higher formation 

at 10 μg/mL. PM-Q and PM-G induced small but 

significant decreases in absorption at 100μg/mL. 

Both PM-ST and PM-S had significant dose 

responses for all concentrations tested. No 

difference was observed between PM-G and PM-Q. 

PM-S and PM-ST pretreated with DFX had a lower 

ability to induce ROS formation. 

Endotoxin Content: Only PM-ST showed positive 

results for endotoxin content. 

December 2009 D-58 
Study Pollutant Exposure Effects 

Reference: Liu et al. 

(2005, 088304) 

Species: Human 

Cell Type: HPAECs 

SE: Wood Smoke Extract; generated 

using a stainless steel receptacle 

containing 100g of dry wood dust 

Particle Size: NA 

Route: Cell Culture 

Dose/Concentration: 40 μg/mL 

Time to Analysis: 0-4 h; Mitochondrial 

Membrane Destabilization: 0-60 min; DNA 

Defragmentation: 0-6 h; Cytotoxicity: 24 h 

Viability: SE exposure reduced cell viability dosedependently. Reduction reached ~38% of control. 

Effect on Oxidative Stress/ Antioxidant Enzymes:

SE caused an increase in ROS levels, in particular 

O2- and H2O2 in a time-dependent manner. 

Exposure to SE for up to 4 h caused a decrease in 

GSH levels in a time-dependent manner. Increased 

expression of Cu/Zn SOD mRNA and HO-1 mRNA 

was observed. Catalase or GPx mRNA expression 

was unaffected. Upregulation of Cu/Zn SOD and 

HO-1 occurred in a time-dependent manner 

Mitochondrial Translocation/ CapsaseIndependent Apoptosis/DNA fragmentation:

Exposure for up to 60 min caused an increase in the 

percentage of annexin V-FITC-pos cells but not PIpos cells. At 4 h, FDA-pos cells was unaffected. SE 

exposure caused a loss of mitochondrial membrane 

potential (indicated by the change in JC-1 

fluorescence). Cytosolic bax levels increased after 

exposure for 1 or 2 h and returned to basal level at 

4 h after exposure. Levels of procaspase-3 and 

caspase-9 were unaltered by SE exposure after 4 h. 

Procaspase-3 increased and caspase-9 decreased 

by H2O2 exposure. SE exposure increased levels of 

AIF and EndoG (exposure up to 4 h). At 6 h, 

increased DNA defragmentation was observed. Pretreatment with caspase inhibitors (CMK and Z-VADFMK) failed to suppress SE-induced apoptosis. 

NAC: Treatment with NAC prevented ROS increase 

in cells exposed to SE for 60 min. NAC addition 

prevented the reduction of GSH by SE. NAC 

decreased nuclear levels of AIF and EndoG and 

completely reduced DNA-fragmentation. NAC 

alleviated the SE-induced reduced viability. GSH 

and DNA fragmentation were unaffected by NAC. 

Reference: Long et al. 

(2005, 087454) 

Species: Human 

Cell Types: Human, 

Peripheral blood 

mononuclear cells 

(PBMCs) differentiated 

into MDMs (90-95 % 

CD14+) and T 

lymphocytes 

Synthetic C and C/Fe particles (phenol 

and paraformaldehyde polymers on a 

zeolite template) 

C/Fe analysis Al 1.38 %, Si 0.33 %, Fe 

0.46% 

Particle Size: 1 μm 

Route: Cell Culture (5×106 cells, 2 mL /well 

MDMs) Dose/Concentration: 5 μg/cm2

Time to Analysis: 2-24 h 

ROS release: Oxidative burst form C/Fe maxes out 

at 20 min with no effect from C particles. 

Cellular particulate actions: C particulates were 

present within lysosomes with small clumps forming 

after 24 h outside of lysosomes with no evidence of 

organelle lysis and/or agglomeration. C/Fe 

particulates showed similar initial effects progressing 

at 24-h total organelle lysis extending to the outer 

cell membrane. 

T cell effects: No effects from C or C/Fe particles 

Medium Effect: Particle agglomeration appears to be 

a direct result of serum present within a cell free 

medium 

Hydroxyl radical formation: C/Fe particles showed 

an order of magnitude of higher hydroxyl formation 

as compared to C particles 

December 2009 D-59 
Study Pollutant Exposure Effects 

Reference: Ma et al. 

(2004, 088417) 

Species: Mouse 

Cell Line: JB6P+ 

(Epidermal Cell Line) 

DEP: SRM 1975 

Particle Size: 0.5 μm 

Route: Cell Culture 

Dose/Concentration: Non-cytotoxic: 5, 10, 

20 μg/mL; Cytotoxic: 0, 10, 20, 40, 80, 100, 

160 μg/mL 

Time to Analysis: 24, 48 h; 

NF-κB and AP-1: 12 h 

Phosphorylation of Akt: 5- 120 min. 

Effect of LY294002 on DEP: Cells pretreated 

with LY294002 (0 or 10 μM) for 30 min and 

then exposed to DEP for 0-60 min. 

Viability: Below 20 μg/mL, DEP had no effect. At 

concentrations greater than 20 μg/mL, DEP caused 

apoptosis. 

NF-κB and AP-1: DEP stimulated NF-κB activity at 

5 and 10 μg/mL. At 20 μg/mL, NF-κB activity 

decreased, but was still greater than the control. 

DEP had no effect on AP-1 activity. 

PI3K/Akt Signaling Pathway: DEP induced 

phosphorylation of Akt on both Thr-308 and Ser-473. 

LY294002 (an inhibitor of P13K) blocked 

phosphorylation of Akt, p70/p85 s6 kinase and GSK 

3b. LY294002 eliminated DEP-mediated 

phosphorylation of Akt. Inhibition of P13K by 

expressing p85 also blocked DEP-induced Akt 

phosphorylation. DEP induced phosphorylation on 

GSH-3B on Ser-9 without affecting tyrosine 

phosphorylation and enhanced phosphorylation of 

p70/p85 S6 kinase on Thr-389. DEP had no effect 

on phosphorylation of FKHR. 

SAPK/JNK Pathway: DEP slightly activated the 

pathway. Increased transient activation of MKK4 (a 

signal component of the SAPK/JNK pathway) and 

thus enhanced phosphorylation of SAPK/JNK. DEP 

promoted phosphorylation of c-Jun and ATF-2. DEP 

did not affect p38 MAPK or ERK phosphorylation. 

LY294002: Treatment with LY294002 (P13K 

inhibitor) eliminated DEP-induced NF-κB activity. A 

similar effect was observed with the use of another 

P13K inhibitor, wortmannin. TDZD-8 (GSK-3B 

inhibitor), D-JNKI(a JNK inhibitor), SB202190 

(inhibitor for p38 MAPK) or PD98059 (inhibitor for 

MEK1) had little effect on DEP-mediated NF-κB 

activation. 

Reference:

Maciejczyk and Chen 

(2005, 087456) 

Species: Human 

Cell Type: BEAS-2B 

CAPs: PM2.5 

Collected via cyclone inlet on side of 

building in Tuxedo, NY. Weekdays 9-3 

March 4 to September 5, 2003 

Mass contributions of the Regional 

Sulfate, Soil, Oil- Combustions and 

Unknown/other categories to CAPs are: 

Regional Sulfate- 65%, Soil- 20%, 

Unknown/Other- 13% and Oil 

Combustion- 2%. 

Composition: 

* Regional Sulfate characterized by 

high concentrations of S, Si and . 

* Soil characterized by high 

concentrations of Ca, Fe, Al and Si. 

* Oil-Combustion characterized by high 

concentrations of V, Ni and Se. 

Particle Size: PM2.5

Route: Cell Exposure (subchronic 

exposures); Cell Culture (NF-κB) (9×104

cells/well) 

Dose/Concentration: CAPS 109 ± 178 

μg/m3

 (air exposure); 300 μg PM/ml (culture)

Time to Analysis: 24 h 

NF-κB: NF-κB response most notably correlated 

with V and Ni - elements associated with oil 

combustion source category (oil combustion makes 

up the group that is the smallest percentage of CAP 

mass). 

December 2009 D-60 
Study Pollutant Exposure Effects 

Reference: Madden et 

al. (2003, 198877) 

Species: Human 

Cell Type: NHBE 

DEP(SRM 2975) 

Diesel Exhaust Extracts from a High 

load (HL~75% engine load) or Low load 

(LL 0% engine load): 

Obtained from Caterpillar diesel engine, 

4 cycl, 4 stroke, model 3304 

Particle Characterization: LL extract 

has greater amount of low-molecularweight carbonyls (2-5 carbons). HL had 

more intermediate size carbonyls (6-9 

carbons). Largest carbonyls analyzed 

(11-12 carbons) found in similar ratios 

in the two types of extract (number of 

carbons is indicative of differences in 

boiling points). 

Particle Size: NR 

Route: Cell Culture 

Dose/Concentration: 0, 10, 50, 100, 250, 

500 μg/well 

Time to Analysis: 24 h (after 2 h of 

treatment, 0.5 ml of BEGM added to each 

well and cells incubated for an additional 22 

h) . 

Cytotoxicity: LL, HL and SRM had no effect on 

LDH release. 

51Cr: Incubation of cells with LL or SRM (10 to 500 

μg/well) had no effect. 500 μg/well of HL induced a 

significant increase in 51Cr release. 

IL-8: HL induced a 5-fold increase in IL-8 at 500 

μg/well. A decrease was observed at the highest 

dose of LL extract. SRM did not significantly alter IL8 production. 

PGE2: Production of PGE2 (inflammatory/immune 

mediator) increased in cells treated with HL extract 

at 500 μg/well. LL had no effect. Stimulation with 

melittin caused LL extract to have inhibitory effect on 

PGE2 at 500 μg/well. SRM had no effect. 

Reference: Matsuo et 

al. (2003, 198879) 

Species: Human 

Cell Type: NHBE, 

NHPAE, TIG-1, TIG-7 

(normal human lung 

embryonic fibroblasts) 

DEP: prepared at National Institute for 

Environmental Studies (Tsukuba, 

Japan) 

RDEP: residual DEP (after sequential 

extraction with hexane (NOS), 

benzene, dichloromethane, methanol, 

1N ammonium hydroxide) 

Particle Size: 0.4 μm (MMAD) 

Route: Cell Culture (NHBE: 5×104 cells/cm2; 

NHPAE: 3×103

 cells/cm2; TIG-1 and TIG -7: 

3×103

 cells/cm2; Apoptosis: 2×105 cells/cm2; 

ROS/NO: 2×104

 cells/cm2; Cytotoxicity 

Modulating Agent: 3×104

 cells/cm2; GSH: 

3×104 cells/cm2) 

Dose/Concentration: 25, 50, 100, 200, 300, 

400, 500 μg/mL 

Time to Analysis: 1 h 

Cytotoxicity in NHBE: Both DEP and RDEP 

exhibited dose-dependent cytotoxicity at 

concentrations beginning from 50 μg/mL and higher. 

RDEP was less cytotoxic than DEP. DEP exposure 

resulted in necrosis, not apoptosis. 

Comparative Cytotoxicity: The order of LC50 

values (50% lethal concentration) was: NHBE (118 

μg/ml), NHPAE (137 μg/ml), TIG (270 μg/ml). 

NHBE’s susceptibility was higher than the 

susceptibility of NHPAE and TIG cells. 

ROS/NO: DEP induced dose-dependent increases 

at 25 and 50 μg/mL. 

Reduced Glutathione: DEP induced dosedependent decreases. At 200 or 300 μg/mL, GSH 

levels decreased by 55.2 or 97.3%, respectively. 

Antioxidant Effects: Various antioxidants either 

decreased DEP cytotoxicity (PMC, Ebselen, EUK-8) 

or had no effect on DEP cytotoxicity (SOD, catalase, 

GSH, α-tocopherol) 

Chelating Agents: DEP became less cytotoxic 

when Ion-chelating agents were preincubated for 

24 h. No effect on DEP cytotoxicity was observed 

when chelating agents were administered to cells 

immediately after sonication. 

Endocytosis inhibitors: Decreased DEP toxicity 

was observed in a dose-dependent manner. 

Reference: Matsuzaki 

et al. (2006, 199517) 

Species: Human 

Cell Type: Peripheral 

neutrophils 

Gender: Male and 

Female 

Age: 20-40 yrs 

DEP: generated from a 4JB1-type, 4 

cyl Isuzu diesel engine 

me-DEP: methanol extract of DEP (40 

% of DEP by dry weight) 

Particle Size: 0.4 μm 

Route: Cell Suspensions (5×105 cells/mL) 

Dose/Concentration: all me-DEP 

f-actin: 1, 5, 10 μg/mL 

CD11b: 5, 10, 30 μg/mL 

IL-8: 5, 10, 30 μg/mL 

H2O2: 5, 10, 30, 60 μg/mL 

MMP-9, LTB-4: 5, 10, 30, 60 μg/mL 

Time to Analysis: f-Actin: 15 min 

CD11b: 2 h 

IL-8: 2 or 24 h 

H2O2: 30 min 

MMP-9, LTB-4: 2 or 24 h 

F-Actin: Treatment with me-DEP showed a dosedependent increase in the f-actin content of 

neutrophils and this increase was significantly higher 

at 5 and 10 μg/mL. 

CD-11b: Treatment increased CD-11b expression 

two-fold at 30 μg/mL. 

IL-8: Minimal response was observed after 2 h. A 

significant increase was observed (243%) at 24 h 

with 30 μg/mL. 

LTB-4: At 2 h, LTB4 increased to 115% and 119% 

with 30 and 60 μg/mL me-DEP respectively. At 24 h 

with 60 μg/mL me-DEP, LTB-4 increased to153%. 

H2O2: Exposure to 30 and 60 μg/mL of me-DEP 

induced large dose-dependent increases of 563% 

and 1220%, respectively. 

MMP-9: A significant increase at 2 and 24 h were 

observed. In both exposure periods, 30 μg/mL 

induced larger increases than 60 μg/mL. 

December 2009 D-61 
Study Pollutant Exposure Effects 

Reference: Molinelli et 

al. (2006, 198949) 

Species: Human 

Cell Type: NHBE, 

BEAS-2B 

PMH: PM10 extracts in hexane 

PMA = PM10 extracts in acetone of 

residue after hexane extraction 

-G: Guaynabo(Urban) and 

-F: Fajardo (Preservation Area) 

Particle Size: PM10

Route: Cell Culture (3×103 cells/well) 

Dose/Concentration: NHBE exposed to 0-

100 μg/mL of PM10

BEAS-2B exposed to 10,100, 250 μg/mL of 

PM10 

Time to Analysis: 48 h 

Metal analysis: Hexane extracts Cu, V, Ni all higher 

in winter than summer. For hexane extracts within 

the same season, metal concentrations were higher 

in the Fajardo extracts. On the other hand, the 

acetone extracts from Guaynabo generally had 

higher metal concentrations than Fajardo. 

Cytotoxicity NHBE: The order of most to least toxic 

for PM extracted with hexane is: winter-G, winter-F, 

summer -G , summer-F. The order of most to least 

toxic for PM extracted with acetone is: summer-G, 

summer-F, winter-g. 

Cytotoxicity BEAS-2: For PM extracted with 

hexane, the cytotoxicity order is: winter-G, winter-F, 

summer-G, summer-F. The order for acetone 

extracted PM is: summer-G , summer-F, winter-F, 

winter-G. Effects trend similar to metal levels (no 

analysis). Summer extracts showed linear doseresponse curves. Winter extracts exhibited more 

equivocal results, especially for Fajardo. Results 

suggest that NHBE cells are more sensitive than the 

BEAS-2B cells to PM extracts. 

Reference: Moller et 

al. (2002, 036589) 

Species: Canine, 

Mouse 

Cell Type: Beagle-Dog 

Alveolar Macrophages 

(BD-AM), J774A.1 

fTiO2 (origin NR) 

ufTiO2 (origin NR) 

ufP-G: carbon black (Printex-G, 

Degussa, Frankfurt, Germany) 

ufP90: carbon black (Printex90, 

Degussa, Frankfurt, Germany) 

ufEC90: EC (produced by electrical 

spark generator under standardized 

conditions with low impurities) 

DEP (SRM 1650) 

UrbD: Urban Dust (SRM 1649a) 

Particle Size: (in diameter) TiO2: 220 

nm; ufTiO2: 20 nm; ufP-G: 51 nm; 

ufP90: 12 nm; ufEC90: 90 nm; 

DEP: 120 nm; UrbD: NR 

Route: Cell Suspension 

Dose/Concentration: 10, 32, 100, 320 

μg/mL 

Time to Analysis: 24 h 

Cytoskeleton of J774A.1: At doses of 32 μg/mL or 

less, the particles did not significantly influence 

relaxation and stiffness. fTiO2and ufP90 had no 

effect at any dose. ufTiO2 at 320 μg/mL induced 

retarded relaxation and significant stiffening. ufEC90 

induced dose-dependent retardation of relaxation 

and increased stiffening. DEP and UrbD induced 

similar results. 

Cytoskeleton of BD-AM: ufTiO2 and fTiO2 both 

induced some retarded relaxation and increased 

stiffening at 100 μg/mL dose. ufTiO2 appears to 

increase stiffening in a dose-dependent manner. 

ufEC90 induced dose-dependent acceleration of 

relaxation due to the carbon content of ufEC90. DEP 

also induced acceleration of relaxation as well as a 

decrease in stiffness. 

Phagocytosis: At 24 h, ufTiO2 and fTiO2

significantly reduced phagocytic ability in J774A.1 

but not in BD-AM. All carbonaceous particles 

induced significant impairment in J774A.1. All 

ultrafine carbon particles inhibited BD-AMs. 

Cell Proliferation: ufTiO2 significantly inhibited 

proliferation compared to the control and fTiO2 at 

100 μg/mL in J774A.1. ufEC90 and ufP90 inhibited 

proliferation slightly with ufEC90 inducing slightly 

greater inhibition than ufP90. UrbD and DEP also 

significantly reduced proliferating. 

Apoptosis: All particles induced decreased viability 

at 100 μg/mL in both cell types. With ufTiO2 inducing 

greater apoptosis than fTiO2, ufEC90 than ufP90 

and ufEC90 than ufP-G. 

Reference: Mutlu et 

al. (2006, 155994) 

Species: Human, Rat 

Cell Type: A549 

PM10

(Collected by baghouse from ambient 

air in Dusseldorf, Germany) 

Particle Size: PM10

Route: Cell Culture 

Dose/Concentration: 0.05, 0.5, 5. 50 

μg/cm2

 

Time to Analysis: 24 h 

Na, K-ATPase Plasma Membrane Protein: PM10

induced a decrease of protein in the plasma 

membrane of A549 cells. Total Na,K-ATPase levels 

were unaffected. 

ROS: Pretreatment with EUK-134, superoxide 

dismutase and catalase mimetic, inhibited the 

decrease of GSH. Furthermore, it attenuated the 

decrease of NA,K-ATPase in A549 cells. 

NA, K-ATPase Activity: PM10 induced a dosedependent decrease in ouabain-sensitive liberation 

of 32P from AT32P in primary rat alveolar type II 

cells. This effect was inhibited with pretreatment with 

EUK-134. 

December 2009 D-62 
Study Pollutant Exposure Effects 

Reference: Nam et al. 

(2004, 198887) 

Species: Human 

Cell Type: A549 

PM2.5 

Collected from hospital rooftop, Seoul, 

South Korea 

Particle Size: PM2.5 

Route: Cell Culture 

Dose/Concentration: 0.5, 1, 10, 25, 50 

μg/cm2 

Time to Analysis: 6, 24 h 

NF-κB/IκBα: 50 μg/cm2 DEP induced IκBα

degradation which peaked at 2 h and recovered 

after 4 h. Treatment with increasing amount of PM2.5

resulted in a dose-dependent decrease in IκBα. 

PM2.5 increased NF-κB in a dose-dependent manner 

up to 10 μg/cm2. NF-κB induction peaked at 12 h. 

IL-8: PM2.5 treatment increased protein level more 

than 3 fold with 100 μg/cm2

 PM2.5. mRNA levels also 

increased. 

iNOS Inhibitor: PM2.5 induced IL-8 elevation was 

completely blocked by iNOS inhibitor. iNOS inhibitor 

also negated PM2.5 induction of NF-κB activity. 

Antioxidants and iNOS inhibitor reduced PM-induced 

IκBα degradation. 

Reference: Nozaki et 

al. (2007, 097862) 

Species: Mouse 

Cell Line: LA-4 

(Alveolar Epithelial 

Cells) 

PM: Rooftop of 5 story building, urban, 

Japan 

PME: dichloromethane extract of PM 

filtered 

P90: Printex 90 (carbon black) 

(Degussa) 

Particle Size: PM: 0.22 μm; PME: 2.5 

μm; P90: 14 nm 

Route: Cell Culture (1.4×104 cells/cm2) 

Dose/Concentration: 1.1 μg/cm2

Time to Analysis: 24, 28, 72 h 

Cytotoxicity: P90 had no effect. PM and PME were 

cytotoxic at similar levels. 

Protein Expression: All particles affected protein 

expression (no specific protein- 2D gel 

electrophoresis). 

Reference: Obot et al. 

(2002, 042370) 

Species: Mouse 

Cell Line: BALB/c 

Cell Type: AM 

PM: SRM 1648 

PM-100: PM heated to 100° C 

PM-500: PM heated to 500° C 

PM-PH: PM acid digestion 

PMAC: Acetone extraction 

PMCH: Cyclohexane extraction 

PMH2O: Water extraction 

All extract fraction used as residual 

particles 

Particle Size: NR 

Route: Cell Culture (5×105 cells/mL)

Dose/Concentration: PM: 200 μg/mL; PM100: 188 μg/mL; PM-500: 130 mg/l; PMPH: 94 μg/mL; PMAC: 173 μg/mL; 

PMCH: 171 μg/mL; PMH2O: 188 μg/mL 

Fraction doses adjusted for mass loss during 

fraction treatment 

Time to Analysis: 4 h 

Cytotoxicity: All 7 fractions had cytotoxic effects. 

PM had highest cytotoxicity. PM-500, PM-PH, PMAC 

less toxic than PM. 

Apoptosis: All 7 fractions significantly increased 

apoptosis. The PM fractions that induced the 

greatest apoptosis in descending order are: PM, 

PMH2O, PM-100, PM-500, PMAC, PMCH and PMPH. PM-induced apoptosis (only PM, PM-500 and 

PMAC tested) was blocked by poly I or 2F8 antibody 

(scavenger receptors). 

Particle Characterization: Untreated PM and PM100 did not have measurable amounts of transition 

metals on its surface. Measured components include 

carbon, O2, N, S, Si, Ca, Al, P, Cl. PM-PH mostly 

contained O2 and Si. PM-500 had increased O2, Si 

compared to PM and measurable amounts of Na, K., 

Zn, Co, Pb, Fe. Included increased surface density 

of S, P, Al. PMCH lacked nonpolar organic 

compounds. 

Reference: Obot et al. 

(2004, 095938) 

Species: Mouse (7-

9wk), Human 

Cell Line: MouseBALB/c 

Cell Type: AM 

PM: SRM 1648 (collected by baghouse in St. Louis, MO). 

PM-100: PM heated to 100 ̊ C 

PM-500: PM heated to 500 ̊ C 

PM-PH: PM acid digestion 

PMAC: Acetone extraction 

PMCH: Cyclohexane extraction 

PMH2O: Water extraction 

All of the 6 extract fractions from 

PM1648 

PM2.5: Collected in Houston, TX 

Particle Size: PM1648: NR; PM2.5 

Route: Cell Culture (5×105 cells/mL)

Dose/Concentration: PM: 200 μg/mL; PM100: 188 μg/mL; PM-500: 130 mg/l; PMPH: 94 μg/mL; PMAC: 173 μg/mL; 

PMCH: 171 μg/mL; PMH2O: 188 μg/mL 

Fraction doses adjusted for mass loss during 

fraction treatment 

PM2.5 = 50, 100, 150, 200 μg/mL 

Time to Analysis: Mouse-4 h; Human-24 h. 

Human AM Viability: Only PM, PM-100, PMAC and 

PMH2O decreased viability. 

Human AM Apoptosis: PM, PM-100 and PMH2O 

increased apoptosis. PM induced greater apoptosis 

than PM-100 and PMH2O. 

Regression Analysis Mouse vs Human: Although 

individual fractions differed somewhat, cell viability 

and apoptosis of all 7 fractions showed linear 

regression 

Human and Mouse AM Viability with PM2.5:

Nearly identical dose-dependent decrease was 

exhibited starting at 50 μg/mL 

Human and Mouse AM Apoptosis with PM2.5:

Nearly identical dose-dependent increases were 

exhibited with human AM responses peaking at 150 

μg /mL and declining at 200 μg/mL (no mouse data 

for 200 μg/mL). 

Regression Analysis with PM2.5: Excellent 

correlation of mouse and human responses for 

viability and apoptosis was exhibited. 

December 2009 D-63 
Study Pollutant Exposure Effects 

Reference: Okeson et 

al. (2003, 042292) 

Species: Rat 

Cell Type: RLE-6TN 

(Type II Alveolar 

Epithelial Cells) 

CG: Coal ash, Germany 

CU: Coal ash, USA 

5C: PM # 5 Oil fly ash coarse 

5F: PM #5 Oil fly ash fine 

6MSC: PM #6 Oil med sulfur fly ash 

coarse 

6HSC: PM # 6 Oil high sulfur fly ash 

coarse 

6HSF: PM # 6 Oil high sulfur fly ash 

fine 

Particle Size: CG, CU: NR; 5C, 6MSC, 

6HSC: >2.5 μm; 5F, 6HSF: <2.5 μm 

Route: Cell Culture 

Dose/Concentration: Coal Fly Ash 12.5, 

25, 50, 125, 250 μg/mL 

Oil Fly Ash - 100 μg/mL 

Time to Analysis: 24 h 

Oil PM Characterization: Generally, the fine 

fractions had higher metal levels than the coarse 

fractions except for Zn. High sulfur had a higher 

metal content than med sulfur. Carbon percent 

weight was stable across all 5 fractions. 

Coal Ash Cytotoxicity: CG treatment exhibited 

similar cytotoxic results as CU. Cytotoxic effects 

were exhibited at concentrations of 12.5 μg/mL and 

above. Effects remained steady at concentrations 

above 50 μg/mL. 

Oil Ash Cytotoxicity: Cytotoxic effects were 

induced by all. The order of PM fractions inducing 

the most cytotoxicity to the least is the following: 5F, 

6HSF, 6HSC, 5C, 6MSC. 

Correlation of Metal Content and Cytotoxicity:

Fe, V showed a reasonable correlation. Zn had no 

correlation. 

Cell Metabolism: An inhibitory effect was observed 

with 100 μg/mL coal ash after 6 h. After 12 h of 

exposure, CU, unlike CG, does not continue to 

inhibit cell metabolism. Oil ash was generally less 

effective than coal ash. The order of PM fractions 

inhibiting metabolism the most to the least is the 

following: 5F, 6HSC, 5C, 6MSC. 6HSF not tested. 

Reference: Okeson et 

al. (2004, 087961) 

Species: Rat 

Cell Type: RLE-6TN 

(Type II Alveolar 

Epithelial Cells) 

Zn, V, Fe chloride as salts (valence 

state not reported) 

Particle Size: NR 

Route: Cell Culture (50000 cells/well) 

Dose/Concentration: 0.001, 0.01, 0.1, 1.0, 

10 mM 

Time to Analysis: 24 h 

Cytotoxicity: All metals cytotoxic at concentrations 

greater than 0.1 mM. V is 5 times less cytotoxic than 

Zn, and Fe is 7 times less cytotoxic than Zn with a 

EC50 of 3mM and 4mM, respectively. At 10 mM of 

each metal, no surviving cells were present. 

NCS: Incubation with NCS (5 or 10 %) decreased 

toxicity of Zn, especially at 0.1 mM, but had no effect 

on Fe or V toxicity. 

Albumin: BSA decreased Zn toxicity at equivalent 

concentrations but to a lesser extent than NCS. 

Reference: OsornioVargas et al. (2003, 

052417) 

Species: Mouse 

Cell Line/Type: 

J774A.1, L929 

(Mesenchymal Cells) 

PM10 

PM2.5 

-N = Northern (industrial) 

-SE = Southeastern (lake basin dust) 

sites, both heavy vehicular traffic, 

Mexico City, Mexico 

Particle Size: PM10; PM2.5 

Route: Cell Culture (J774A.1: 15000 

cells/cm2; L929: 30,000 cells/well) 

Dose/Concentration: 20, 40, 80 μg/cm2

Time to Analysis: 24-72 h 

PM Characterization: Elements similar in particle 

types with elements in PM10 more abundant. 

Northern particles contained more Co, Zn, Ni, Pb. 

Endotoxin: All PM samples had detectable amounts 

of endotoxin. PM2.5 -N had 22 EU/mg. PM10-N had 

30 EU/mg. PM2.5 -SE had 12 EU/mg. PM10-SE had 

59 EU/mg. 

Cytotoxicity (J774A.1): The two northern samples, 

PM2.5 and PM10, both induced similar cytotoxic 

effects at 40% survival. PM10-SE and PM2.5 -SE 

induced dose-dependent responses. In general, the 

northern samples had a higher cytotoxic effect than 

the southern samples. 

Apoptosis (J774A.1): Northern samples induced 

more apoptosis than did the southeastern samples. 

There was no difference between PM10 and PM2.5

induced apoptosis. 

TNF-α and IL-6 (J774A.1): TNF-α and IL-6 induced 

dose-dependent increases. At 80 μg/cm2

, PM10 -SE 

induced the most production of IL-6 followed by 

PM2.5 -SE, PM10-N , and PM2.5 -N. 

J774A.1 Supernatant Toxicity (L929): Conditioned 

medium from J774A.1 pre-exposed to each PM type 

reduced cell viability in L929 cells. This was 

correlated with TNF-α level in supernatants. 

December 2009 D-64 
Study Pollutant Exposure Effects 

Reference: Penn et al. 

(2005, 088257) 

Species: Human 

Cell Type: BEAS-2B 

BDS: Butadiene soot (created on-site 

by passing BD through a back-flash 

protected stainless steel two-stage 

regulator to a stainless steel Bunsen 

burner) 

-P1: <2.5 μm 

-P2: 2.5-10 μm 

-P3: >10 μm 

BDS-W: solvent washed 

Graphite 

Composition: <2.5 μm = 92%, 2.5-10 

μm = 5%, >10 μm = 3% 

Particle Size: BDS-P1: <2.5 μm; BDSP2: 2.5-10 μm; BDS-P3: >10 μm 

Route: Cell Culture (1-1.5×106 cells) 

Dose/Concentration: 3 mg BDS 

Time to Analysis: 5 min-72 h 

Particle Characterization: By weight, EC makes up 

94% of BDS, hydrogen 2%, nitrogen and sulfur 1%, 

and oxygen less than 0.1%. 

PAH Components of BDS: 13 prominent PAHs:

acenaphthylene, fluorene, anthracene, 

cyclopentaphenanthrene, fluoranthene, 

acephenanthrylene, pyrene, benzofluorenes, 

acepyrene, chrysene, benzopyrenes, perylene, 

benzoperylene. 

BDS Activity: At 60-120 min, BDS was observed in 

the cells. At 4 h, fluorescence observed in 

cytoplasmic vesicles and increased during the first 

24 h then plateaued for the next 72 h. BDS-W 

appeared in vesicles sooner than BDS. 

Reference: Pozzi et 

al. (2005, 088610)

Species: Mouse 

Cell Type: RAW 264.7 

PM: Collected continuously for 15 days, 

8-10 m from street, Sept 1999, Rome, 

Italy 

-F = Fine particulate 

-C = Coarse particulate 

CB (Degussa Huber NG90) 

Particle Size: PM-F: 0.4-2.5 μm; PMC: 2.5-10 μm; CB: 200-250 nm 

Route: Cell Culture (1.3×105 cells/well) 

Dose/Concentration: 30 μg/mL; 14 μg/cm2

120 μg/mL; 54 μg/cm2

Time to Analysis: 5, 24 h 

Cytotoxicity: For 24 h, lower levels of PM-F, PM-C, 

and CB had no effect on cell viability. Higher levels 

of PM-C and CB induced a significant release of 

LDH. 

Arachidonic Acid (AA): Both fractions of PM 

increased AA release in a dose-dependent manner 

at 5 h. CB increased a release only at the higher 

concentrations although, in terms of magnitude, the 

CB-induced release was much less than the ambient 

PM-induced release. Pretreatment with 

deferoxamine was not effective in decreasing AA 

release. 

TNF-α: TNF-α levels increased significantly for both 

concentrations and time periods for PM. PM-C at 

24 h was significantly lower than at 5 h for both 

concentrations. PM-C at 30 μg/mL induced a much 

greater TNF-α release than PM-F at 5 h. 

IL-6: PM-F significantly increased at 5 h for both 

concentrations. Elevated IL-6 levels were exhibited 

at both PM-C doses at 24 h. At 5 h, only the high 

dose elevated IL-6 levels. CB was devoid of an 

effect on IL-6. LPS-induced IL-6 response was 

similar to coarse PM at the high dose, with the 

response being greater at 24 h than at 5 h. 

Reference: Prophete 

et al. (2006, 156888) 

Species: Rat 

Cell Type: NR8383

AMs 

Ambient PM2.5 

NYC: 1st and 26 St, NYC 

LA: San Gabriel foothills, Claremont, 

CA 

SEA: 15th Ave S and S. Charleston, 

Seattle, WA 

V, Mn, Al, Fe levels in PM 

added metals to cells 

V: Na3VO4

Al: AlCl3•6H2O 

Mn: MnCl2•4 h20 

Fe: FeCl3•6H20 

Particle Size: PM2.5 

Route: Cell Culture (2×105 cells/mL) 

Dose/Concentration: Fe(III) 16 μmol 

V, Mn, and Fe(III) mixtures with V or Mn in 

molar ratios 0.02, 0.08, 0.2 and 0.4 × Fe(III) 

Al and Fe(III) mixtures with Al in molar ratios 

0.37, 0.75, 2, 7.5 × Fe(III) 

Time to Analysis: 20 h 

Particle Characterization: Fe and metal to F ratios 

based on ratios observed in PM2.5 from LA, SEA and 

NYC sites. V: Fe ratios remarkably similar among 

sites. Fe levels fixed at NYC level of 16 μm 

(highest). 

IRP: Coexposure with 3 metals increased IRP 

binding activity relative to Fe(III) alone, by up to 3.5 

fold for Al (1.5-3 ratio), 2 fold for Mn (0.08-0.2 ratio) 

and 7 fold for V (0.2 ratio). IRP activity dropped at 

higher ratios. A drop in IRP activity at higher ratios 

may be result of cytotoxicity for Al, but not for V and 

Mn. 

iNOS: Al induced iNOS expression dosedependently. There was no observed effect for Mn 

and V. 

Induction of Hypoxia-inducible Factor (HIF-1α):

Only V and Al induced HIF-1α. 

Activation of ERK1 and -2: V and Al induced 

pERK1, but only V induced pERK2. Mn had no 

increasing effects, but data indicated a decreasing 

induction. 

December 2009 D-65 
Study Pollutant Exposure Effects 

Reference: Ramage 

and Guy (2004, 

055640) 

Species: Human 

Cell Type: A549 

PM10: Collected in Wolverhampton, UK 

ufCB: Ultrafine Carbon Particles 

(Origin not reported) 

Particle Size: PM10, ufCB: <100 nm 

(diameter) 

Route: Cell Culture 

Dose/Concentration: 80 μg/mL 

Time to Analysis: 0, 0.5, 3, 6, 18 h 

CRP: Treatment with ufCB or PM10 produced an 

increase in CRP expression with similar effects 

noted after 6 h. PM10 induced greater increases than 

ufCB. Both the cytoplasm and nucleus contained 

CRP. 

Hsp70: PM10 and ufCB induced increased levels at 

all time points with ufCB inducing greater levels than 

PM10. Hsp70 expression was observed in the 

cytoplasm and nucleus. 

Antioxidants of CRP and Hsp70: Coincubation of 

ufCB with Nacystelin and Trolox caused a small 

reduction in CRP and Hsp 70. 

Reference: Rao et al. 

(2005, 095756) 

Species: Rat 

Strain: SD 

Cell Type: AMs and 

cultured lung 

fibroblasts 

DEP: SRM 2975 (NIST) 

Particle Size: 0.5 μm 

Route: Cell Culture 

Dose/Concentration: 200 μg/mL

Time to Analysis: 4 h 

mRNA Expression: No change in IL-1β or iNOS 

were observed. Data suggests that the lung 

fibroblasts is the main source of IL-6 and MCP-1 in 

BAL fluid because of their comparatively high 

message levels. Due to the extreme variability in 

results, the cause of an increase on co-culture with 

AMs and/or DEPs was not assessed. 

Reference: Reibman 

et al. (2003, 156905) 

Species: Human 

Cell Type: HBEC, 

BEAS-2B 

UFPM: Ultrafine PM 

FPM: Fine PM 

IPM: Intermediate PM 

CPM: Coarse PM 

CB: Carbon black 

All PM collected 8th floor, 26th St and 

1st Ave, New York City, NY 

Particle Size: UFPM: <0.18 μm; FPM: 

0.18 - 1.0 μm; IPM: 1.0 - 3.2 μm; CPM: 

>3.2 μm 

Route: Cell Culture 

Dose/Concentration: 11 μg/cm2; 100 μg/mL

Time to Analysis: 6, 18 h 

Cytotoxicity: After treatment, cells were more than 

90% viable. UFPM and FPM caused no gross 

alterations in cell morphology or adhesion. 

MIP3α/CCL20 mRNA (6 h): Stimulation of mRNA 

released by HBEC upon exposure to UFPM 

appeared similar to that provided by TNF-α (5 

μg/mL) and IL-1β (10 mg/mL). 

MIP3β/CCL20 protein in HBEC (18 h): TNF-α and 

IL-1β induced a dose-dependent increase in 

MIP3α/CCL20 protein (0-10 ng/mL), whereas Il-4 

and IL-13 induced MIP3α/CCL20 protein release 

that reached maximum levels at 1 ng/mL. No 

release of MIP1α/CCL3 nor RANTES/CCL-5 was 

observed upon stimulation with cytokines. 

Secretion of MIP3α/CCL20 in response to PM (18 

h): All PM fractions less than 2.5 μm resulted in the 

release of MIP3α/CCL20 protein in HBEC roughly 

equivalent amounts. CB similar in size to UF/fine PM 

did not result in the release of MIP3α/CCL20, nor did 

LPS (0.01-1.0 μg/mL). No release of MIP1α/CCL3 

nor RANTES/CCL 5 was observed upon stimulation 

by PM fractions. 

Activation of MAPK (ERK1/2 and p38): ERK1/2 

and p38 was activated by TNF-α, IL-1β, IL-4 and IL13 within 15 min and was sustained for at least 60 

min. Erk1/2 and p38 inhibitors reduced 

MIP3α/CCL20 release in BEAS-2B cells in response 

to cytokines. 

December 2009 D-66 
Study Pollutant Exposure Effects 

Reference: Riley et al. 

(2003, 053237) 

Species: Rat 

Cell Type: RLE-6TN 

(Type II Alveolar 

Epithelial Cells) 

Zn: ZnCl2

Cu: CuCl2

Fe: FeCl2

V: VCl4

Ni: NiCl2

Particle Size: NR 

Route: Cell Culture 

Dose/Concentration: 0.01, 0.1, 1.0, 10 mM 

Time to Analysis: 2, 4, 24, 72 h 

Cytotoxicity (SDH): All particles were cytotoxic in a 

dose-dependent manner. Zn and V were cytotoxic at 

0.05 mM, Cu at 0.5 mM, Ni at 0.8 mM and Fe at 2 

mM. For Zn, cell death (LDH) had a biphasic 

response: a slow logslope until approx 0.1 mM at 

which point it rapidly accelerated to a peak at 5 mM 

with a small decline at 10 mM. Most of Zn 

cytotoxicity was not due to apoptosis. LPS did not 

affect either Zn or Cu cytotoxicity. 

Metabolism Inhibition Time Course Response 

(Cu and Zn only): At high (1 mM) concentrations, 

Zn toxicity peaked at 36-48 h followed by a 2-fold 

recovery by 72 h. Cu showed a faster, steady 

decline plateauing after 36 h. At low concentrations 

(0.1 mM), Cu showed a steady slow decline. At 48 h, 

Zn decreased faster to max activity and returned to 

control by 72 h. 

IL-6 Secretion: Zn and Cu both decreased IL-6 

secretion. Decreases were very similar for both 

metals and concentrations when expressed as 

secretion per viable cell ratio except for Zn at 1.0 

mM. 

Metal Combinations: Zn and Cu gave variable 

results. Zn protected against V cytotoxicity. Zn and 

Cu had an additive response. Zn did not affect Fe 

toxicity. 

Reference: Riley et al. 

(2005, 096452) 

Species: Rat, Human 

Cell Type: RLE-6TN, 

NR8383 Alveolar 

Macrophages, A549 

Fe: FeCl2

Ni: NiCl2

Cu: CuCl2

V: VCl2 

Particle Size: NR 

Route: Cell Culture (5×104 cells/well 

Alveolar Cells; 1.2×105 cells/well NR8383) 

Dose/Concentration: AMs: 0.02, 0.05, 

0.07, 0.08 mM; RLE-6TN: 0.1, 0.2, 0.6, 1.0, 

6.0 mM; A549: 0.5, 0.8, 4.4, 4.8 mM 

Time to Analysis: 2-48 h 

Relative Sensitivity of Cell Strains to Metal 

Chloride: NR8383 was more sensitive than RLE6TN and A549 except for V where 

NR8383 and RLE-6TN were both more sensitive 

than A549. 

Relative sensitivity of Cell Strains to Metal 

Chloride vs Sulfate: With the exception of Cr, 

sulfate was generally more cytotoxic than chloride 

(note V valence state). 

A549 Cytotoxicity Time Course: Zn cytotoxicity 

takes 24 h to develop whereas Cu cytotoxicity 

develops within 2 h. LDH release for Cu, however, 

develops in 24 h. 

RLE Cytotoxicity Time Course: Zn starts at 2 h 

and develops until 24 h. Cu develops within 2 h and 

continues until 24 h where it is less toxic than Zn. 

Both release equivalent amounts of LDH after 24 h. 

NR8383 Cytotoxicity Time Course: Both Zn and 

Cu exhibit time dependent toxicity beginning as early 

as 4 h. LDH release maximizes at 12 h and either 

remains steady or declines. 

Reference: Ritz et al. 

(2007, 198901) 

Species: Human 

Cell Type: BEAS-2B, 

NHBEC 

DX: Extract of DEP (generated from a 

light duty four-cylinder diesel engine 

4JB1 type Isuzu Automobile) 

Particle Size: <1 μm (diameter) 

Route: Cell Culture 

Dose/Concentration: 0, 20, 50, 100 μg/mL 

Time to Analysis: 24 h 

NQO1 (Sentinel Phase II Enzyme): Cells 

transfected with NQO1 reduced induction of IL-8 by 

DX exposure. 

Sulfurophane: Increased gene expression of phase 

II enzymes, particularly NQO1, was observed in both 

cell types. Gene expression in BEAS-2B was greater 

than that of NHBEC. 

Sulfurophane did not upregulate GSTM1 in BEAS2B but induced a 2-fold increase in NHBEC. 

Pretreatment also inhibited DX-induction of IL-8 in 

both cell types. 

Cytokines: DX induced significant increase of IL-8 

in both cell types at concentrations of 10 μg/mL or 

higher. GM-CSF and IL-8 remained unaffected in 

BEAS-2B. GM-CSF and IL-8 Increased in NHBECs 

and reached statistical significance at 25 μg/mL. 

December 2009 D-67 
Study Pollutant Exposure Effects 

Reference: Rosas 

Perez et al. (2007, 

097967) 

Species: Mouse 

Cell Type: J774A.1 

PM10 

Collected in Mexico City, Mexico from 

January-June, 2002 

North: Iztacala, manufacturing industry; 

Center: Merced, heavy traffic; 

South: Ciudad Universitaria, residential 

Principal Component Analysis of Air 

Pollution Data: 

Group 1:S/K/Ca/Ti/Mn/Fe/Zn/Pb (43% 

of variance); 

Group 2: Cl/Cr/Ni/Cu (16%); 

Group 3: Endotoxins/OC/EC (14%). 

For all 3 sites: Averages of Group 1 is 

statistically different among the center, 

north and south sites with the central 

site producing the highest values. 

Group 2 is similar among the sites and, 

for Group 3, the north had a lower 

average than the center and south 

sites. 

Particle Size: PM10 

Route: Cell Culture (1.5×104 cells/cm2) 

Dose/Concentration: 20, 40 or 80 μg/cm2 

Time to Analysis: 72 h 

Cytotoxicity: Responses were dose-dependent; 

there was no observed site interaction. Cytotoxicity 

seems to be a result of the following components: 

S/K/Ca/Ti/Mn/Fe/Zn/Pb. 

IL-6: Only the center site at 40 μg/cm2 induced an 

increase. Induction of higher IL-6 levels seems to be 

related to high values of S/K/Ca/Ti/Mn/Fe/Zn/Pb and 

endotoxins/OC/EC. 

TNF-α: Production was induced by all samples in a 

dose-dependent manner. Similar to IL-6, induction of 

higher TNF-α levels seems to be a result of high 

values of S/K/Ca/Ti/Mn/Fe/Zn/Pb and 

endotoxins/OC/EC. 

p53: Only south PM had effect. Induction of p54 

seems to depend on high levels of Cl/Cr/Ni/Cu and 

low levels of S/K/Ca/Ti/Mn/Fe/Zn/Pb. 

Reference: Sakamoto 

et al. (2007, 096282) 

Species: Human 

Age: 58-82 yr 

(Smokers) 

Cell Type: HBEC 

PM10: EHC-93 (Obtained from Health 

Canada, Canata) 

Particle Size: PM10

Route: Cell Culture 

Dose/Concentration: 100, 300 and 500 

μg/mL 

Time to Analysis: Calcium responses: up to 

60 min; cytokines: 6 or 24 h 

Intracellular [Ca2+]: [Ca] concentration slowly 

increased, elevating after 10 and 30 min for 500 and 

300 mg/mL, respectively. The response plateaued at 

35 min for 500 μg/mL. 

Extracellular [Ca2+]: Starting at 20 min, the 

removal of extracellular Ca decreased the PM10

response significantly. Calcium channel blocker 

(10μM or 1mM) LaCl3 and (5mM) NiCl2 significantly 

blocked the PM-induced intracellular Ca. Lacl2 

administration (1mM) inhibited the PM-induced 

Ca2+ response in a dose-dependent manner. 

Mode of Action: Intracellular Ca induced by ATP 

declined more slowly in the cells exposed by PM10. 

This indicates that PM10 blocks Ca clearance via the 

calcium pumps. 

Cytokines: PM10 induced a dose-dependent 

increase in cytokine mRNA levels and cytokines IL1β, LIF, IL-8 and GM-CSF. Cytokine expression was 

unaffected by the reduction of extracellular Ca2+ . 

Preincubation with the calcium chelator reduced 

responses for IL-1β and IL-8 but not LIF or GM-CSF.

Reference: Salnikow 

et al. (2004, 087469) 

Species: Human 

Cell Line: 1 hAEoFeSO4

FeCl3

NiSO4

Particle Size: NR 

Route: Cell Culture 

Dose/Concentration: 0.25 and 0.5 mM 

Fe exposures also contained 60 μg/mL 

apotransferrin 

Time to Analysis: 24 h 

Cytotoxicity: Both Fe had no effect. NiSO4 caused 

marginal cytotoxicity (75%). 

Hypoxic Stress: At 20 h, NiSO4 (at concentrations 

of 0.25 or 0.5 mM) induced NDRG-1/Cap43 protein 

production indicating hypoxic stress. DFX and 

DMOG induced a similar effect. 

IL-8: NiSO4 induced IL-8 time-dependently for up to 

48 h. At 48 h, the increase was 6+ fold. 

Coexposure (Ni + Fe) on Fe uptake: Fe(III) uptake 

was greater than Fe(II) uptake. NiSO4 had no effect. 

Ni uptake was greater than Fe uptake but was 

decreased by coexposure to Fe. Coexposure also 

did not effect hypoxic stress. Coexposure with Fe 

did reduce Ni-induced IL-8 production. 

December 2009 D-68 
Study Pollutant Exposure Effects 

Reference: Salonen et 

al. (2004, 187053) 

Species: Mouse

Cell Type: RAW 264.7 

PM10 (urban traffic) Finland 

Pooled as winter (W), spring I (SI), or 

spring II (SII) based on component/time 

considerations 

Particle Size: PM10: 0.12-10 μm 

Route: (2×106 cells/well) 

Dose/Concentration: 15, 50, 150, 500, 

1000 μg/mL 

Time to Analysis: 0, 24 h 

Air quality parameters: Winter and spring I did not 

differ. SII much lower PM2.5 

Metal data equivocal as well as highly variable 

resuspension rates. 

Total PAHs: W=303; SI=233; SII=204 ng/mg 

Inflammation (IL-6, TNF-α, NO)/Cytotoxicity: A 

dose-dependent increase was observed for TNF-α, 

IL-6 and NO except for SI. The IL-6 levels, of those 

particles exposed to SI, decreased at 1000 μg/mL. 

TNF-α, IL-6: SI = SII>>W>control. 

NO production: W≥SI≥SII 

Cell Viability: W=SI=SII toxic at 500 and 1000 

μg/mL 

Water-soluble vs Insoluble: TNF-α and IL-6 were 

nearly entirely the result of insoluble components of 

PM10. Cytotoxicity was driven by both soluble and 

insoluble components. 

Metal Chelation: The addition of metal chelators did 

not modify IL-6, TNF-α or cytotoxicity 

LPS inhibitor: Treatment with the LPS inhibitor 

eliminated the IL-6 response and, perhaps, slightly 

reduced the TNF-α response but not cytotoxicity 

Hydroxyl radicals: A dose-dependent induction of 

hydroxyl radicals and induction of hydroxyl radical 

lesions (at 500 and 1000 μg/m3) in the calf thymus 

DNA were observed. 

Reference: Samet et 

al. (2003, 113782) 

Species: Human 

Cell Type: A431 

(Epidermoid Cells) 

As: NaASO3

V: VOSO4

Zn: ZnSO4 

Particle Size: NR 

Route: Cell Culture 

Dose/Concentration: 500μM 

Time to Analysis: 20, 30 or 90 min 

EGFR Dimerization: Zn, V or As did not induce 

EGFR dimerization in a cell free system i.e., no 

direct crosslinking. Zn did not induce dimerization in 

whole cells either. 

Phosphorylation of EGFR: Zn induced 

phosphorylation at 3 sites similar to EGF. As and V 

had no effect. 

EGFR Kinase Inhibitor: While EGF action was 

blocked, Zn continued to induce phosphorylation 

and was independent of EGFR kinase activity. 

c-Src: Blocking of c-Src tyrosine kinase 

(transactivator of phosphorylation) negated all Zninduced phosphorylation but only had a slight effect 

on EGF stimulated cells. 

ERK1/2 Phosphorylation: Zn increased levels of 

ERK1/2. Pretreatment with EFGR kinase inhibitor 

reduced both Zn and EGF effect. This effect was not 

blocked by the c-Src blocker. 

Reference: Santini et 

al. (2004, 087879) 

Species: Mouse 

Cell Type: RAW 264.7 

DEP: Collected adjacent to moderate 

traffic in Rome, Italy 

Particle Size: PM2.5 

Route: Cell Culture (2.5×105 cells/mL) 

Dose/Concentration: 0.01, 0.1, 1.0 μg/mL 

Time to Analysis: 24 h 

500 MHz Results (no 1 μg/mL): DEP induced a 

dose-dependent increase in choline compounds, αand βgamma- glutamine/glutamate (0.01 >0.1 

μg/mL), lactate, and CH2, CH3 moieties of fatty 

acids. DEP decreased inositol and 

phosphoreatinine. 

700 MHz Results (no 1 μg/mL): DEP induced 

similar results, except α-, βgamma-glutamine were 

dose-dependent. Inositol showed no effect. Taurine 

slightly increased. Results were confirmed after 

eliminating biological interferences via perchloric 

acid. 

Growth Curves/Cell Cycle Analyses/Cell 

Morphology: DEP had no effect. 

Cytokines: IL-6 levels increased at 0.1 and 1 

μg/mL. TNF-α was unaffected. 

December 2009 D-69 
Study Pollutant Exposure Effects 

Reference: Saxena et 

al. (2003, 096986) 

Species: Mouse 

Cell Type: RAW 264.7 

DEP: SRM 1650 

CO: Crude Organic Extract of DEP 

Fractionated into 

asphaltene (pentane/hexane), 

saturated hydrocarbon, 

less polar (aromatic) hydrocarbon, 

more polar (aromatic) hydrocarbon, 

resins, residual (resins) 

Particle Size: NR 

Route: Cell Culture (2.5×104 cells/mL) 

Dose/Concentration: DEP, CO 5, 10, 15, 

20, 25 μg/mL 

IFN-y: 10 ng/mL 

LPS: 1 mg/mL 

Time to Analysis: 1-3 days 

Cytotoxicity: No cytotoxic effects were observed. 

NO: DEP alone induced NO in a dose-dependent 

manner which peaked after 1 day and plateaued for 

days 2 and 3. IFN-y + DEP showed dose- and timedependency. LPS + DEP showed no effect at 1 day, 

but dose-dependently reduced NO production on 

days 2 and 3. Addition of Bacillus Calmette-Guerin 

(BCG) eliminated the effect of DEP at 2 days but 

showed a dose-dependent decrease at 3 days. 

Effectiveness of Particulate Components: The 

carbonaceous core of DEP did not affect BC Gstimulated NO production. CO significantly inhibited 

BCG-stimulated NO production. Study indicated that 

the extract of aromatic hydrocarbons and resins 

caused an inhibitory effect in a dose-dependent 

manner. 

Reference: Seagrave 

et al. (2007, 097549) 

Species: Human 

Gender: Male (3 

donors) 

Age: 16, 23 yr 

Cell Type: A549 

DE: Generated by DE 5500 watt 

generator using #2 certification oil 

performed under 5000w load. 

Emissions diluted to 3 mg/m3

 total 

particulate matter. 

Particle Size: 0.14-0.5 μm 

Route: Air Liquid Interface 

Dose/Concentration: 8.33 mL/min/well 

Time to Analysis: 3 h exposure; 1 or 21 h 

post-exposure 

Particle Deposition: 140 and 500 nm microspheres 

demonstrated uniform deposition of approx. 10%. 

Transepithelial Electric Resistance: No effect of 

DE; rather, more effect was observed from air 

controls. 

Macromolecular permeability: DE caused an 

increase 1 h but returned to control at 21 h. 

LDH/Cytotoxicity: DE had a highly variable(donor 

specific) effect at 1 h and returned to control levels 

at 21 h 

Mitochondrial activity (WST): DE reduced activity 

at 1 h and possibly increased activity at 21 h (high 

donor-to-donor variability) 

Mucus Like Substance Excretion: There was high 

donor to donor variability; no overall effects were 

observed. 

Alkaline Phosphatase (AP): DE decreased at 1 h 

and perhaps increased at 21 h 

Glutathione: DE caused a large decrease at 1 h but 

returned to normal at 21 h. 

HO-1: After DE exposure, levels increased but were 

still lower than air exposed controls 

Cytokines: No differences for IL- 8 or 12, TNF-α, 

GM-GSF, IL-1α, or IFN-γ were observed. IL-4 and -6 

were decreased upon DE exposure. 

Reference: Seagrave 

et al. (2004, 087470) 

Species: Human 

Cell Type: A549 

DPM: SRM2975 (NIST) 

DPM-O: DPM organic extract 

(acetone/DCM) 

CB: Carbon Black (Elftex-12, Cabot) 

Particle Size: CB: 37 nm; Stokes 

diameter 198 nm 

Route: Cell Culture (1×105 cells/well) 

Dose/Concentration: 0.03 -1,000 μg/cm2

Time to Analysis: 0, 18 h 

IL-8 release: DPM increased semi dosedependently (perhaps steady based on error range) 

up to 1 μg/cm2

 after which IL-8 declined dose 

dependently to zero (control = 100%) at 300 and 

1000 μg/cm2

. LDH release was steady which 

indicates no cytotoxicity. 

DPM interaction with IL-8: DPM depletes IL-8 from 

solution in a dose-dependent manner (cell free). 

BSA preincubation reduced the slope of the dose 

response but not the final result. CB has no effect. 

DPM-O residuals act identical to DPM. Increasing 

NaCl concentrations reduced DPMs depletion of IL-8

Neutrophil responses: DPM and bound IL-8 

together caused a marked aggregation of cells 

resulting in spindle shapes. DEM or IL-8 alone did 

not cause this aggregation although DEP did recruit 

neutrophils 

December 2009 D-70 
Study Pollutant Exposure Effects 

Reference: Seagrave 

et al. (2003, 054979) 

Species: Human, Rat 

Cell Line: F344/Crl BR 

(mouse) 

Age: 11 wk (mouse) 

Weight: 250 g 

Cell Type: A549, AMs 

PM filter collection 

Collected from diesel or gasoline 

powered vehicles as follows: 

BG: BS Gasoline 

G30: Normal Emitter gasoline (30F) 

G: Normal emitter gasoline (72F) 

HD: High Emitter Diesel 

D30: current technology diesel (30F) 

D: current technology diesel (72F) 

WG: White Smoke Gasoline 

Particle Size: NR 

Route: Cell Culture (1×105 cells/well) 

Dose/Concentration: 0.03-10,000 μg/cm2

Time to Analysis: 16-18 h 

Cytotoxicity: LDH activity increased in A549 cells. 

The types of pollutants that are most toxic, in 

decreasing order of cytotoxicity, are the following: 

BG, G30, and G which are significantly different from 

HD, D30, D, WG which are also significantly 

different from DS. LDH activity also increased in rat 

macrophages. G, G30, and BG were the most toxic. 

HD and D30 were intermediately toxic and D, WG, 

and DS were the least toxic. In both cell types, 

gasoline was more cytotoxic than diesel. 

Cytokines: All particle types except DS increased 

IL-8 levels in A549 though not all increases were 

statistically significant. Also, many particle samples 

at high concentrations produced an apparent 

suppression of IL-8 release. 

Alkaline Phosphatase: G30 and G were more 

potent than the other particle samples in A549. WG 

and D30 induced no significant effects. For A549 

cells, activity increased at low concentrations and 

was suppressed at higher concentrations. 

Macrophage Peroxide Production: In rat AMs, 

peroxide production was often the highest at the 

lowest concentrations and the lowest production 

caused by the highest concentrations. D30 followed 

this trend and induced the highest production as well 

as the greatest suppression. Using two different 

statistical methods, D30 >6 others which in turn 

>DS. Using the second method D30 and D >all other 

6. Order of potency between two methods 

completely different. Authors noted that in vitro 

potency quite different from in vivo potency 

(previous paper). 

Reference: Seaton. et 

al. (2005, 198904) 

Species: Human 

Cell Type: A549 

PM2.5 from London 

PM10 from Manchester (positive control)

PM from Holland Park, Hampstead and 

Oxford Circus stations (HP, HR and 

OC) 

Particle Size: PM2.5, PM10, Holland 

Park, Hampstead and Oxford Circus 

PM had a median diameter of 0.4 μm. 

80% of the particles had a diameter 

less than 1 μm. 

Route: Cell Culture 

Dose/Concentration: 1-100 μg/mL 

Time to Analysis: Cytotoxicity: 24 h; IL-8: 8 

h; Generation of hydroxyl radicals: 8 h 

Cytotoxicity: Dust from all three tunnels (Holland 

Park, Hampstead and Oxford Circus) were able to 

cause cell death (LDH). The release of LDH 

indicated a dose-dependent relationship. The 

highest dose of Holland Park PM induced the ~17% 

release of LDH, Hampstead triggered ~ 13% and 

Oxford Circus ~3% (no different than control). PM10

from Manchester caused a 7% LDH release at the 

highest dose. The negative control (TiO2) caused no 

response (2% release at highest dose). 

IL-8: All three tunnel PMs induced a dosedependent release of IL-8. At the highest dose, all 

three tunnel dusts induced IL-8 stimulation more so 

than the control site PM2.5. HP induced a 3 fold 

increase. Also, the highest TiO2 concentration 

caused the least IL-8 stimulation. 

Hydroxyl Radical Generation/ DNA Plasmid 

assay: The plasmid assay indicated that the tunnel 

dusts induce more free radical activity than the 

Manchester PM10 and TiO2. 

HP nearly doubled the percentage of DNA damage 

with intermediate results for HR and OC. Results for 

PM10, TiO2 and control were identical 

December 2009 D-71 
Study Pollutant Exposure Effects 

Reference: Singal and 

Finkelstein (2005, 

198905) 

Species: Human, 

Mouse 

Cell Type/Line: 

A549Luc1 lung 

adenocarcinoma 

epithelial cell line 

(human), MLE15Luc1 

and MLE15Luc2 

(mouse) 

All cells contain human 

cytokine IL-8 

controlling firefly 

luciferase 

AE2: Aerosil 200, amorphous silica 

(Degussa) 

CI: Carbon iron particles (25% Fe) 

Particle Size: AE2: 12 nm surface area 

~200 ± 25 m2

/g; CI: ~40 nm 

Route: Cell Culture (5×105 cells/well) 

Dose/Concentration: 18 μg/mL, 36 μg/mL, 

72 μg/mL all in 1 mL /well 

Time to Analysis: 24 h 

Luc Activity: Luciferase enzyme activity is 

significantly less in MLE15Luc2 cells than in 

MLE15Luc1 cells. For both cells, luciferase activity is 

time- and dose-dependent peaking at 4-8 h. 

Aerosil 200: AE2 induced dose- and timedependent Luc response which peaked at 3 h and 

decreased thereafter in a similar way as TNF-α. 

Contrary to TNF-α, AE2 induced much cytotoxicity 

starting at 6 h. 

Effect of Proteasomal Inhibitors (MG-132):

Inhibitor reduced AE2 Luc activity to near control 

levels. Similarly, LDH-cytotoxicity was halved 

A549 Human Cell Response: AE2 acted similarly 

to the MLE response. CI particles showed slightly 

less activity without peaks. AE2 increased 

cytotoxicity after 12 h, whereas CI had no effect. 

Contrary to MLE mouse, MG_132 did not affect Luc 

activity but PD98059 (selective noncompetitive 

inhibitor of the MAP pathway) and SN50 (NF-κB 

inhibitor) reduced AE2 and CI-induced activity. 

Reference: Song et al. 

(2008, 156093) 

Species: Rat 

Cell Type: RAW 264.7 

DEP collected from a 4JB1-type, lightduty (2740 cc), four-cylinder diesel 

engine operated using standard diesel 

fuel at speeds of 1500 rpm under a 

load of 10 torque. 

Particle Size: 0.4 μm (mean diameter) 

Route: Cell Culture (5×105 cells seeded on 

a 24-well plate) 

Dose/Concentration: 50 μg/mL 

Time to Analysis: 72 h 

Nitrite Production: 50 μg/mL of DEP induced 

production when compared to the control. Over the 

72 h period, a general trend was not observed, but 

maximal induction of nitrite occurred at 4 h after 

stimulation. 

Reference: 

Steerenberg et al. 

(2006, 088249) 

Species: Rat, Human 

Cell Type: AM (rat), 

Type 2 cells (rat), A549 

PMC: PM Coarse 

PMF: PM fine 

Ambient air samples collected from 

Rome, Italy; Oslo, Norway; Lodz, 

Poland; Amsterdam, the Netherlands; 

De Zilk, the Netherlands. 

Particle Size: PMC: 2.35-8.5 μm; PMF: 

0.12-2.35 μm 

Route: Cell Culture 

Dose/Concentration: NR 

Time to Analysis: 20 h 

Crustal material (metals and endotoxin but not Ti, 

As, Cd, Zn, V, Ni, Se) were positively associated 

with AM IL-6 and TNF-α and Type 2 MIP-2 and IL-6. 

Sea spray (Na and Cl) was also correlated with AM 

IL-6. 

Reference: Tal et al. 

(2006, 108588) 

Species: Human 

Cell Type: HAEC 

100 mM Zn(II) or V(IV) stock solutions 

Particle Size: NR 

Route: Cell Culture 

Dose/Concentration: 500 μmol 

Time to Analysis: 5, 20 min 

Zn-mediated EGFR Phosphorylation: EGFR 

kinase activity was required but not EFGR ligand 

binding. EGFR Kinase inhibition reduced Zn 

mediated EGFR activation. (authors NOTE: 

complete reverse of results in B82L and A431 cells). 

Src Kinase is not required. Zn inhibiting Src kinase 

was nearly total after 20 min. 

EGFR-Specific Protein Tyrase Phosphatase 

(PTP): Zn inhibited PTPs, similar to V(IV) resulting in 

a decrease of exogenous EGFR dephosphorylation 

Reference: Tamaoki et 

al. (2004, 157040) 

Species: Human 

Cell Type: HBEC 

UFCB: Ultrafine Carbon Black - (Tokai 

Carbon, Japan) 

FCB: Fine Carbon Black (Tokai Carbon, 

Japan) 

Particle Size: UFCB: 11 ± 0.5 nm 

(mean diameter) 

FCB: 250 ± 16 nm (mean diameter) 

Route: Cell Culture (104 cells/well) 

Dose/Concentration: 6.1, 12.3, 18.4, 24.5, 

30.7 μg/cm2

Time to Analysis: Up to 72 h 

DNA Synthesis/ Protein Synthesis: Synthesis 

increased by UFCB (30.7) for up to 72 h and 

flattened after 48 h. FCB had no effect. UFCB also 

showed a dose-dependent response beginning at 

12.3 μg/cm2

 up to 24.5 after which the response 

plateaued. The addition of Cu/Zn Super oxide 

dismutase (SOD) or a NADPH oxidase inhibitor 

completely inhibited the UFCB effects. Similarly, two 

different EGFR tyrosine kinase inhibitors, and a Me 

inhibitor all reduced UFCB response to control 

levels. 

ERK activation: UFCB caused phosphorylation of 

ERK beginning at 2 min, peaking at 5 min and 

decreasing at 10 min. ERK activation was inhibited 

by EGFR tyrosine kinase inhibitor Cu/Zn SOD and 

neutralizing body for HB-EGF but not by PDGF-R 

kinase inhibitor. 

HB (polyclonal heparin binding)-EGF release:

UFCB induced rapid cell surface loss with recovery 

after 20 min and nearly full recovery at 360 min. 

Metalloproteinase inhibitor and Cu/Zn SOD both 

prevented HB-EGF release. 

December 2009 D-72 
Study Pollutant Exposure Effects 

Reference: Tao and 

Kobzik (2002, 157044)

Species: Rat 

Cell Type: RLE-6TN 

(Alveolar Type II 

Epithelial Cells), Fetal 

Lung Fibroblasts 

(RFL), AMs 

UAP: Urban Air Particles (SRM 1649) 

TiO2

SiO2

ROFA 

Particle Size: TiO2: ~1 μm; SiO2: ~1 

μm; ROFA: NR 

Route: Cell Culture (1×105 cells AM 

1.4×105

 cells RLE/RFL) 

Dose/Concentration: 1-50 μg/mL 

Time to Analysis: 24 h 

Cytokines: TNF-α and MIP-2 in RLE was 

unaffected by any particle samples. TNF-α and MIP2 in AM significantly increased with 25 μg/mL UAP. 

TNF-α and MIP-2 in the co-culture of AM + RLE 

increased with each particle. The order of particles 

in decreasing order are as follows: SiO2 at 25μg/mL, 

UAP at 12.5 μg/mL, ROFA at 25 μg/mL, and TiO2 at 

50 μg/mL. Except for SiO2, the blocking of effects 

caused by LPS absorbed on the particles did not 

affect the cytokine response. For SiO2, the response 

was reduced but still above the control. 

Co-culture: Physically separating AM and RLE cells 

and adding PM completely negated the co-culture’s 

response to PMs. This indicates that cell to cell 

contact is required for co-culture potentiation of PM 

effects. 

Inhibitors: Various inhibitors of cell adhesion 

molecules (heparin, β -1, 2 or 3 integrin) had no 

effect on UAP-induced cytokine release. 

Reference: Veranth et 

al. (2007, 090346) 

Species: Human 

Cell Type: BEAS-2B, 

A549, NHBE 

Artificial particles and PMs 

N-Al: nano alumina Al2O3

M-Al: Micro Al2O3

N-Ce: nano CeO2

M-Ce: micro CeO2

N-Fe: nano Fe2O3

M-Fe: micro Fe2O3

N-Ni: nano NiO 

M-Ni: micro NiO 

N-Si: nano SiO2

M-Si: micro SiO2

N-Ti: nano TiO2

M-Ti: micro TiO2

KLN: kaolin 

MUS: Min-U-Sil (ground crystalline 

silica) 

DD: desert rural soil Utah PM2.5 

JE: Juarez, urban street PM2.5 

MNC: Mancos, rural Utah PM2.5 

LPS: lipopolysaccharide 

V: VOSO4 (soluble) (19 μg/mL) 

Particle Size: (Surface mean diameter)

N-Al: 6 nm (261 m2

/g) 

M-Al: 210 nm (7.7 m2

/g) 

N-Ce: 14 nm (71 m2

/g) 

M-Ce: 1500 nm (0.6 m2

/g) 

N-Fe: 5 nm (221 m2

/g) 

M-Fe: 100 nm (12 m2

/g) 

N-Ni: 6 nm (145 m2

/g) 

M-Ni: 16 nm (57 m2/g) 

N-Si: 19 nm (127 m2

/g) 

M-Si: 440 nm (5.4 m2

/g) 

N-Ti: 6 nm (242 m2/g) 

M-Ti: 410 nm (3.5 m2

/g) 

KLN: 100 nm (24.3 m2

/g) 

MUS: (NOS <5 μm) 

DD: 400 nm (6.2 m2

/g) 

JE: (NOS <3 μm) 

MNC: 200 nm (13.0 m2

/g) 

Route: Cell Culture (35,000 cells/cm2 BEAS; 

2500 cells/cm2

 NHBE; 20,000 cells/cm2

A549) 

Dose/Concentration: 0.53, 5.3 and 53 

μg/cm2

 (= 1, 10, 100 μg/mL) 

Time to Analysis: 24 h 

Cell Viability: Except for Ni and V no cytotoxicity 

was observed at the highest concentration. 

IL-6 Secretion in BEAS-2 B Cells: Nano and micro 

sizes of the same metal showed no differences in 

response (high experiment to experiment variability). 

In general, the soil-derived dusts (JE, DD, MNC) 

were more potent than the metal and ceramic oxide 

particles. In KGM media, BEAS-2B cells are more 

responsive to vanadium and other soluble metals 

and less responsive to LPS, but this relationship is 

reversed in LHC-9 media. 

IL-8 Secretion in BEAS/LHC vs NHBE in BEGM 

Cells: Levels were much higher in NHBE cells than 

BEAS-2B cells. For BEAS-2B, the nano size Si and 

both sizes of Ni induced levels statistically greater 

than the control. For NHBE, only Si and Ni (for both 

sizes) were statistically greater than control. 

IL-6 in NHBE: The nano and micro sized particles of 

Al, Ce, Fe and nano sized Si all induced statistically 

significant increases. Control levels of IL-6 were 

much higher in NHBE cells than in BEAS-2B cells. 

Secretion induced by pure oxide particles was small 

for both the mid and high concentration levels (5.3 

and 53 μg/cm2

). 

BSA/ Bovine Serum Addition Effect: In a fixed 

solution nano-Ni, nano-Ti and KLN all reduced the 

measured IL-6 by 60+ percent. Addition of BSA or 

bovine serum dose dependently reduced the action 

of the particles to near control levels. 

PM Effects (without added protein) on IL-6 In 

Solution: Increasing metal concentration did not 

affect a fixed IL-6 concentration until the 100 or 316 

μg/mL levels. 

December 2009 D-73 
Study Pollutant Exposure Effects 

Reference: Veranth et 

al. (2007, 090346) 

Species: Human, 

mouse, rat 

Cell Type: A549, 

BEAS-2B (types E and 

U), RAW 264.7, 

Primary macrophages 

S: desert dust (collected from unpaved 

desert road in Utah, PM2.5 enriched) 

V: vanadium soluble (prepared from 

VOSO4, Alfa Aesar, Ward Hill, MA) 

C: Coal fly ash (PM2.5 enriched and 

derived from commercial power plant 

burning Utah bituminous coal) 

D: Diesel PM (tail-pipe particles 

collected from high emitting BSr onroad light duty truck) 

L: Lipopolysaccharide 

T: Titanium dioxide (Alfa Aesar) 

K: Kaolin (purchased from Capitol 

Ceramics, UT) 

Particle Size: BET surface (m2/g) 

S: 6.2 (PM2.5 enriched) 

V: NA 

C: 5.4 (PM2.5 enriched) 

D: NR 

L: NA 

T: 3.5 (1-2 μm) 

K: 24 (<200 mesh = 74 μm) 

Route: Cell Culture 

Dose/Concentrations: Maximum 

concentrations: 

S = 100 μg/cm2

V = 100 μg/cm2

C = 100 μg/cm2

D = 32 μg/cm2

L = 1000 EU/mL 

T = 100 μg/cm2

K = 100 μg/cm2

Time to Analysis: 24 h 

Viability: Generally, cell viability was greater than 

75% of the control post treatment. Vanadium, at the 

highest concentration, induced less than 50% of 

control viability whereas kaolin, also at the highest 

concentration, induced cell death. 

IL-6: BEAS-2B E or U in LHC-9 showed a response 

to S and L. BEAS-2B (U) was in LHC-9 medium with 

added serum (FBS). This resulted in a doubling of 

response coupled with at least an 8 fold increase in 

control levels. BEAS-2B (E) showed response for S 

and V but not L. A549 showed response to S and K. 

RAW 264.7 and Rat macrophages showed 

responses to S(very low) and L. In general, the IL-6 

responses in A549 and RAW 264.7 were similar and 

significantly lower than the responses in rat 

macrophages or BEAS-2B. 

Effect of Culture Media Composition (BEAS-2B):

Varying ratios of LHC-9 and KGM media resulted in 

a near 10 fold increase in control rate once LHC was 

33% or more of the media. Upon Soil Dust (NOS) 

exposure IL-6 increased linearly with % LHC-9 in 

culture/exposure media. Addition of calf serum (0.1-

10 %) raised control IL-6 levels at least 40 fold. At a 

steady PM concentration, the addition of serum 

resulted in a log-linear increase in IL-6 release which 

blocked any PM effect. 

Reversibility of Media Effect: Changing media with 

every passage showed that media effects do not 

persist once media are changed. 

Culture Well Size: Going from a 6 well to 96 well 

plate (decreasing well size) increased IL-6 control 

values about ten fold, while the positive control 

(TNF) response increased 3 fold. Hence the 

sensitivity of the test (i.e., positive/control response) 

declined from 11 fold to 3 fold with increasing well 

number / decreasing well size. Because cell seeding 

density and the like were held constant, these 

changes suggest that edge effects are the cause of 

the IL-6 changes. 

Reference: Veranth et 

al. (2006, 087479) 

Species: Human 

Cell Type: BEAS-2B 

PM2.5 samples from 28 samples from 8 

locations in Utah, New Mexico and 

Texas (rural, industrial, road side, 

military) 

2 coal fly ash samples (a product of 

combustion using Utah bituminous coal 

and New Mexico bituminous coal) 

TiO2 

kaolin clay 

Particle Size: PM2.5; TiO2: 1-2 μm 

Route: Cell Culture (35,000 cells/ cm2) 

Dose/Concentration: 10, 20, 40, 80 μg/cm2

Time to Analysis: 24 h 

Cell Assays: In sample soils viability declined dose 

dependently while IL-6 increased dose-dependently. 

IL-8 was highly variable (peak at 20 μg/l, dosedependent increase or flat response.)

IL-6 Assays for All Soil PMs: Soils ranged across 

an order of magnitude greater than LPS, coal fly 

ash, TiO2 or kaolin samples. One soil even 

exceeded the pos V control at equal concentrations 

Correlation with Cell Viability: Correlation was 

strong for Mn (p<0.001) and weak for EC3, K, Se, 

and Hg (0.01<p<0.05). 

IL-6, 10 μg/cm2: Correlation was medial for OC1(OC) and P at 0.001<p<0.01. 

IL-6, 80 μg/cm2

: Correlation was strong for OC3, 

OP (pyrolized Carbon), OC, EC1, TC and 

intermediate for OC2, OC4, Zn and weak for Ca2+, 

EC2, Si, Ca, Ca: Al. 

IL-8, 10 μg/cm2: Correlation was weak for EU 

(Endotoxin), CO3, Si, and Br.

IL-8, 80 μg/cm2

: Correlation was medial for CO3, Sr 

and weak for K+, EC3, Mg, Si. 

IL-8 trend (corr over 10-80 range): Correlation was 

strong for EC, intermediate for OC4, EC1, EC2, 

EC3, TC, Ni and weak for OP, OC, Cr, and Sr. IL-6 

and Il-8 were not correlated nor were IL-6 and cell 

viability. Authors noted that weak correlations 

(0.01<p<0.05) contained false positives. 

December 2009 D-74 
Study Pollutant Exposure Effects 

Reference: Veranth et 

al. (2004, 087480) 

Species: Human 

Cell Type: BEAS-2B 

PM2.5 enriched soil samples 

DD: desert dust, unpaved road, Utah 

WM: West Mesa, sandy grazing site, 

NM 

R40: Range 40 gravel soil, TX 

UN: Uinta, sandy soil, UT 

Particle Size: 0.4-3 μm 

Route: Cell Culture (20,000/cm2) 

Dose/Concentration: 10, 20, 40, 80, 160 

μg/cm2

Time to Analysis: 24 h 

Elemental Analysis of PM: Major differences UN 

generally lower in major minerals but high Fe 

content and high EC. High Mn. Low Pb and Zn 

Cytotoxicity: UN and WM were the most cytotoxic 

at all dose levels, followed by R40 and DD. All 

particles showed a dose-dependent cytotoxic 

response. 

IL-6 Release: DD and R40 (up to the 160 μg/cm2

) 

showed dose-dependent responses and induced an 

8-fold increase at the highest concentration levels. 

WM peaked at 40 μg/cm2

 and UN induced similar 

responses above 10 μg/cm2

.

IL- 8 Release: DD induced a dose-dependent 

response. WM peaked at 10 μg/cm2

. Release 

induced by DD and WM seemed to be limited by 

toxicity. There was no treatment with R40. 

TNF-α: DD, WM and UN induced release was not 

detected at the 40 or 80 μg/cm2

 concentrations. 

LPS: LPS was the primary factor in inducing IL-6 

release when exposed to LPS-containing mixtures. 

LPS alone induced lesser responses than treatment 

to the environmental dust particles. TiLPS induced a 

less than two-fold increase in IL-6 versus the over 

seven-fold increase induced by soil dust positive 

control. LPS treatments were less cytotoxic than DD. 

Limited IL-6 and IL-8 responses were observed at 

2000 EU/mL compared with DD at 80 μg/cm2

Endotoxin: Inverse relationship between endotoxin 

content and IL-6 release was observed. 

Viability vs Physical Modification of Dust Sample 

(no UN): Only leaching in a variety of water based 

vehicles increased viability minimally (generally <25 

%). Heat treatment (150-, 300, 550° F) and 

methanol extraction had no effect 

IL-6 Release vs Physical Modification of Dust 

Sample (no UN): One hour thermal treatment at 

150 ̊ F had no effect on IL-6 response. All other 

treatments reduced IL-6 release (heat 350 ̊, 500 ̊

and extractions). 

Reference: Veronesi 

et al. (2002, 024599) 

Species: Human 

Cell Type: BEAS-2B 

Ambient PM 

- St. Louis: Urban particulates 

- Ottawa: Urban particulates 

-MSH: Volcanic dust from Washington 

state’s Mt. St. Helen 

-Woodstove: Woodstove particles from 

conventional fireplace burner 

-CFA: Coal fly ash from western U.S. 

power plant 

-OFA: Oil fly ash from Niagara, NY 

- A: Total Fractions 

- B: Soluble Fractions 

- C: Washed Fractions 

Particle Size: PM >2.5 μm; PM: 2-10 

μm; PM >10 μm 

Route: Cell Culture 

Dose/Concentration: 50 μg/mL; 30 μg/cm2

100μg/mL; 60 μg/cm2

Time to Analysis: 4, 16 h 

Ca: Calcium increased significantly with all particles 

types. 

IL-6: At 50 and 100 μg/mL, IL-6 increased with all 

particle types at 4 and 16 h. Overall, fraction -A was 

the most potent. 

Surface charge: Surface charge correlated strongly 

with increases in both Ca2+ and IL-6 levels. OFA, 

however, was unmeasurable due to technical 

difficulties. 

December 2009 D-75 
Study Pollutant Exposure Effects 

Reference: Vogel et 

al. (2005, 087891) 

Species: Human 

Cell Type: U937 

(ATCC) monocytes 

(macrophage 

differentiation) 

UDP: SRM 1649 (NIST) 

UDP-OE: DCM extract of SRM-1649, 

0.45 μm filter 

sUDP: stripped particles UDP 

DEP: SRM 2975 (NIST) 

DEP-OE: DCM extract of SRM-2975, 

0.45 μm filter 

sDEP: stripped particles DEP 

CB95: Carbon Black (Degussa) 

Particle Size: UDP, DEP: NR; 

CB95: 95 nm 

Route: Cell Culture (2×105 - 2×106 cells/mL) 

Dose/Concentration: DEP, UDP: 2.5, 10 or 

40 μg/cm2

(eq to 12.5, 40, 200 μg/mL) 

DEP-OE, UDP- OE: 10 μg/cm2

 (particle 

equivalent) 

Time to Analysis: 24 h 

Effect On mRNA Expression (COX-2, TNF-α, IL-6, 

IL-8, C/EBPβ, CRP, CYP1a1): All DEP and UDP 

induced dose-dependent increases. IL-6 tended to 

plateau at 10 μg/cm2. Generally, with the exception 

of COX-2, UDP effects on genes were stronger than 

DEP. 

Cytotoxicity: Both DEP and UDP were cytotoxic at 

40 μg/cm2

Fractionation and mRNA Expression: For COX-2, 

TNF-α, IL-8 mRNA fractions were much more active 

than parent particles and consequently stripped 

particles were much less active than parent 

particles. CB95 had no effect. The reverse effect 

occurred for IL-6 and CRP mRNA expression. The 

particles that induced mRNA expression in 

decreasing order are: sUDP, UDP, UDP-OE. 

Inhibition Of mRNA Expression: CRP: 

pretreatment with IgG and wortmannin (Fcγ receptor 

binding and ingestion dependent inhibitors resp) 

blocked the effects of DEP, UDP and sDEP and 

sUDP. Luteolin (AhR inhibitor) had no effect. 

COX-2: Only luteolin inhibited COX-2 expression for 

DEP, DEP-OE, UDP, and UDP-OE. 

CYP1a1: Luteolin also inhibited OE-DEP and OEDUP effects (only those two particles tested). 

Cholesterol Accumulation: DEP, UDP and UDPOE and DEP-OE at 10 μg/cm2

 all increased 

cholesterol accumulation by at least 2 fold 

Reference: Wang et 

al. (2003, 157106) 

Species: Rat 

Cell Type: Lung 

Myofibroblasts 

V2O5: (Aldrich Chemical Co., 

Wisconsin) 

Particle Size: NR 

Route: Cell Culture (1×105 cells/100 mm 

dish; 3.2×104

 cells/cm2) 

Dose/Concentration: 400 μm 

Time to Analysis: 0.5, 1, 4, 24 h 

H2O2 Drives STAT-1 Activation: Pretreatment with 

NAC or catalase reduced V2O5-induced STAT 

activation by more than 90% and completely 

abolished H2O2-induced STAT activation. Within 5 

min of V2O5 treatment, H2O2 was significantly 

decreased in the supernatants of cultured 

myofibroblasts and suppression of H2O2 levels 

continued for up to 24 h post V2O5 treatment. This 

supports the findings that myofibroblast-generated 

H2O2 is required for V2O5-induced STAT activation. 

Temporal STAT-1 Activation: H2O2 induced rapid 

activation within minutes whereas activation by V2O5

occurred more slowly (beginning 8h post treatment). 

p38, ERK, EGFR: p38 and EGFR are required for 

H2O2- or V2O5-induced STAT-1 activation whereas 

ERK is not required 

Reference: Whitekus 

et al. (2002, 157142) 

Species: Mouse 

Cell Line: RAW 264.7

DEP (light-duty, four-cylinder engine4JB1 type, Isuzu Automobile, Japan; 

standard diesel fuel) (extracts) 

Particle Size: NR 

Route: Cell Culture 

Dose/Concentration: 50 μg/mL 

Time to Analysis: 5 h 

DEP significantly reduced the GSH:GSSG ratio. This 

effect was prevented by adding thiol antioxidants 

NAC or BUC. DEP increased lipid peroxide levels, 

but the addition of all antioxidants decreased these 

levels. DEP increased carbonyl groups. NAC, BUC, 

and luteolin reduced HO-1 expression. 

December 2009 D-76 
Study Pollutant Exposure Effects 

Reference: Wilson et 

al. (2007, 097268) 

Species: Mouse 

Cell Type: J774 

CB: Carbon Black, Printex 90 

(Degussa) 

FeCl3

ZnCl2

Particle Size: CB: 14 nm 

Route: Cell Culture (4×105 cells/mL at 

1mL/well) 

Dose/Concentration: CB 1.9 -31 μg/mL; 

FeCl3, ZnCl2 0.01-100 μmol 

Time to Analysis: 4 h 

ROS Production in Cells: CB alone increased 

ROS. Coexposure with ZnCl2 did not affect ROS. 

ROS Production - Cell Free: CB induced a 

significant increase in ROS. ZnCl2 had no effect. 

Coexposure CB/Zn also had no effect. 

TNF-α Production (Fe -Zn 0.01-100 μmol): 

Coexposure of CB over a range of metals gave no 

change over CB alone for Fe. For Zn, only at the 

concentration of 100 μmol was there a small 

interaction between Zn and CB. 

Similar results were seen at metal concentrations 

between 20 -100 μmol. Synergism was observed 

between Zn and CB and no observed effect of Fe. 

Macrophage Cytoskeleton: CB resulted in black 

vacuoles. Co-treatment of cells with Zn and CB 

increased the severity of Zn effects. Fe exhibited no 

synergism. 

Apoptosis /Necrosis: No synergism of CB with 

either Fe or Zn. 

Phagocytosis: Only at 31 μmol CB and 50 μmol Zn 

did a synergistic effect occur; it resulted in a 4-fold 

reduction. 

Reference: Wottrich et 

al. (2004, 094518) 

Species: Human 

Cell Type: A549, THP1, Mono Mac 6 

Fe: hematite α-Fe2O3

Si60: silicasol (SiO2, amorphous silica) 

Si100: silicasol 

Q: crystalline quartz DQ12 

Particle Size: Fe: 50-90 nm; Si60: 60 

nm; Si100: 80-110 nm; Q <5 μm 

Route: Cell Culture (2×104 cells/well. Coculture: 2×104

 A549 and 2×103

Macrophages) 

Dose/Concentration: A549 light 

microscopy hematite 100μg/mL (23 μg/cm2

) 

TEM hematite 50 μg/mL (16 μg/cm2

) 

Cytotoxicity 10, 50, 100 and 200 μg/mL (6.1, 

30, 61 and 121 μg/cm2

) 

Cytokines 50 and 200 μg/mL 

Time to Analysis: 24 h 

Particle Uptake: Hematite agglomeration was 

observed in all 3 cell lines. TEM confirmed cytosol 

aggregates as well as single particles, which 

includes particles transported intracellularly to 

basolateral membrane of epithelial cells. 

Cytotoxicity: LDH increased significantly in A549. 

In decreasing order, Q , Fe, S60, and S100 (which 

exhibited levels similar to controls) all induced 

cytotoxicity. THP-1 cells appeared the most sensitive 

with Q, Fe, S60, S100, control inducing cytotoxicity 

in decreasing order. Mono Mac 6 cells were the least 

sensitive with Fe, S60, Q, S100. 

Cytokines: IL-6 and IL-8 released from A549 cells 

upon exposure to all particles. No response was 

observed in Mono Mac 6 or in THP-1 cells. 

Co-cultures: Mix of A549 with either Mono Mac 6 or 

THP-1 led to a large (ten fold) increase in response 

to particles. Ten fold increases were observed in IL-6 

and IL-8 levels with the Mono Mac 6 co-culture and 

the THP-1 co-culture, respectively. 

December 2009 D-77 
Study Pollutant Exposure Effects 

Reference: Wu et al. 

(2007, 098412) 

Species: Human 

Cell Line: B82L 

Cell Type: B82L- par 

(parental fibroblasts), 

B82L-wt (wild type 

EGFR), B82L-K721M 

(kinase defective 

EGFR), B82L-c’958 

(COOH-terminally 

truncated EGFR at 

Tyr-958) 

ZnSO4 (Sigma) 

Particle Size: NR 

Route: Cell Culture 

Dose/Concentration: Zn: 500 μmol 

EGF: 100 ng/mL 

Time to Analysis: 20 min 

EGFR Mutations: EGFR-wt has a 

functional tyrosine kinase domain, intact Src 

phosphorylation (Tyr 845) and 5 tyrosine 

autophosphorylation sites. EGFR-c’958 lacks all 5 

tyrosine autophosphorylation sites. EGFRK721M lacks tyrosine kinase (ATP binding). EGFRY845F lacks Src autophosphorylation (Tyr 845) and, 

instead, has a receptor at Tyr 845 that is 

phosphorylated by nonreceptor Tyrosine kinase Src.

Zn Induced Ras (MAPK signaling protein): No 

effect was observed in B82L-par cells. Zn had an 

effect in -wt, -c’958, and -K721M which confirms the 

need for EGFR. This indicates that neither tyrosine 

kinase nor autophosphorylation sites were required 

for Zn effects. No observed increase for Y845F 

indicated that EGFR tyrosine 845 (phosphorylated 

by c-Src) is required for Zn effects. However, it was 

not required for EGF effects. 

Src Kinase Requirement: Using a Src blocker 

drastically reduced Zn effect but not the EGF effect. 

Src activation occurred independent of EGFR Tyr845. 

Zn Induced Association of EGFR with Src: Zn 

induced a physical association in all 4 mutants; EGF 

did not. 

Zn Induced Phosphorylation of EGFR at Tyr-845:

Zn induced phosporylation of EGFR at Tyr-845 in 

B82L-wt,-c’958 and -K721M. EGF exhibited similar 

effects. Src blockers significantly reduced 

phosphorylation induced by Zn but not for EGF. 

Neither Zn or EGF induced phosphorylation in B82LY845F cells. 

Reference: Wu et al. 

(2003, 199749) 

Species: Human 

Cell Type: BEAS-2B 

Zinc Ion: Zn2+

Particle Size: NR 

Route: Cell Culture 

Dose/Concentration: 10, 25, 50 μmol 

Time to Analysis: 0-8 h 

Cytotoxicity: Exposure to 50 μmol Zn2+ for 8 h did 

not result in significant alterations in cell viability. 

PTEN Protein Levels: 50 μmol Zn2+ for 4 and 8 h 

significantly decreased levels in a dose-dependent 

manner. Exposure to 50 μM vanadyl sulfate 

(tyrosine phosphatase inhibitor) had minimal effects 

on PTEN. 100 ng/mL of non-specified EGF receptor 

ligand for 1-8 h did not exhibit any significant effects 

on PTEN levels. 

P13K/Akt: Zinc induced Akt activation in a doseand time- dependent fashion. Active Akt levels were 

the highest at 1 h post exposure to Zn2+, 

corresponding with the time period when there was 

a minimal effect on PTEN protein level. When 

treated with LY294002 (inhibitor of P13K activity), 

Akt phosphorylation was significantly inhibited. 

PTEN mRNA Levels: Decreased PTEN mRNA 

expression was observed in cells exposed to 50 

μmol Zn2+ for 8 h whereas PTEN protein levels 

declined as early as 4 h. 

Proteasome-mediated PTEN Degradation: Use of 

MG132 (proteasome inhibitor) had no significant 

effect on Zn2+ induced PTEN mRNA expression. 

Therefore mRNA expression may not play a critical 

role in PTEN protein reduction. Instead data 

suggested that 26 S proteasome played a vital role 

in Zn2+ induced PTEN degradation. PI3K inhibitor 

blocked Zn-induced PTEN degradation, but failed to 

prevent significant Zn-induced down-regulation of 

PTEN mRNA. 

December 2009 D-78 
Study Pollutant Exposure Effects 

Reference: Wu et al. 

(2004, 096949) 

Species: Human 

Cell Type: NHBE 

Zinc Ion: Zn2+

Particle Size: NR 

Route: Cell Culture 

Dose/Concentration: 100 μmol 

Time to Analysis: 2 h 

Cell Viability: After 2 h of exposure, Zn2+ induced 

effects in NHBE cells at 100 and 200 μmol levels 

(but not 50 μmol). Continuing exposure to 100 μmol 

Zn2+ for 4 and 6 h did not significantly alter cell 

viability. Thus, in all subsequent studies, NHBE cells 

were treated with 100 μmol Zn2+. 

Induced EGFR Phosphorylation: Exposure to 

100μM Zn2+ for 1-4 h induced phosphorylation of 

EGFR in NHBE cells. EGFR kinase inhibitor 

PD153035 (to determine if phosphorylation of EGFR 

was the result of autophosphorylation of activated 

EGFR tyrosine kinase activity) caused Zn2+ -induced 

phosphorylation to subside. Zn2+ activity requires 

tyrosine kinase activity. 

EGFR Phosphorylation Pathway: To test whether 

Zn2+ exposure results in ligand release, which in turn 

can activate phosphorylation, NHBE cells were 

pretreated with LA1 blocking antibody. Results 

showed significant suppression of Zn2+ induced 

phosphorylation, therefore Zn2+ phosphorylation 

might be initiated by the release of EGFR ligands. 

HB-EGF, TGF-α, EGF: To examine the involvement 

of specific ligands (HB-EGF. TGF-α and EGF) in the 

phosphorylation pathway, cells were exposed to 

anti-HB-EGF, anti-TGF-α and anti-EGF. Results 

showed that anti-HB-EGF reduced Zn2+ induced 

phosphorylation significantly, anti-TGF-α produced 

partial inhibition and anti-EGF had no inhibitory 

effect. Exposure with blocking antibody LA1 was 

tested to determine if it caused an increase in 

soluble HB-EGF. HB-EGF mRNA expression was 

also elevated in cells exposed to Zn2+. Previous 

studies indicate metalloproteinase (MMP) 

involvement in cleaving ligand precursors. It was 

found that MMP-3 inhibitor partially blocks Zn2+

induced HB-EGF release. (MMP-2 and MMP-9 did 

not show similar inhibition patterns) Zn2+ exposure 

increased the release of MMP-3 from HNBE cells. 

December 2009 D-79 
Study Pollutant Exposure Effects 

Reference: Wu et al. 

(2005, 097350) 

Species: Human 

Cell Line: Subclone 

S6 

Cell Type: BEAS-2B 

Zinc Ion: Zn2+

Particle Size: NR 

Route: Cell Culture 

Dose/Concentration: 50 μmol 

Time to Analysis: 4 or 8 h; EGFR 

phosphorylation: 30, 60, 120, 240 min 

Cell Viability: Exposure to 50 μmol Zn2+ for 8 h did 

not result in significant alterations in cell viability 

(assessed by LDH release). 

P13K/Akt Signaling Pathway: To evaluate P13K’s 

on COX-2 Zn2+ induced expression, LY-294002 (a 

P13 inhibitor) and another unnamed P13 inhibitor 

were used. Exposed cells indicated suppressed 

levels of Zn2+ induced COX-2. To determine Akt role, 

ad-DN-Akt (AAA) was used. Infected cells indicated 

over-expression of Akt and significant reduction of 

Zn2+ induced GSK-3α/β phosphorylation. Over 

expression of DN-Akt(AAA) blocked Zn2+ induced 

COX-2 expression. 

PTEN’s Role in Blocking Zn2+ Induced COX-2 

mRNA Expression: PTEN is an antagonist of 

P13/Akt pathway. Overexpression of wildtype PTEN 

blocked Zn2+-induced mRNA COX-2 expression, 

suggesting PTEN inhibits PIP3 signal transduction to 

Akt. 

Analysis of the Src/EGFR Signaling Pathway: 

Zn2+ induced a time-dependent increase in Src and 

EGFR phosphorylation in cells. Blockage of Src 

activity via PP2 (Src inhibitor) decreased Zn2+

induced EGFR phosphorylation. The EGFR tyrosine 

inhibitor completely blocked Zn2+-induced EGFR 

phosphorylation. EGF (a ligand of EGFR signaling) 

induced COX-2 expression, suggesting that EGFR 

regulated Zn2+ -induced COX-2 expression. 

p-38 and EGFR Kinase Activity: Use of PD153035 (EGFR inhibitor) and PP2 (Src inhibitor) and 

SB-203580 (p38 inhibitor) all blocked Zn2+-induced 

Akt phosphorylation of Src., EGFR and p38. It is 

thought that p38 is a critical kinase in regulation of 

Zn2+ -induced COX-2 protein expression. 

Reference: Yacobi et 

al. (2007, 156166) 

Species: Rat 

Cell Type: L2 (Lung 

epithelial cells) 

PNP: Polystyrene nanoparticles, 

negatively charged (Molecular Probes, 

Eugene, OR) 

PNPA: Amidine modified PNP, 

positively charged 

SWCNT: Single-wall carbon nanotubes 

(Carbon Nanotech, Houston, TX) 

QDC: Chitosan coated (CdSe/ZnS) 

Quantum dots, positively charged 

(made) 

QDA: Alginate coated QD, negatively 

charged 

UAPS: Ultrafine Ambient particulate 

suspensions (VACES) (48 % OC) 

Particle Size: PNP20: 20 nm; 

PNP100: 100 μm; SWCNT: 0.8-1.2 nm 

(diameter); SWCNT: 100-1000 nm; 

QD: 30 nm; UAPS: <150 nm 

Route: Cell Culture (1.2×106 cells/cm2) 

Dose/Concentration: PNP up to 706 μg/mL 

QD up to 176 μg/mL 

SWCNT up to 88 μg/ mL 

UAPS up to 36 μg/mL 

Time to Analysis: on days 4, 5 or 6 by 

replacing monolayer apical fluid with PM in 

suspension for up to 1440 min. 

Intermediate measurements at 15, 30, 60, 

120, 240 and 1440 min. 

UAPS and Rt (transmonolayer resistance): Rt 

declined up to 60% within 1 h at 36 μg/mL. Rt 

plateaued (or exhibited a very slight upgradient) for 

up to 24 h (last measurement). No cytotoxicity was 

observed. Replacement of apical fluid with fresh 

media after 2 h of exposure restored Rt to near 

control values within 24 h. 

UAPS and Leq (short-circuit current): Peak 

decline of 30% after 4 h followed by gradual 

recovery over 24 h. Replacing media after 2 h 

exposure returned Ieq to control values within 24 h. 

UAPS and Apparent Permeability: Permeability 

measured via C14 mannitol and inulin showed no 

effect of UAPS. 

QD and Rt: QD depressed Rt by nearly 55% at 4 h 

for positively charged and 30% for negatively 

charged QDs. Recovery towards control values 

started at 4 h and was near complete at 24 h 

SWCNT and Rt: SWCNT depressed Rt by ~ 40% at 

1 h (same for 22, 44, and 88 μg/mL). Recovery was 

near complete at 4 h and complete at 24 h. 

PNP and Rt: No statistically significant effects were 

observed. 

December 2009 D-80 
Study Pollutant Exposure Effects 

Reference: Yun et al. 

(2005, 088302) 

Species: Human 

Cell Type: A549 

DEP: Collected using a 6 cyl 11L, 

heavy duty (2001 yr) bus engine (South 

Korea) 

Particle Size: NR 

Route: Cell Culture (3×104 cells/well) 

Dose/Concentration: 1, 10, 100, 250, 500 

and 1000 μg/mL; main testing 250 μg/mL 

Time to Analysis: 12 h 

NF-κB Transcription Activation: DEP induced 

dose-dependent activity up to 250 μg/mL. After 

peaking at 250 μg/mL, concentrations above 250 

induced dose- dependent declines. Activity peaked 

at 12 h for 250 μg/mL and declined to control at 24 

or 48 h. The mechanism of DEP action was the 

degradation of IκBα which is an intracellular inhibitor 

of nuclear translocation of NF-κB. 

TAK1 and NIK Required for NF-κB Activation by 

DEP: Dominant negative mutants of TAK1 and NIK 

reduced DEP induced response to basal level. TAK1 

was phosphorylated after DEP exposure and was 

sustained for at least 90 min. 

Reference: Zhang et 

al. (2007, 156179) 

Species: Human, Rat 

Cell Type: A549, RLE6TN 

PM2.5: Collected by baghouse from 

Dusseldorf, Germany 

Particle Characterization: Carbon 20%, 

Hydrogen 1.4%, Nitrogen <0.5%, 

Oxygen 14.1%, Sulfur 2.1%, Ash 

63.2%. 

Particle Size: PM2.5 

Route: Cell Culture 

Dose/Concentration: 100 μg/cm2

Time to Analysis: 24 h 

Apoptosis: At 100 μg/mL for 24 h, PM induced a 

2.5 fold increase in apoptosis in A549. 

Mitochondrial Membrane Potential: A significant 

reduction in AEC mitochondrial membrane potential 

was observed. 

Caspase -3 & -9: Increased activity of both 

enzymes in both cell types was observed. More 

specifically, a 2- to 2.5-fold increase of caspase -3 

and -9 in A549 and an 8-fold increase of caspase-9 

and 4-fold increase of caspase-3 in RLE-6TN were 

observed. 

BIM: Downregulation of BIM by RNA interference 

inhibited PM-induced apoptosis. An inhibited 

decrease in mitochondrial membrane potential and 

activation of both caspases were observed. 

Reference: Zhang et 

al. (2004, 157183) 

Species: Mice 

Cell Line/Type: C10 

(alveolar Type II-like 

epithelial cell line) 

DEP: SRM 1650a 

Particle Size: NR 

Route: Cell Culture 

Dose/Concentration: 5 or 25 μg/mL 

Time to Analysis: 30-360 min 

fra Expression: DEP induces fra-1 but not fra-2 

expression. mRNA induction peaks around 180 min 

DEP affects fra-1 mRNA expression at the 

transcriptional level. 

ERK/JNK/p38 MAPK signaling pathways: 3 

inhibitors (PD-98059, SB-202190 or SP-600125) all 

reduced DEP stimulated fra-1 induction to near 

control levels. DEP stimulated phosphorylation of 

the MAPKs which peaks at 60 min but stays 

elevated at 180 min. 

MMP-9 promoter activity: fra-1 upregulation may 

play a role in DEP induced increases in MMP-9 

promoter activity as fra-1 appears to bind at the -79 

TRE sequence of the MMP-9 promoter. 

Table D-3. Respiratory effects: in vivo studies. 

Reference Pollutant Exposure Effects 

Reference: 

Adamson et al. 

(2003, 087943) 

Species: Rat 

Gender: Male 

Strain: SD 

Weight: 150 g 

PM10: EHC-93W (whole dust) 

EHC-93S (soluble) 

EHC-93L (leached) 

EHC-2KW, -S, -L 

Measured components Zn, Mg, 

Pb, Fe, Cu, Al 

Particle Size: EHC-93W, -93S, -

93L, -2KW, -2KS, -2KL: PM10 

Route: IT Instillation 

Dose/Concentration: 5 mg/rat; 33.3 mg/kg 

Time to Analysis: 4 h, 1 day, 3 day, 7 days, 14 

days 

BALF Cells: The greatest increase in cell numbers 

was observed with EHC-93W. Activity peaked at 1 day 

with a return to normal levels by 7 days. EHC-93L also 

induced an increase in cell numbers, more so than 

EHC-93S, but both particles induced statistically 

significant increases. However, these increases were 

mostly attributable to an increase in the AM and PMN 

populations. 

BALF Inflammatory/Injury Markers: Metalloproteinase (MMP) 2 and 9 both increased, peaking at 1 

day and 4 h respectively. MMP2 activity appears 

related to the soluble fraction whereas MMP-9 activity 

appears to be related to the leachable fraction. 

December 2009 D-81 
Reference Pollutant Exposure Effects 

Reference: Ahn et 

al. (2008, 156199) 

Species: Mouse 

Gender: Male 

Strain: BALB/C1 

Age: 6 wk 

Weight: 19-24 g 

DEP: Collected using a turbocharged, intercooler, 6-cylinder, 

heavy-duty, diesel engine (model 

year 2000 

DPBS: control 

Particle Size: NR 

Route: Oropharyngeal Aspiration 

Dose/Concentration: 1, 10, 25 mg/kg per day; 

Those receiving 25 mg/kg DEP also received pretreatment of Dex (1, 5 mg/kg) 1 h prior

Time to Analysis: 5 consecutive days; 72 h post 

final exposure 

BALF Inflammatory/Injury Markers: Lung injury was 

more severe in mice exposed to 25 mg/kg of DEP than 

when compared to mice exposed to 1 mg/kg DEP. 

However, lung injury caused by exposure to 25 mg/kg 

DEP could be completely prevented with pre-treatment 

of 5mg/kg Dex. Treatment with 1 mg/kg Dex prior to 

exposure to 25 mg/kg DEP depicted partial reduction in 

lung injury. 

BALF Cells: Treatment with DEP over a 5 day period 

caused an increase in total number of cells 

(macrophages, neutrophils and lymphocytes) when 

compared to control. 

Total Cells: Control - 5.33 ± 0.44 cells 

1 mg/kg DEP - 6.26 ± 0.87 cells 

10 mg/kg DEP - 14.40 ± 1.90 cells 

25 mg/kg DEP - 47.20 ± 3.40 cells 

COX-2 Expression: Exposure to DEP lead to a dosedependent increase in COX-2 levels; specifically, 

treatment with 25 mg/kg significantly increased COX-2 

levels. This effect was completely reduced by treatment 

with 5mg/kg of Dex. 

Reference: Ahsan 

et al. (2005, 156200) 

Species: Mouse 

Gender: Male and 

Female 

Strains: hTrx-1-

transgenic and 

C57BL/6 (control) 

Age: 8-8.5 wk 

DEP: Obtained from Dr. Masaru 

Sagai (Amori, Japan) 

Particle Size: NR 

Route: IT Instillation 

Dose/Concentration: Lung Damage: 0.1 

mg/mouse; Survival Analysis: 0.2 mg/mouse; ESR: 

0.05 mg/mouse 

Time to Analysis: 24 h 

ESR: hTrx-1 induced 0.05 mg generation of hydroxyl 

radicals in the lungs (mid thorax ESR spectra) 

compared to control. 

BAL Inflammatory/Injury Markers: hTrx-1 attenuated 

cellular damage from 0.1mg DEP. Control mice showed 

massive edema with neutrophilic infiltration, 

hemorrhagic alveolar damage and collapsed air 

spaces. hTrx-1 mice showed mild/moderate edema 

with clear demarcation of air spaces. 

Viability: After 4, 12 and 24 h, survival was 32, 24 and 

12% respectively as compared to 80, 52 and 40% for 

hTrx-1 mice. 

Reference: Andre et 

al. (2006, 091376) 

Species: Mouse 

Gender: Female 

Strain: BALB/cJ 

Age: 10-12 wk 

UFCP: Ultra Fine Carbon 

Particles (electric spark 

generator, Model GFG 1000; 

Palas, Karlsruhe, Germany) 

Measured Component: 

UFCP>96% EC 

Particle Size: 49 nm 

Route: Whole-body Inhalation 

Dose/Concentration: 380 μg/m3

Time to Analysis: 4 and 24 h; 0 and 24 h postexposure 

BALF Cells: A small increase in PMN number 

suggests a minor inflammatory response after 24 h 

exposure. Number of macrophages did not increase. 

BAL Inflammatory/Injury Markers: Total protein 

concentration significantly increased post 24 h 

inhalation. Post 4 h, heat shock proteins were induced. 

Post 24 h, immunomodulatory proteins (osteopontin, 

galectin-3 and lipocalin-2) significantly increased in 

alveolar macrophages and septal cells. 236 (1.9%) 

genes was increased and 307 (2.5%) genes were 

decreased with upregulated genes being primarily 

related to the inflammatory process. 

Reference: 

Antonini et al. 

(2004, 097199) 

Species: Rats 

Gender: Male 

Strain: SD 

Weight: ~250 g 

ROFA-P: Precipitator 

-S: Soluble (0.22 μm filter), 

Components: Fe, Al, Ni, Ca, Mg, 

Zn 

-I: insoluble, Components: Fe, 

Al, Ni, Ca, Mg, Zn, V 

-T: total 

ROFA-AH: Air Heater 

-S: Soluble (0.22 μm filter), 

Components: Fe, V, Ni, AL 

-I: Insoluble, Components: Fe, V, 

Ni, AL 

-T: Total 

Particle Size: < 3 μm (mean 

diameter) 

Route: IT Instillation 

Dose/Concentration: 1mg/100g bw in 300 μl 

saline; 60 mg/kg

Time to Analysis: 24 h; Clearance Experiment: 

two single exposures day 0 and 3 observed at day 

6, 8 and 10 

ESR: Only ROFA-P contained free radicals, primarily in 

ROFA-P-S. 

BALF Cells: No effects on alveolar macrophages were 

observed, but all ROFA-P fractions increased lung 

neutrophils. ROFA-P-S and ROFA-P-I effects combined 

roughly equaled ROFA-P-T. 

BAL Inflammatory/Injury Markers: ROFA-AH-T and 

ROFA-AH-I increased LDH. ROFA-P and -AH 

increased albumin for T and I fractions. 

Pulmonary Clearance (Listeria Monocytogenes):

ROFA-P-T and ROFA-P-S significantly slowed bacteria 

clearance from lungs. ROFA-AH and ROFA-P-I had no 

effect. 

December 2009 D-82 
Reference Pollutant Exposure Effects 

Reference: Arimoto 

et al. (2007, 097973) 

Species: Mouse 

Strain: ICR 

Gender: Male 

Age: 6 wk 

Weight: 29-33 g 

DEP (collected using a 4JB1 4-

cyl, 2.74L Isuzu diesel engine) 

DEP-OC: organic chemical 

extracts 

LPS 

DL = DEP + LPS 

DOL = DEP-OC + LPS 

Particle Size: 0.4μm 

Route: IT Instillation 

Dose/Concentration: DEP or DEP-OC: 4 mg/kg; 

LPS: 2.5 mg/kg; DL or DOL: NR 

Time to Analysis: 24 h 

Cytokines: DEP-OC or DEP alone did not change 

levels of MIP-1α, MCP-1 or MIP-2. DL induced 

significant increases in MIP-1, MIP-2 and MCP-1. 

LPS: LPS and DOL induced increases in MCP-1 

though the increase induced by DL was greater. No 

effect on MIP-1α or MIP-2 was observed. 

Reference: 

Bachoual et al. 

(2007, 155667) 

Species: Mouse 

Strain: C5B17

Gender: Male 

Age: 7 wk 

Weight: 22.3 ± 

073 g 

RER: PM10

Paris, France subway 

CB 

TiO2

DEP 

Particle Size: RER: 79% < 0.5 

μm; 20%: 0.5-1 μm 

CB: 95 nm 

TiO2: 150 μm 

DEP: NR 

Route: IT Instillation 

Dose/Concentration: 5, 50, 100 μg/mouse, 0.22, 

2.2, 4.5 mg/kg

Time to Analysis: 8 or 24 h 

BALF Cells: 100 μg RER and 100 μg DEP increased 

total cell count and neutrophil influx after 8 h and 

returned to normal by 24 h. Smaller doses of RER and 

DEP induced no effect. CB induced no effect. 

BAL Inflammatory/Injury Markers: 100 μg RER 

increased BALF protein after 8 h. No effect was 

observed after 24 h nor with smaller doses of PM. RER 

significantly increased MMP-12 mRNA level after 8 h 

and HO-1 total lung mRNA content. No effects on 

MMP-2 or -9 or TIMP-1 or -2 expression were 

observed. No effects from CB or DEP were observed. 

Cytokines: 100 μg RER increased BAL, TNF-α and 

MIP-2 protein content after 8 h. 

Reference: Batalha 

et al. (2002, 088109) 

Species: Rat

Gender: Male

Strain: SD 

Age: NR 

Weight: 200-250 g

CAPs (Harvard Ambient Particle 

Concentrator) 

Particle Size: Mean: 2.7 μm 

Route: Whole-body Inhalation 

Dose/Concentration: Range: 73.5-733 μg/m3

Time to Analysis: CAPs exposure 5 h/day, 3 days 

(consecutive). SO2 exposure to induce CB 5 h/day, 

5 days/wk, 6 wk. Killed 24 h postexposure.

Histopathology: CAPs slightly increased the wall 

thickness of small pulmonary arteries and edema in the 

adventitia and hyperplasia of the terminal bronchiole 

and alveolar ducts epithelium. 

L/W ratio: The L/W ratio decreased in CAPs-exposed 

rats as particle mass, Si, Pb, SO42-, EC and OC 

increased. Univariate analyses showed significant 

negative correlations between the L/W ratio and Si and 

SO42- in normal rats and Si and OC in CB rats. 

Multivariate analysis showed only Si to be significant in 

both groups.

Reference: Becher 

et al. (2007, 097125) 

Species: Mouse 

Strain: Crl/Wky 

(iNOS(-/-)) and 

C57Bl/6 

Gender: Male 

Age: 8-14 wk 

Weight: 25 g 

Suspended PM: SRM-1648 

Particle Size: NR 

Route: IT Instillation 

Dose/Concentration: 1.6 μg/lung; 64 mg/kg

Time to Analysis: 20 h 

Cytokines: In both wild and KO strains, all particles 

caused increases of IL-6, MIP-2 and TNF-α levels. 

NADPH-oxidase KO mice showed significantly lower 

levels of IL-6 and MIP-2 responses to SPM comparatively to wildtype. iNOS KO mice showed significantly 

reduced IL-6, TNF-α, MIP-2 responses to SPM 

comparatively to wildtype. 

Free Radicals: SPM induced significant increases in 

free radical formation in alveolar type 2 cells but could 

be inhibited by DPI. 

Reference: 

Bhattacharyya et al. 

(2004, 088095) 

Species: Mouse 

Strain: SD 

Weight: 200-250 g 

Douglas Fir Wood Smoke 

(generated by burning wood at 

400 ̊C in crucible oven) 

Particle Size: NR 

Route: Nose-only Inhalation 

Dose/Concentration: 25 g/mouse 

Time to Analysis: Various exposure periods (0, 5, 

1 0, 15, 20 min). Parameters measured after 24 h 

recovery period. 

Biochemical Parameters: Lipid peroxidation 

increased after 20 min of wood smoke inhalation as did 

Myeloperoxidase at 20 min. No effects were observed 

at other times or for total antioxidant status, reduced or 

oxidized glutathione. 

Antioxidant Enzyme Activities: No effect was 

observed. 

Histology: Dose-dependent damage progressing from 

loss of cilia (5 min), degeneration of mucosal 

epithelium, loss of mucosal epithelium to disrupted 

mucosal epithelium with submucosal edema and 

inflammation. Changes persisted for up to 4 days. 

December 2009 D-83 
Reference Pollutant Exposure Effects 

Reference: Cao et 

al. (2007, 097491) 

Species: Rat 

Strain: SH and 

WKY 

Age: 12 wk 

PM2.5 (Shanghai, China) 

Components: As, Cd, Cr, Cu, Fe, 

Ni, Pb, Zn, V, Ba, Se, Mg, Co, 

Mn 

Particle Size: PM2.5

Route: IT Instillation 

Dose/Concentration: 1.6, 8.0 and 40 mg/kg

Time to Analysis: Exposed 1/day for 3 days, 

sacrificed 24 h following last exposure 

BALF Cells: PM decreased macrophages and 

increased neutrophils and lymphocytes in a dosedependent manner. For the same exposed dose, WKY 

rats had a higher percentage than SH but a smaller 

percentage of neutrophils and lymphocytes. 

BAL Inflammatory/Injury Markers: LDH activity and 

TBARs increased a in dose-dependent manner. 

Notably, activity in SH rats was much higher than WKY 

at the same dose exposed for each dose level. 

Cytokines: PM induced pro-inflammatory cytokine 

release (IL-1β, TNF-α, CD44, MIP-2, TLR-4, OPN). 

Again, SH cytokine level was greater than WKY at all 

dose levels. PM induced anti-inflammatory cytokines 

CC16 and HO-1 in a similar manner but at much lower 

rate. 

Reference: Carter 

et al. (2006, 095936) 

Species: Rat, 

Mouse, Hamster 

Gender: Female 

(all) 

Strain: F-344 (rat), 

B6C3F1 (mouse), 

Syrian Golden 

(hamster) 

Age: 7-10 wk 

CB: Printex 90 

Particle Size: primary size: 17 

nm; 1.2-1.6 μm (aerosol 

aerodynamic diameter) 

Route: Whole-body Inhalation 

Dose/Concentration: 1, 7, 50 mg/m3

Time to Analysis: 6 h/day for 5 days/wk for 13 wk; 

1 day, 3 m, 11 m post-exposure 

Superoxide: Levels rose in all species at 50 mg dose. 

Hamsters had no increase at 7 and 1 mg doses. Mice 

also increased at 7 mg. Rats significantly increased at 

all dose levels. Rats maintained elevation except for 

the 50 mg dose at 11 mo postexposure; it declined but 

was still higher than control. Mice maintained elevation 

at 50 mg while 7 mg returned to control levels by 3 mo 

postexposure. 

H2O2: At 50 mg, increased levels in all species, with the 

highest in rat, were observed. At 7 mg, increased levels 

in rats and mice were initially seen but levels returned 

to baseline by 11 mo. Hamster levels were not 

significant. At 1 mg, no significant changes were 

observed. 

NO: Induced similar reactions as H2O2. Rat response 

continued through the study while mice and hamsters 

returned to baseline by 11 mo postexposure. Rats 

produced significantly higher levels at all times than 

other species. 

BALF Cells: CB induced significant increases in 

neutrophils at 7 and 50 mg for all species. Rats had the 

highest and most prolonged PMN response. Mice and 

hamsters had very similar reactions. 

Cytokines: TNF-α, MIP-2 and IL-10 increased in a 

dose-dependent manner in rats and mice. Hamsters 

increased for IL-10 only. MIP-2 levels were highest in 

rats. TNF-α level were similar in all three species at 50 

mg, but hamsters started with a markedly higher basal 

level. 

Glutathione Peroxidase: Hamsters were the most 

responsive with significant increases at all levels. Rats 

and mice increased at 50mg and continued to increase 

for up to 11mo. Hamster levels declined with time but 

continued to be higher than control. 

Glutathione Reductase: Rats increased only at 50mg 

and remained elevated for up to 11mo. Mice increased 

at 7 and 50mg and remained elevated for up to 11mo. 

Hamsters increased at all levels at 11mo, but at 50mg, 

levels only increased post 1 day. 

Superoxide Dismutase: All species reacted in a dosedependent manner. Rats were the least responsive. 

Rat SOD activity increased over time while rat and 

mouse activity decreased at 50mg. Data were 

consistent with cytokine data. 

Summary: Rats appear to produce proinflammatory 

responses while mice and hamsters produce 

antiinflammatory responses. 

December 2009 D-84 
Reference Pollutant Exposure Effects 

Reference: Cassee 

et al. (2005, 087962) 

Species: Rat 

Gender: Male 

Strain: Wistar Kyoto 

and SH/NHsd 

Age: 7 wk and 8-12 

wk 

CAPs: PM2.5 

Netherland suburban, industrial 

and freeway tunnel site 

collections 

Wistar rats pre-exposed to O3

SO4, NO3 and NH4 ions: 54 ± 4% 

suburban, 53 ± 7% industrial and 

35 ± 5% freeway site conc. of 

total CAPS mass 

Particle Size: PM2.5

(0.15<PM<2.5 μm) 

Route: Nose-only Inhalation 

Dose/Concentration: PM 365-3720 μg/m3 

(results from 16 different exposures 2000, 2002); 

O3: 1600 μg/m3 (0.8 ppm)

Time to Analysis: 8 h O3 pre-exposure; 6 h CAPS 

exposure; 48 h post-exposure 

BALF Cells: Wistar exhibited increased protein, 

albumin, NAG and decreased ALP activity and 

macrophage numbers. Wistar showed increased PMNs 

due to O3, but was not significantly increased with 

additional CAPs exposure. SH showed no effect of 

CAPS except for the increased PMNs. 

BAL Inflammatory/Injury Markers: No effect on AL, 

LDH, Glutathione, GSSG, GSH, Uric Acid was 

observed. 

Cytokines: No effect on IL-6, MIP-2 or TNF-α was 

observed. CAPs induced an increase in CC16 plasma 

of SH rats. 

Hematology: CAPS induced an increase in RBC, HGB 

and HCT of Wistar rats and fibrinogen of SH rats. 

Histology: Wistar and SH rats had no obvious lung 

abnormalities. Small changes include increased 

macrophages and cellularity of centriacinar septa of O3-

only rats. Both O3-only and O3+CAPS showed 

bronchial epithelium hypertrophy and perivascular 

influx of PMNs. 

BrdU Labeling Index of Terminal Bronchiolar 

Epithelium: No CAPs effects were observed. 

Reference: Chang 

et al. (2005, 097776) 

Species: Mouse 

Gender: Male 

Strain: ICR 

Age: 5 wk 

Weight: 25-30 g 

UFCB: Ultrafine Carbon Black - 

Printex 90 (Degussa) 

Particle Size: 14 nm 

Route: IT Instillation 

Dose/Concentration: 200 μg/100 uμ/mouse 

Time to Analysis: Parameters measured 4, 16, 

21, 42 h post single exposure 

BALF Cells: Neutrophil number was at control level at 

4 h, increased after 16 h, peaked at 21 h and returned 

to normal at 42 h. No effect was observed for the 

macrophage count. 

BAL Inflammatory/Injury Markers: UfCB increased 

total protein with peak at 21 h. TNF-α increased at 4 h 

and returned to normal at 16 h. 

VEGF (Vascular Endothelial Growth Factor):

Increased at 4 h and peaked at 16 h but remained 

elevated at 21 and 42 h. VEGF and total protein in 

BALF were correlated (R2 = 0.7352). 

ROS: Pretreatment with NAC (ROS inhibitor) 

decreased induction of BALF VEGF and total protein 

by UfCB but did not fully block its effect. 

Histology: Thickened alveolar walls in lungs of UfCBtreated mice 16 h post-IT was observed. 

Reference: Chang 

et al. (2007, 097475) 

Species: Mouse 

Gender: Male 

Strain: ICR 

Age: 5 wk 

Weight: 25-30 g 

UFCB: Ultrafine Carbon Black - 

Printex 90 (Degussa) 

Particle Size: 14 nm diameter 

Route: IT Instillation 

Dose/Concentration: 200 μg/mouse; 

8 mg/kg 

Time to Analysis: Pretreatment with NAC (Nacetylcysteine) ip 320 mg/kg, 2 h before UFCB IT 

instillation. Parameters measured 24 h post 

exposure. 

BALF Cells: Increased relative lung weight, total 

protein (2 fold), total cells (11 fold) and number of 

neutrophils were observed. BALF AM count was not 

affected. 

BAL Inflammatory/Injury Markers: Of the 33 

identified proteins, the following 6 were confirmed and 

validated: Cp (ceruluplasmin), albumin, EGFR, LIFR 

(leukemia inhibitory factor receptor), α2M and β-actin. 

All were increased following UFCB exposure. The 

following were also identified: 3 membrane proteins, 3 

intracellular proteins, 10 protease inhibitors and 6 

antioxidants. UfCB increased LIFR and EGFR in BALF. 

UfCB significantly reduced EGFR and LIFR in lung 

homegenate. UfCB did not affect EGFR protein but 

down-regulated LIFR in A549 cells treated with UfCB. 

Antioxidant: Pretreatment with NAC reduced the 

intensity of albumin and α2M bands in BALF as well as 

most other proteins. Statistical analysis showed 

positive correlation between VEGF and albumin 

(R2 = 0.796) and VEGF and α2M (R2 = 0.7331) in BAL. 

December 2009 D-85 
Reference Pollutant Exposure Effects 

Reference: Cho et 

al. (2005, 156344) 

Species: Mouse 

Gender: Male 

Strains: DBA/2J, 

129P3/J, C57BL/6J, 

BALB/cJ, A/J, 

C3H/HeJ, 

C3H/HeOuJ

Age: 6-8 wk 

ROFA: Obtained from Power unit 

4, Boston, MA 

Absent of LPS 

Particle Size: NR 

Route: IT Instillation 

Dose/Concentration: 6 mg/kg bw (150 μg in 50μl/ 

25 g) 

Time to Analysis: 24 h; Additional HeJ and OuJ 

mice: single: 1.5, 3 and 6 h (compare TLRmediated molecular events) 

BALF Cells: Significant genetic effects on number of 

macrophages and PMNs after ROFA challenge. For 

PMNs, DBA/2J, C57BL/6J, BALB/cJ, and 129P3/J all 

induced increases significantly higher than C3H/HeJ. 

For macrophages, only the A/J strain induced 

increases significantly higher than C57BL/6J. Total 

protein, PMNs and macrophages all increased with 

HeOuJ inducing increases significantly different from 

HeJ. 

BAL Inflammatory/Injury Markers: Significant genetic 

effect on mean total protein concentration was 

observed. In decreasing order, DBA/2J, 129P3/J and 

C57BL/6J all induced increases significantly higher 

than C3H/HeJ. 

TLR4 mRNA Expression: A significant decrease was 

observed in TLR4 transcript level in HeJ- ROFA 

exposed mice post 1.5 h. Post 6 h, TLR4 levels were 

greater than the control levels. OuJ expression 

increased beginning 1.5 h post exposure. 

TLR4 Protein Level: Protein level of OuJ mice 

significantly exceeded (~2-3 fold) HeJ mice at 1.5, 3 

and 6 h. 

Activation of Downstream Signal Molecules:

Greater activation of MYD88, TRAF6, IRAK-1, NF-KB, 

MAPK, and AP-1 was observed in OuJ mice than in 

HeJ mice before the development of ROFA- induced 

pulmonary injury. 

Cytokines: IL-1β, LT-β, IL-1α, IL-7, IL-13, IL-16 

increased in both strains (OuJ and HeJ). Levels of all 

cytokines above were significantly higher in OuJ than in 

HeJ. 

Reference: Churg et 

al. (2003, 087899) 

Species: Human 

Gender: Female 

(Mexico City); Male, 

Female (Vancouver) 

Age: 66 ± 9yr 

(Mexico City); 76 ± 

11yr (Vancouver) 

Weight: NR

PM (Mexico City- high PM 

region, Vancouver- low PM 

region) 

Particle Size: Geometric mean 

size of individual particles in 

tissue: 0.040-0.067 μm; 

Aggregates in tissue: 0.34-0.54 

μm; Mexico City: 2.5, 10 μm 

Route: Ambient Air Exposure. Autopsy Tissue. 

Dose/Concentration: 10 - >1000×106 g dry 

tissue; Mexico City: PM10: 66 μg/m3, Vancouver: 

PM10: 25 μg, PM2.5: 15 μg 

Time to Analysis: Lung samples taken from 

deceased lifelong Mexico City residents and 

Vancouver residents >20 yr. Subjects were neversmokers, did not work in dust occupations or cook 

with biomass fuels.

The lungs from Mexico City residents showed 

increased muscle and fibrous tissue in the 

membranous bronchioles and respiratory bronchioles 

compared to the Vancouver residents. Pigmented dust, 

lumental distortion and carbonaceous aggregates of 

UFPs were present in the Mexico City lungs. 

Reference: Costa et 

al. (2006, 088438) 

Species: Rat 

Gender: Male 

Strain: SD 

Age: 60 day 

ROFA 

FP&L plant #6 oil, 1% sulfur 

Particle Size: ~1.95 μm 

Route: IT Instillation, Nose-only Inhalation (IH) 

Dose/Concentration: IT instillation = 110 μg/rat 

IH = 12 mg/m3

Time to Analysis: IT instillation: single; IH: 6 h 

24, 48, 96 h (histopathology 24 and 48 only) 

ROFA distribution: IH and IT instillation resulted in 

equivocal distribution (μg/g lung tissue) in 5 different 

lung lobes. 

Airway Hyperactivity: IT instillation resulted in 

doubled airway hyperreactivity at 24 h which was 

sustained for 96 h. IH hyperreactivity did not reach 

statistically significant level. 

BALF Cells: Neutrophils peaked at 24 h and slowly 

declined at 48 and 96 h. 

BAL Inflammatory/Injury Markers: IH and IT 

instillation showed very similar responses (R2 = 0.98). 

Time-dependent increases were observed for protein 

and LDH. 

Lung Pathology: IT instillation showed more alveolitis, 

bronchial inflammatory and fibrinous fluid infiltrate. IH 

showed relatively more congestion of small airways 

and alveolar hemorrhage. 

December 2009 D-86 
Reference Pollutant Exposure Effects 

Reference: Courtois 

et al. (2008, 156369) 

Species: Rat 

Gender: Male 

Strain: Wistar Kyoto 

Age: 12-14 wk 

Weight: NR

 PM (SRM 1648; 63% inOC, 4-

7% OC, >1% mass fraction- Si, 

S, Al, Fe, K, Na) 

Carbon black (FW, P60) 

UF, fine TiO2

Particle Size: PM mean 

diameter: 0.4 μm; Carbon black: 

FW- 13 nm, P60- 21 nm; TiO2

mean diameter: 0.14 μm 

Route: IT Instillation 

Dose/Concentration: 5 mg PM or TiO2

Time to Analysis: 6-72 h 

Particles were present in lung parenchyma that was 

removed 12 and 72 h post-instillation. 

Reference: Dick et 

al. (2003, 036605) 

Species: Mouse 

Gender: Female 

Strain: CD1 

Age: 8-10 wk 

Weight: 20-25 g 

CO: PM Coarse 

FI: PM Fine 

FU: PM ultrafine 

PM collected in RTP, NC 

Particle Size: CO: 3.5-20 μm;

FI: 1.7- 3.5 μm; FU: <1.7 μm 

Route: IT Instillation 

Dose/Concentration: 10 μg, 50 μg, 100 

μg/mouse; 0.5, 2.5, 5.0 mg/kg 

Time to Analysis: DMTU 500 mg/kg bw 30 min 

pre-exposure for some mice. Parameters 

measured 18 h post-exposure. 

Particle Characteristics: S increased (CO-33.20 

μg/mg, FI- 49.44 μg/mg FU- 122.79 μg/mg) with 

decreasing particle size (mostly in the water-soluble 

fraction). Fe and Cu higher in coarse and fine fractions 

(mostly present in the insoluble). CO PM contained 

more nickel (in both soluble and insoluble) than FI or 

FU particles. Also, endotoxin levels similar in CO and 

FI; much lower in FU (0.165 EU/mg). 

BALF Cells: PMN increased with exposure for all 3 

fractions except 100 μg FI. 

BAL Inflammatory/Injury Markers: Albumin increased 

only at 100 μg FI. No differences in NAG or LDH 

observed. 

Cytokines: IL-6 increased at 100 μg dose for all 3 

fractions with similar responses. TNF-α increased a 

100 μg dose of fine PM vs control. 

Effect of PM After Pre-treatment w/DMTU: Systemic 

administration of DMTU alone depicted a two-fold 

increase in total antioxidant capacity. 

DMTU halved neutrophil response observed with 

PMs alone: No fractions were increased over DMTU 

alone which was at least two-fold saline control. IL-6 

concentrations were drastically reduced in the DMTU 

group for the mice exposed to coarse particles (all 

fractions were reduced but only coarse had a 

significant response). TNF-α levels were decreased 

after treatment with particles and DMTU but treatment 

with particles and saline (control) produced similar 

results. 

Reference: Dybdahl 

et al. (2004, 089013) 

Species: Mouse 

Gender: Female 

Strain: BALB/CJ or 

trans-genic 

(MutaMouse) 

Age: 9-10 wk 

Weight: ~20 g 

DEP: SRM 1650 (NIST) 

Particle Size: DEP: NR; Control: 

PM 0.13 μm diameter 

Route: Nose-only Inhalation 

Dose/Concentration: I: 20, 80 mg/m3

II: 5, 20 mg/m3

Time to Analysis: I: single exposure 90 min; II: 90 

min/day for 4 days; I & II: parameters measured 1, 

3, or 22 h post exposure 

Cytokines: A single 90 min DEP exposure increased 

IL-6 gene level dose-dependently in the lung. For 80 

mg/m3

 DEP, significantly higher IL-6 gene level was 

observed, both 1 and 22 h post exposure. For 20 

mg/m3

 DEP, a significantly higher IL-6 level was 

observed at 1 h post exposure but normalized at 3 h. 

BALF Cells: Inhalation of DEP did not decrease 

viability of BALF cells. For mice exposed to 20 mg/m3

DEP, at 1 h post exposure in BAL fluid there was 3 fold 

increase in total cell number. 

DNA Damage: Level of 8-oxodG increased post single 

exposure with 80 mg/m3

 inducing levels significantly 

higher than controls. Repeated exposures were 

associated with significantly higher DNA strand breaks. 

Reference: Elder et 

al. (2004, 055642) 

Species: Rat 

Gender: Male 

Strain: F344, SH 

Age: 23 m (Fisher), 

11-14 m (SH) 

UFP: argon-filled chamber with 

electric arc discharge (TSI, Inc., 

St. Paul, MN) 

Particle Size: 36 nm 

Route: Whole-body Inhalation. 

Dose/Concentration: UFP: 150 μg/m3 bw; LPS: 2 

mg/kg 

Time to Analysis: 6 h, 18 h 

BALF Cells: Neither inhaled UFP nor LPS cause a 

significant increase in BALF total cells or percentage 

of neutrophils in either rat strain. No significant 

exposure-related alteration in total protein 

concentration was observed. In both rat strains LPS 

induced a significant increase in the amount of 

circulating PMNs. When combined with inhaled UFP, 

PMNs decreased; for F-344 rats, this decrease was 

significant. 

ROS in BALF: In F-344 rats, both UFP and LPS have 

independent and significant effects on DCFD oxidation. 

Effects were in opposite directions; particles decreased 

ROS whereas LPS increased ROS. 

December 2009 D-87 
Reference Pollutant Exposure Effects 

Reference: Elder et 

al. (2004, 087354) 

Species: Rat 

Gender: Male 

Strain: F344 

Age: 21 mo 

Freshly generated vehicle 

exhaust emissions from I-90 

between Rochester and Buffalo, 

NY 

Particle Size: NR

Route: Whole-body Inhalation; IT Instillation 

(Influenza) 

Dose/Concentration: Vehicle exhaust: 0.95-

3.13×105

 particles/cm3

Endotoxin: 84 EU 

Influenza (IV): 10, 000 EID 50 in 250 μl

Time to Analysis: 1×6 h, 3×6 h or both. 

Parameters measured 18 h post-exposure. 48 h 

prior to on-road exposures, instilled intratracheally 

with IV. Immediate pre-exposure of priming agent 

endotoxin. 

EXPERIMENTS 

1: LPS + PM 6 h 

2: LPS + PM 6 h, 3×6 h 

3: IV + PM 6 h 

4: IV + PM 6 h, 3×6 h 

No departures from normal baseline cellular or 

biochemical values were observed, suggesting that onroad exposures were well tolerated by the rats. 

BAL Inflammatory/Injury Markers: Increase in total 

protein concentration, LDH and B-glucuronidase 

activities were observed. 

Specific results according to groups 1-4 are as 

follows: 

Experiment 1: No endpoints revealed significant 

differences between groups of rats exposed to gas 

phase only versus the gas-phase/particle mixture. 

Experiment 2: Combination of endotoxin and particles 

produced greater inflammatory responses than those 

treated with saline and particles post 1 day. After 3 

days, no statistically significant changes were noted. 

Experiment 3: Influenza virus significantly increased 

ROS release in BALF cells. 

Experiment 4: Influenza virus significantly increased 

both percentage of PMNs in BALF and BALF cell ROS 

release. 

Reference: Elder et 

al. (2005, 088194) 

Species: Rat, 

Mouse, Syrian 

Golden Hamster 

Gender: Female 

Strain: F-344, 

B6C3F1, FIB 

HSCb: Printex-90 high surface 

area carbon black, DegussHuels (Trostberg, Germany). 

LSCb: Sterling V, low surface 

area carbon black, Cabot 

(Boston, MA) 

Particle Size: HSCb = 14 nm, 

LSCb = 70 nm 

Reference: Whole-body Inhalation 

Dose/Concentration: 0, 1, 7, 50 mg/m3 HSCb; 50 

mg/m3

 LSCb (rats only)

Time to Analysis: 6 h/day, 5 daus/wk for 13 wk. 

Parameters measured 1 day, 3 mo, 11 mo postexposure 

Body Weight: Environmental changes pre and postexposure affected test subjects’ life spans, particularly 

hamsters. Hamsters also experienced significant loss 

of body weight when exposed to high doses of HSCb. 

Effects of Carbon Black: In rats, lung weight of the 

high dose HSCb doubled. After 11mo, analysis of all 

lungs showed no significant difference. Mice had the 

highest relative lung burdens at the end of exposure 

time but also cleared particles faster at high doses than 

rats. However, clearance slowed over the 11mo 

recovery period, especially in high dose mice. 

Hamsters showed significant elevations in lung carbon 

black burden for all exposures at all time points. 

Hamsters exposed to high dose HSCb exhibited 

impaired clearance. 

BALF Cells: Presence of PMNs was limited to the mid 

and high dose groups. Overall maximal response was 

reached in mice and hamsters, but not in rats with 

increasing mass dose of HSCb. 

Reference: Evans 

et al. (2006, 097066) 

Species: Rat 

Gender: Male 

Strain: SD 

DEP: collected under dry, 

outdoor, ambient conditions from 

tractor exhaust pipe (1985, 

Japanese ISEKI 1500 cc tractor) 

burning Esso 2000 diesel and 

20/30 mixture of Esso light 

engine oil. 

10% UF, 90% fine 

Cabosil: amorphous silicon 

dioxide 

16% UF, 84% fine 

Particle Size: DEP: 30 nm; 

Cabosil: 7 nm 

Route: IT Instillation 

Dose/Concentration: 1 mg/rat DEP; 1 mg/rat 

Cabosil 

Time to Analysis: Pretreatment with 0.5 unit of 

bleomycin; IT 3 or 7 days 

after pre-treatment; 1wk post-IT 

Lung permeability: In bleomycin-treated group, 

obvious inflammatory status and edema within the lung 

was observed. This was shown by significant increases 

in acellular protein and free cells. 

Changes in lung: Body weight ratio, lung surface 

protein content, free cell counts, and apical surface 

protein of rat type I cells were only altered by 

bleomycin treatment and not particle exposure. 

December 2009 D-88 
Reference Pollutant Exposure Effects 

Reference: Finnerty 

et al. (2007, 156434) 

Species: Mouse 

Gender: Male 

Strain: C57BL/61 

Age: 12 wk 

Weight: 24.3 ± 0.3 g 

Coal Fly Ash (generated at U.S 

EPA National Risk Management 

Research Laboratory by burning 

Montana subbituminous coal 

under conditions simulating fullscale utility boiler conditions) 

Transition metals of Coal Fly 

Ash: Fe, Mg, Ti, Mn, V 

Particle Size: >PM2.5

Route: IT Instillation 

Dose/Concentration: PM: 200 mg/mouse; 9.1 

mg/kg 

PM+LPS10: 200 mg PM+10 mg LPS 

PM+LPS100: 200 mg PM+100 mg LPS 

LPS: 100 μg 

Time to Analysis: 18 h 

BALF Cells: No significant differences in platelet 

concentration or white blood cell count in any groups 

were observed. The percentage of neutrophils increased significantly with PM+LPS100. PMN rose in 

PM groups and increased further with LPS treatment. 

Increases in PM+LPS were groups statistically 

significant. More leukocytes were present in the 

alveolar space in PM+LPS10 compared to the PM 

group. The most severe response was in the 

PM+LPS100 group. 

Cytokines: Plasma TNF-α and IL-6 significantly 

increased for the PM+LPS100 group. An additive effect 

of LPS and PM for IL-6 was observed. For saline and 

PM groups, pulmonary TNF-α was below detection 

range. A synergistic effect for TNF-α was observed. A 

less than additive effect for IL-6 was observed. 

Pulmonary TNF-α significantly increased in the PM+ 

LPS100 group. Pulmonary IL-6 significantly increased 

in both PM+LPS groups. 

Reference: Fujimaki 

et al. (2006, 096601) 

Species: Mouse 

Gender: Male 

Strain: IL-6(-/-) and 

WT: B6J129Sv 

(control) 

Age: 5-6 wk 

DEP: collected from a 4-cylinder, 

2.74 L, Isuzu diesel engine 

Particle Size: 0.4 μm 

Route: Whole-body Inhalation 

Dose/Concentration: 1.0, 3.0 mg/m3 

Time to Analysis: 12 h/dayfor 4 wk. Parameters 

measured 1 day post-exposure 

BALF Cells: Treatment significantly increased BAL 

cells from WT mice at both dose levels. The increase of 

macrophages and neutrophils were dose-dependent. 

An increase in lymphocytes were present in WT mice 

with the low dose. No significant increase in cells were 

observed from IL-6 (-/-). 

Cytokines: TNF-α largely increased in IL-6(-/-) mice 

exposed to 3 mg/m3

 compared to WT mice. IL-6 

production increased in WT mice exposed to 3 mg/m3. 

CCL3 increased in both WT and IL-6(-/-) at high dose. 

IL-1β remained at the control level. 

Reference: GerlofsNijland et al. (2005, 

088652) 

Species: Rat 

Gender: Male 

Strain: SH/NHsd 

Age: 11-12 wk 

Weight: 250-350 g 

RTD: road tunnel dust (obtained 

from a Motorway tunnel in 

Hendrik-Ido-Ambacht, Netherlands) 

EHC-93 

(Ottawa, Canada) 

Particle Size: Coarse: 2.5- 10 

μm; fine: 0.1- 2.5 μm 

Route: IT Instillation 

Dose/Concentration: 0.3, 1, 3, 10 mg/kg; EHC93: 10 mg/kg

Time to Analysis: 4, 24, 48 h 

BALF Cells: PMN significantly increased in RTD (3 

and 10 mg/kg dose) and EHC-93 exposed animals at 

24 h and decreased by 48 h but remained statistically 

significant. AM numbers decreased for 3 mg/kg RTD 

group at 4 h. 

BAL Inflammatory/Injury Markers: Myeloperoxidase 

(measured at 24 h in 1, 3, 10 mg/kg RTD groups) was 

elevated in a dose-dependent manner. RTD induced 

time-dependent increases in LDH activity at 24 and 48 

h, although these increases were less than EHC-93 

values at the same time points. Alkaline phosphatase 

increased dose-dependently for RTD at 48 h. GSH 

decreased at 24 h to approximately the same levels in 

0.3, 1, and 3 mg/kg RTD dose groups. Uric acid only 

decreased in 1 mg/kg RTD group at 24 h. 

Cytokines: IL-6 levels were elevated only at 10 mg/kg 

for RTD and EHC-93 at 4 and 24 h; it remained 

elevated for EHC-93 at 48 h. A dose-dependent 

increase in TNF-α at 4 h for RTD was observed. TNF-α

levels remained elevated only for the 10 mg/kg groups 

at 24 h and returned to control levels by 48 h. A dosedependent increase in MIP-2 for all RTD dose groups 

were observed and remained elevated through 48 h for 

both PM types (although values were returning to 

control levels). 

Pulmonary Histopathology: A dose-dependent 

increase in the number of inflammatory foci at 24 and 

48 h for 3 and 10 mg/kg RTD groups was observed. 

The response was even greater for the EHC-93 

exposed group at similar time points. 

December 2009 D-89 
Reference Pollutant Exposure Effects 

Reference: GerlofsNijland et al. (2007, 

097840) 

Species: Rat 

Gender: Male 

Strain: SH/NHsd 

Age: 13 wk 

Weight: 250-350 g 

PM samples collected from: 

1. MOB high traffic density 

2. HIA high traffic density 

3. ROM high traffic density 

4. DOR moderate traffic density 

5 MGH low traffic density 

6 LYC low traffic density 

Particle Size: Coarse: 2.5 - 10 

μm; Fine: 0.1 - 2.5 μm 

Route: IT Instillation 

Dose/Concentration: 3, 10 mg/kg 

Time to Analysis: 24 h 

BALF Cells: Pulmonary inflammation was induced in a 

significant and dose-dependent manner for both dose 

levels. Inflammation in the BALF included airway 

neutrophilia, increased macrophage numbers and mild 

lymphocytosis. Both coarse and fine PM caused dosedependent alveolitis. Fine PM from LYC (10 mg/kg 

dose) also caused some bronchiolitis. 

BAL Inflammatory/Injury Markers: LDH was 

significantly increased for all doses of coarse PM and 

for the high dose of fine PM. BALF protein 

concentration was observed predominantly at the high 

dose of coarse PM. Location ROM had evidence of 

attenuated responses with fine PM. Ascorbate concentrations were reduced but were only significant for rats 

exposed to the highest dose of coarse PM fractions 

from the locations MOD, HIA, and LYC. 

Cytokines: TNF-α concentrations increased for all 

coarse samples with the exception of DOR and LYC. 

Fine PM induced similar responses for all sites. MIIP-2 

concentrations increased only at certain sites for 

coarse but not fine PM. 

Location-related Differences: Coarse PM from MOB, 

HIA and MGH induced higher LDH responses than 

other locations. Coarse PM from HIA produced BALF 

protein concentrations higher than LYC and ROM. 

MGH induced greater amounts of BALF protein than 

ROM. Coarse PM from LYC lowered fibrinogen values 

more than PM from location MOB, HIA, and MGH. Fine 

PM showed less differences among the various sites. 

Particle Correlation: Fine PM exhibited significant 

correlation between zinc content and BALF cytotoxicity 

markers protein and LDH - mainly from HIA. Fine PM 

also exhibited positive correlations with copper and 

barium. Coarse PM showed positive correlation with 

barium and copper, mainly from MOB. 

Reference: GerlofsNijland et al. (2009, 

190353) 

Species: Rat 

Gender: Male 

Strain: SH 

Age: 12 wk 

Weight: 200-300 g

PM (Prague, Czech Republic; 

Duisburg, Germany; Barcelona, 

Spain) (Prague and Barcelona 

coarse PM organic extracts) 

Particle Size: Coarse: 2.5-10 

μm, Fine: 0.2-2.5 μm 

Route: IT Instillation 

Dose/Concentration: 7mg/kg 

Time to Analysis: 24 h 

Cytotoxicity (LDH, protein, albumin) and inflammation 

(NAG, MPO, TNF-α were increased by PM, and were 

greatest in the coarse PM fraction. Metal-rich PM had 

greater inflammatory and cytotoxic effects. PAH content 

influenced greater inflammation (including neutrophils), 

and cytotoxicity. Generally, whole PM and coarse PM 

were more potent than organic extracts and fine PM, 

respectively.

Reference: Ghio et 

al. (2005, 088272) 

Species: Rat 

Gender: Male 

Strain: N8 b/b 

Belgrade rats and 

N8+ lb Belgrade 

controls 

Oil Fly Ash (Southern Research 

Institute, Birmingham, AL) 

Particle Size: 1.95 ± 0.18 μm 

(MMAD) 

Route: IT Instillation 

Dose/Concentration: 500 μg/rat; 2 mg/kg 

Time to Analysis: 24 h 

BALF Cells: Homozygous Belgrade with mutation 

G185R had higher levels of Fe and V 24 h postexposure. This may demonstrate a decreased ability to 

remove Fe and V from the lower respiratory tract than 

heterozygous +lb littermates. This also indicates that 

DMT1 is normally responsible for at least some Fe and 

V uptake; thus, a defective DMT1 transports less. 

BAL Inflammatory/Injury Markers: Increased protein 

and LDH concentrations in the homozygous strain were 

observed when compared to control 

Reference: Ghio et 

al. (2005, 088275) 

Species: Rat 

Gender: Male 

Strain: SD 

Age: 60 day 

Weight: 250-300 g 

Ferric ammonium citrate (FAC) 

Vanadyl sulfate (VOSO4) 

Particle Size: NR 

Route: IT Instillation 

Dose/Concentration: 0.5 mL 100 μm FAC/rat; 0.5 

mL 10 μm VOSO4/rat; 500 μg oil fly ash; 2 mg/kg 

Time to Analysis: Single or double exposure with 

24 h rest period. Parameters measured 15, 30, 60 

min, 24 h post-exposure. 

DMT1 Immunohistochemistry and Lung Injury: FAC 

increased and VOSO4 decreased -IRE DMT1 staining. 

Same exposures had no effect on +IRE DMT1. -IRE 

DMT1 expression in macrophages, airway and alveolar 

epithelial cells increased with increased Fe exposure. 

Vanadium nearly eliminated staining except in alveolar 

macrophages. Increased metal clearance with preexposure to FAC. Less metal clearance with preexposure to VOSO4. Pre-exposure to iron diminished 

lung injury whereas pre-exposure to vandium increased 

lung injury after oil fly ash instillation. Lung injury 

measured by concentration of protein and LDH in BAL. 

December 2009 D-90 
Reference Pollutant Exposure Effects 

Reference: Gilmour 

et al. (2007, 096433) 

Species: Mouse 

Gender: Female 

Strain: BALB/c 

Age: 10-12 wk 

Weight: 20-22 g 

PM - CO, FI, UF 

(obtained from U.S. Seattle (S), 

Salt Lake City (SL), South Bronx 

(SB), Sterling Forest (SF)) 

SB: included 35% sulfate, 22% 

gasoline, diesel and brake wear. 

SF: 48% sulfate. 

SL: 34% wood combustion and 

28% sulfate 

S: 39% wood combustion and 

29% sulfate 

Residual oil combustion and soil 

dust less than 5% for all sites. 

Particle Size: CO: 2.5-10 μm; 

FI: ≤ 2.5 μm; UF: ≤ 0.1 μm 

Route: Oropharyngeal Aspiration 

Dose/Concentration: 25 μg or 100 μg PM; 1.25 or 

5 mg/kg 

Time to Analysis: 18 h 

BALF Cells: PMN increased with the high dose of CO 

samples from SB, SL, S, but not SF. No significant 

increases from FI were observed, though the high dose 

induced increased PMN. UF from SL caused a highly 

variable response. 

BAL Inflammatory/Injury Markers: Seattle CO 

fractions showed no dose-dependent effect on protein 

concentration. Results for other locations were 

distinctly higher with 100 μg dose than 25 μg and 

saline doses. SL CO high dose induced the most 

significant increase. LDH response was weakly doserelated. Only SB showed a statistically significant 

increase for LDH with the high dose UF. 

Cytokines: MIP-2 was similar to PMN response. SB 

CO induced the most significant response. SL UF was 

highly variable. 

Particle Characteristics: LPS was higher in S (CO, FI, 

UF) and SL (CO, FI, UF). Zn levels were highest in SB 

(CO, FI, UF). Fe was higher in all CO and FI samples 

with SB CO inducing the highest. 

Reference: 

Gilmour et al. (2004, 

057420) 

Species: Mouse 

Gender: Female 

Strain: CD1 

Age: 8-10 wk 

Weight: 20-25 g 

Coal Fly Ash 

MU: Montana Ultrafine 

MF: Montana Fine 

MC: Montana Coarse 

KF: W. Kentucky Fine 

KC: W. Kentucky Coarse 

Particle Characteristics: 

Montana Sulfur 0.83%, Ash 

11.72%. Trace amounts of Ba, P, 

Sr, V, Nb, Cd, Se, Ga, Cu. 

Depleted in Si, Al, Fe, Mg, Ti. 

Kentucky Sulfur 3.11%, Ash 

8.07% 

Particle Size: Coarse: >2.5 μm; 

Fine: <2.5 μm; 

Ultrafine: <0.2 μm 

Route: Oropharyngeal Aspiration 

Dose/Concentration: 25 ug or 100 μg/mouse 

Time to Analysis: 18 h 

BALF Cells: PMN highly increased for MU at both 

doses. The level was comparable to the positive 

control. PMN also increased with KF at high dose. 

Coarse particles caused no significant increase in 

PMN. Number of macrophages did not change, but 

NAG increased significantly with MU for both dose 

levels and with KF and MF at high dose level. 

BAL Inflammatory/Injury Markers: Total protein and 

LDH was not significantly elevated. Albumin 

concentration increased significantly after treatment 

with the fine high dose of both particle types. 

Cytokines: MU particles caused a significant increase 

in TNF-α. MIP-2 increased in all fine and ultrafine PMinstilled animals with the highest in the MU and KF at 

both doses. IL-6 was detectable only in the BALF of 

MU and KF with substantial variability. The IL-6 levels 

were not significant. 

Reference: Gilmour 

et al. (2004, 087948) 

Species: Rat 

Gender: Male 

Strain: SH/NQIBR, 

WKY 

Age: 12 wk 

Weight: 280-340 g 

PM (collected from precipitator 

unit of an oil burning power plant 

in Boston) 

Measured Components of PM: 

S, Zn, Ni, V, Al, Cu, Pb, Fe, Ca, 

Na, K, Mg, Endotoxin 

Particle Size: NR 

Route: IT Instillation 

Dose/Concentration: 0.0, 0.83, 3.3, and 8.3 

mg/kg in SH rats; 0.0 or 3.3 mg/kg in WKY and SH 

rats

Time to Analysis: 24 h 

BALF Cells: No increase in macrophage number was 

observed in either rat strain following saline or PM 

exposure at 24 h. 

BAL Inflammatory/Injury Markers: LDH activity 

increased in a dose-related manner; this was observed 

in SH rats after exposure to 0.83, 3.33 and 8.3 mg/kg 

PM. SH rats showed greater lung permeability following 

PM exposure than WKY rats. SH rats showed acute 

lung inflammatory response after exposure to PM when 

compared to WKY rats. 

Cytokines: MIP-2 mRNA expression increased 

significantly in SH PM exposure group only. No 

significant differences in TNF-α RNA expression in 

either WKY, SH rats or control treatment groups were 

observed. 

CD14: A significant increase in lung CD14 protein was 

observed only in SH rats exposed to PM. 

TLR4: A significant increase in TLR4 protein in SH rats 

exposed to PM was observed. 

NF-κB: A significant increase in NF-κB binding protein 

in the nuclei of SH rats exposed to PM was observed. 

This effect was not observed in the control of PMexposed WKY rats. 

December 2009 D-91 
Reference Pollutant Exposure Effects 

Reference: Gilmour 

et al. (2004, 054175) 

Species: Rat 

Gender: Male 

Strain: Wistar Kyoto 

Age: 12 wk 

ufCB: Ultrafine carbon black 

(Printex 90 (Degussa) 

CB: (Huber 990, HR. Haeffner 

and Co) 

Particle Size: ufCB: 14 nm; CB: 

260 nm (primary particle 

diameter) 

Route: Whole-body Inhalation 

Dose/Concentration: ufCB: 1.66 mg/m3

fCB: 1.40 mg/m3

Number concentrations 

ufCB: 52380 particles/cm3

fCB: 3800 particles/cm3

Time to Analysis: Exposed for 7 h. Sacrificed 0, 

16 or 48 h post-exposure. 

BALF Cells: Total number of cells increased 

significantly in UfCB-exposed rats at 0 and 16 h. 

Recruitment of cells did not occur in response to CB 

exposure. PMNs increased significantly in the BALF of 

ufCB-exposed rats at 16 h. Leukocytes remained 

unchanged following CB exposure but increased 

significantly at 0 and 48 h post exposure to ufCB. 

Cytokine mRNA: A significant increase in BALF MIP-2 

mRNA expression was observed at 48 h. No 

differences in MIP-2 mRNA levels were observed in the 

whole lung tissue. 

Reference: 

Godleski et al. 

(2002, 156478) 

Species: Rat 

Gender: Male 

Strain: SD

Age: NR 

Weight: 200-250 g

CAPs (Boston; Harvard Ambient 

Particle Concentrator) 

Particle Size: 0.27 ± 2.3 μm 

(diameter) 

Route: Whole-body Inhalation 

Dose/Concentration: 73.5-733 μg/m3 

Time to Analysis: Exposed 5 h/days, 3 days 

(consecutive). BAL 24 h post-exposure 

BALF Cells and Inflammatory Markers: PMNs 

significantly increased with CAPs exposure and also in 

relation to CAPs mass, Br, SO4

2-, EC, OC and Pb. An 

overall increase in pro-inflammatory mediators and 

decrease in immune enhancer and evidence of 

vascular endothelial responses occurred with CAPs 

exposure.

Reference: 

Gottipolu et al. 

(2009, 190360) 

Species: Rat 

Gender: Male 

Strain: Wistar 

Kyoto, SH 

Age: 14-16 wk 

Weight: NR

DE (30-kW (40hp) 4-cylinder 

indirect injection Deutz diesel 

engine) (O2- 20%, CO- 1.3-4.8 

ppm, NO- <2.5-5.9 ppm, NO2- 

<0.25-1.2 ppm, SO2- 0.2-0.3 

ppm, OC/EC- 0.3 ± 0.03) 

Particle Size: Number Median 

Diameter: Low- 83 ± 2 nm, High88.2 nm; Volume Median 

Diameter: Low- 207 ± 2 nm, 

High- 225 ± 2 nm 

Route: Inhalation 

Dose/Concentration: Low- 507 ± 4 μg/m3, High2201 ± 14 μg/m3

Time to Analysis: Exposed 4 h/day, 5 days/wk, 4 

wk. Necropsied 1 day post-exposure.

DE increased neutrophils in a concentration-dependent 

manner, and GGT activity at the high dose. Particleladen macrophages were found in DE-exposed rats. 

Reference: 

Gunnison and Chen 

(2005, 087956) 

Species: Mouse 

Gender: Male 

Strain: DK (ApoE-/-, 

LDLr-/-) 

Age: 18-20 wk 

CAPS 

(Northeastern regional background) 

Ambient air copollutants 

measured O3, NO2

Particle Size: 389 ± 2 nm 

Route: Whole-body Inhalation 

Dose/Concentration: CAPS = 131 ± 99 μg/m3

including O3 = 10 ppb and NO2 = 4.4 ppb 

Time to Analysis: 6 h/day, 5 days/wk for 4 mo 

(5/12/03-9/5/03). Sacrificed 3-4 days postexposure. 

Microarray Data: 13 genes in the heart tissue and 47 

genes in the lung tissue were identified as possibly 

affected. Strict standards (1.5 fold response, 10% false 

discovery rate) resulted in responses by only 1/13 

genes (Rex3 - no known heart physiology) in the heart 

tissue and 0/47 genes in the lung tissue. Using more 

liberal response (nonstatistical) standards (1.5 fold 

only) and comparison of each CAPS animal with all 3 

control animals (3x3 array) resulted in possible effects 

on 7 additional genes in the heart tissue and 37 genes 

in the lung tissue. 

Reference: 

Gurgueira et al. 

(2002, 036535) 

Species: Rat 

Gender: Male 

Strain: SD 

Weight: 250-300 g 

CAPs 

(Harvard Ambient Particle 

Concentrator) 

CB 

(C198 Fischer Scientific, Pittsburg, PA USA) 

Composed of 85.9 ± 0.2% 

Carbon, 13.0 ± 0.2% O2, 1.17 ± 

0.2% Sulfur 

ROFA 

(Boston, MA USA oil-fired power 

plant) 

Particle Size: CAPs: 1-2.5 μm; 

CB: <2.5 μm; ROFA: <2.5 μm 

Route: Whole-body Inhalation 

Dose/Concentration: 300 ± 60 μg/m3 

Time to Analysis: 1, 3, 5 h CAPs Exposure 

followed by immediate post-exposure analysis. 

5 h CB, immediate analysis. 

30min ROFA, Immediate analysis. 

In situ Chemiluminescence(CL): Data show a 

significant increase in lung and heart CL at 5 h. Lung 

CL increased linearly with time of exposure. 

Oxidants: CAPs-initiated oxidative stress was not 

detectable in those rats allowed to recover in room air 

after the simulated “peak” in particulate air pollution. 

Rats breathing particle-free filtered air for 3 days had 

significantly lower levels of oxidants. Exposure to inert 

CB did not exert oxidant effects on the heart and lung. 

BAL Inflammatory/Injury Markers: The water content 

of the lung and heart increased significantly upon 

exposure to CAPs but not to filtered air and increased 

as a function of length of exposure. Rats breathing 

CAPs also showed increases in LDH and CPK as a 

function of length of exposure. 

Antioxidant Enzymes: Data showed an increase in 

SOD and catalase activities in both the lung and heart. 

The pattern of increase was tissue specific. 

December 2009 D-92 
Reference Pollutant Exposure Effects 

Reference: Hamoir 

et al. (2003, 096664) 

Species: Rabbit 

Strain: New 

Zealand 

Age: 12-16 wk 

Weight: 2.8 ± 0.5kg 

PSC: Polystyrene particles, Carboxylate modified, 3 types 

PSA: Polystyrene particles, 

Amine modified, 1 type 

Particle Size: PSC: 24, 110 or 

190 nm (PSC24, PSC110, 

PSC190); PSA: 190 nm 

Route: IT Instillation 

Dose/Concentration: PSC24: 0.04 or 4 mg/rabbit 

PSC110, PSC190, PSA190: 4 mg/rabbit 

Time to Analysis: 0, 30, 60, 90, 120 min 

Capillary Filtration Coefficient: A time-dependent 

increase correlating to total number of particles/surface 

area, not particle size, was observed. PSA induced a 

significant increase in microvascular permeability as 

compared to PSC. This suggests that the number of 

particles exposed should be considered an important 

parameter for measuring air quality rather than total 

particle surface area. 

Reference: Happo 

et al. (2007, 096630) 

Species: Mouse 

Gender: Male 

Strain: C57BL/6J 

Weight: 19-30 g 

Age: 10-11 wk 

PMC (Coarse) 

PMF (Fine) 

PMUF (Ultrafine) 

Collected in 6 European cities: 

Duisburg, Prague, Amsterdam, 

Helsinki, Barcelona, Athens 

Particle Size: PMC: PM10-2.5; 

PMF: PM2.5-0.2; PMUF: PM0.2 

Route: IT Instillation 

Dose/Concentration: 1, 3, 10 mg/kg 

Time course: 10 mg/kg 

Time to Analysis: 1. Dose-Response study: 

parameters measured 24 h post exposure. 2. Time 

course study: parameters measured 4, 12, 24 h 

post single exposure (at 10 mg/kg). 

BALF Cells: 1. For the dose-response study, all the 

PMC samples exhibited dose-dependent increases of 

total cell numbers. The 3 and 10 mg/kg doses of PMC 

induced statistically significant increases. At 10 mg/kg, 

only 2/6 samples induced statistically significant 

increases. No PMUF samples induced effects at any 

dose. 2. For the time-response study, no increases in 

cell numbers were shown at 4 h. Though the levels 

induced by PMC at 24 h were lower than at 12 h, both 

levels were statistically significant. PMF induced 

statistically significant increases only at 12 h for 4/6 

samples. PMUF induced only 1 significant increase at 

12 h; the 24 h time point was not tested. 

BAL Injury Markers: 1. The lower doses of 1 and 3 

mg/kg did not induce significant increases in any of the 

PM samples, except for PMUF-Athens. All 6 samples of 

PMC, at 10 mg/kg, induced significant increases. At 10 

mg/kg, 4/6 PMF samples induced significant increases. 

2. At 4 h, none of the samples increased protein 

concentration. The PMC samples, excluding Prague, 

induced significantly higher concentrations at 12 h. At 

24 h, only 3/6 PMC samples induced significant 

increases. Only 2 PMF samples induced significant 

increases at 12 and 24 h. At 12 h, effects induced by 

PMUF were minimal and inconsistent; the 24 h time 

point was not tested. 

Cytokines: 1. Only PMC induced dose-dependent 

responses that reached statistical significance at 10 

mg/kg. PMF and PMUF induced minimal and 

inconsistent responses. 2. TNF-α levels increased 

significantly at 4 and 12 h by PMC. At 24 h, TNF-α

levels returned to near control levels. PMF, at 4 h, 

induced statistically significant increases for 3/6 

samples and significant increases in 2/6 samples at 12 

h. No PMUF samples significantly increased TNF-α

levels. PMC induced the highest IL-6 levels at 4 h. 

Levels at 12 and 24 h were reduced with 6/6 and 3/6 

samples showing statistically significant increases, 

respectively. PMF showed a similar trend with 4 h 

inducing the highest levels that were reduced at 12 and 

24 h. Of the PMUF samples, only the Helsinki and 

Duisburg samples induced statistically significant 

results at 4 and 12 h. Generally, the PMUF responses 

were negligible when compared to PMC and PMF. 2. 

All PMC samples induced the highest levels of KC 

production at 4 h. At 12 and 24 h, levels were reduced 

but 4/6 samples induced statistically significant levels. 

PMF showed a similar trend- the highest levels were 

induced at 4 h (in 3/6 samples). PMUF at 4 h showed 

small, though not significant, increases. At 12 h, only 2 

samples showed statistically significant differences 

from the control; the 24 h time point was not tested. 

Reference: Harder 

et al. (2005, 087371) 

Species: Rat 

Gender: Male 

Strain: Wistar Kyoto 

Age: 14-17 wk 

Weight: NR

Carbon UFP 

Particle Size: 37.6 ± 0.7 nm 

(diameter) 

Route: Inhalation 

Dose/Concentration: 180 μg/m3

Time to Analysis: 24 h exposure. 3 day recovery.

UFP induced mild pulmonary inflammation, significantly 

increased PMN, and increased the total protein and 

albumin concentrations. Particle-laden macrophages 

sporadically accumulated in the alveolar region.

December 2009 D-93 
Reference Pollutant Exposure Effects 

Reference: 

Harkema et al. 

(2004, 056842) 

Species: Rat 

Gender: Male 

Strain: F344, BN 

Age: 10-12 wk 

Weight: NR 

CAPs (Detroit; July-Sept. 2000; 

Harvard Ambient Fine Particle 

Concentrator) 

Particle Size: 2.5 μm (diameter) 

Route: Inhalation, IT Instillation. 

Dose/Concentration: 4 day concentration: 676 ± 

288 μg/m3, 5 day concentration: 313 ± 119 μg/m3, 

July concentration: 16-185 μg/m3

, September 

concentration: 81-755 μg/m3

; IT Instillation- 200 μL 

(soluble and insoluble) 

Time to Analysis: F344 rats sensitized to 

endotoxin, BN rats to OVA. Exposed 10 h/day 1, 4, 

5 days (consecutive). Another group of rats IT 

instilled. Both groups killed 24 h post-exposure. 

The retention of PM in the airways was enhanced by 

allergic sensitization. Recovery of anthropogenic trace 

elements was greatest for CAPs-exposed rats. 

Temporal increases in these elements were associated 

with eosinophil influx, BALF protein content and 

increased airway mucosubstances. A mild pulmonary 

neutrophilic inflammation was observed in rats instilled 

with the insoluble fraction but instillation of total, soluble 

or insoluble PM2.5 in allergic rats did not result in 

differential effects. 

Reference: 

Hiramatsu et al. 

(2003, 155846) 

Species: Mouse 

Gender: Female 

Strain: BALB/c and 

C57BL/6 

Age: 8 wk 

Weight: 17-22 g 

DE: generated by 2369-cc diesel 

engine (Isuzu) at 1050 rpm and 

80% load with commercial light 

oil 

Particle Size: NR 

Route: Whole-body Inhalation 

Dose/Concentration: DEP: 100 μg/m3 or 3 

mg/m3

; SO2<0.01 ppm; NO2 2.2 ± 0.3 or 15 ± 1.5 

ppm; CO 3.5 ± 0.1 or 9.5 ± 0.6 ppm 

Time to Analysis: 7h/d, 5 days/wk for 4 or 12 wk, 

Immediate 

BALF Cells: Alveolar macrophages (AMs) increased 

dose-dependently at 30 and 90 day. High DE exposure 

resulted in bronchus-associated lymphoid tissue 

(BALT) around DEP-AMs; this was less conspicuous in 

C57BL/6 than in BALB/c mice. B- and T-cell 

populations were found in the BALT with no significant 

differences observed between the strains. 

Lymphocytes and neutrophils increased time- and 

dose-dependently with a greater increase in BALB/c 

than C57BL/6 observed. No eosinophils or basophils 

were observed. Mac-1-positive cells exposed to high 

DE levels increased in both strains at 1 month (33.8%) 

and 3 mo (20.3%) vs. low dose group (5.3 and 7% 

respectively). 

Cytokines: At 30 days, TNF-α, IL-12p40, IL-4 and IL10 mRNA increased, IL1b and iNOS decreased. IFN-γ

increased in BALB/c but decreased in C57BL/c. IL-6 

mRNA was not affected. At 90 day, IL-4 and IL-10 

mRNA similarly increased in C57BL/6 mice exposed to 

low DE level but decreased at high DE level. 

Reference: 

Hollingsworth et al. 

(2004, 097816) 

Species: Mouse 

Gender: Male 

Strains: 

C57BL//6TLR+/+, 

C57BL//6TLR-/-

Age: 8-9 wk 

ROFA 

Particle Size: NR 

Route: Oropharyngeal Aspiration 

Dose/Concentration: 50 μl of 1μg/mL suspension 

per mouse 

Time to Analysis: Parameters measured post 

single exposure of 6 and 24 h. 

Methacholine sensitivity: No ROFA effect was 

observed in wild type or knockout mice. 

BALF Cells: ROFA increased total cell number. Total 

number of neutrophils with lavage fluid increased 24 h 

post-exposure in both strains. 

Reference: 

Hutchison et al. 

(2005, 097750) 

Species: Rat 

Gender: Male 

Strain: Wistar Kyoto 

Age: 3 m 

Weight: 250-300 g 

PM10 

United Kingdom 

samples collected before (-B), 

during closure (-C) and 

reopening of steel plant (-R) 

PMT = PM total (aqueous 

sonicate) 

PMS = PM aqueous supernatant 

PMI = PM insoluble pellet 

Particle Size: PM10

Route: IT Instillation 

Dose/Concentration: 112 to 180 μg PM in 500 μl; 

0.44-0.72 mg/kg 

Time to Analysis: 18 h 

BALF Cells: PMT-R neutrophil cell number and 

percentage were significantly higher than PMT-C or 

control. PMS-R and PMI-R were also higher than their 

respective controls. The neutrophil cell numbers 

induced by PMI-R were greater than PMI-C and the 

control. Total cell count unchanged. 

BALF Inflammatory/Injury Markers: Only albumin 

increased after PMT-R. Upon exposure, total protein 

and LDH did not increase. 

Cytokine mRNA expression: Only PMT-R increased 

IL-1ß mRNA expression. No effects on TNF-α and 

TGF-ß expression levels were observed. IL-6, MIP2, 

and GM-CSF mRNA was not detected in BAL cell 

extracts from either the control or treated groups. 

Reference: Inoue et 

al. (2006, 097815) 

Species: Mouse 

Gender: Male 

Strains: C3H/HeJ 

(TLR-4 point mutant) 

and C3H/HeN 

(Control) 

Age: 6 wk 

DEP (derived from 4 cyl, 2.74l 

light duty diesel engine) 

Particle Size: NR 

Route: IT Instillation 

Dose/Concentration: 12 mg/kg 

Time to Analysis: 24 h 

BALF Cells: DEP induced an increase in total cells, 

neutrophils, and mononuclear cells. TLR4 knockout 

mice (C3H/HeJ) showed a much lower response. 

Cytokines: DEP induced a massive increase in MIP1x, IL-1β and KC. However, levels of MIP-1x were 

significantly less in the knockout than the wild type 

while levels of IL-1β and KC were significantly higher in 

knockouts than the wild type. 

December 2009 D-94 
Reference Pollutant Exposure Effects 

Reference: Inoue et 

al. (2005, 097481) 

Species: Mouse 

Gender: Male 

Strain: NC/Nga 

Age: 10 wk 

DEP (derived from 4 cyl, 2.74l 

light duty diesel) 

Particle Size: NR 

Route: IT Instillation 

Dose/Concentration: 100 μg/mouse 

Time to Analysis: 1/wk for 6 wk. Parameters 

measured 24 h after last administration 

BALF Cells: DEP significantly increased total cells, 

neutrophils and mononuclear cells but did not induce 

an effect on eosinophils. 

Cytokines: DEP increased IL-4, KC and MIP-1. The 

increase in IL-5 was not statistically significant. 

Reference: Ishihara 

et al. (2003, 096404) 

Species: Rat 

Gender: Male 

Strain: Wistar Kyoto 

Age: 5 wk 

DE 

(from 2 engines, produced on 

site) 

-L = low level DE 

-M = medium level 

-MG = DE w/o particulates 

-HR = high level 

Measured Components: NO2, 

SO4, SO2, CO, CO2, NOX, NO, 

HTHC, HCHO, O2

Particle Size: L: 0.33 -0.50 μm 

M: 0.35 - 0.40 μm 

HR: 0.42 - 0.45 μm 

Route: Whole-body Inhalation 

Dose/Concentration: L: 0.18 - 0.21 mg/m3

M: 0.92- 1.18 mg/m3

MG: 0.01 mg/m3

HR: 2.57 - 2.94 mg/m3

Time to Analysis: 16 h/day, 6 days/wk, for 6, 12, 

18 & 24 mo. Parameters measured immediately 

following last exposure. 

Morbidity and Mortality: Weight gain in HR group was 

less than other groups at 18 and 24 mo. This indicates 

a significant difference between the HR and C group. 

Mortality during the study was frequent. C group 

experienced an 8% mortality rate, L group 12%, M 

group 15%, MG group 12% and HR group 23%. 

BALF Cells: The HR group showed a significant 

increase in total cell count from 6 to 18 mo. The 

percentage of PMN increased at 6mo in M, MG and HR 

group. M group lymphocytes significantly increased at 

6, 12, and 24 mo of exposure. Macrophages decreased 

at 6 mo for the M and HR groups. 

BAL Inflammatory/Injury Markers: Significant 

differences were seen among groups with respect to 

number of total cells and percentages of cell 

differential, total protein, fucose, sialic acid, 

phospholipid and prostoglandin E2. Total protein 

increase was observed in both M and HR dose groups 

with the HR group increasing time-dependently. 

Mucus and Surfactant: The HR group showed a 

significant increase from 12 to 18 mo. 

Reference: Jones et 

al. (2005, 198883) 

Species: Rabbit 

Strain: New 

Zealand 

Weight: 2.5- 3.5 kg 

ASP: Amorphous silica particles 

(Hypersil) 

MCSP: Microcrystalline silica 

particles 

Particle Size: ASP: 5 μm; 

MCSP: 5 μm 

Route: Intrapulmonary Instillation (Right upper 

lobe of lung) 

Dose/Concentration: 50mg in 0.5 mL saline 

Time to Analysis: Parameters measured at 

varying times from 6 h to 91 days post treatment. 

MCSP: At 6 h, neutrophils increased. Macrophages 

increased 3 fold. At 60 h, neutrophils were pyknotic and 

the lungs displayed a thickened interstitium containing 

silica particles. At 5 days, collagen deposition 

appeared. At 8 days, fibroblastic activity and necrosis 

were observed. At 15 days, aggregation of silica 

particles and necrotic debris were apparent. At 8 wk, 

fibroblasts were still present. At 13 wk, active scarring 

and raised neutrophil macrophage counts were still 

present. 

ASP: At 15 h, neutrophils increased. Macrophages 

tripled and remained increased for 3wk. At 4 day, 

macrophages bore particles. At 13 day, neutrophils 

decreased significantly. By 25 day, silica spheres were 

gradually removed from lungs. 

PET Scanning: 18F-fluoroproline showed increased 

activity beginning at 14 days and peaking at 41-54 

days. 

Microautoradiography: 3 h-proline at 13 wk showed 

radiolabel localization to fibroblasts in the challenged 

lung. 

December 2009 D-95 
Reference Pollutant Exposure Effects 

Reference: Kato 

and Kagawa (2003, 

089563) 

Species: Rat 

Gender: Male 

Strain: Jcl Wistar 

Age: 5 wk 

Roadside air 

(Prefectural Tokyo-DanishiYokohama highway, YokohamaHaneda Airport Metropolitan 

expressway and SatsukibashiMizuecho city road, Japan) 

Particle Size: NR 

Route: Whole-body Inhalation 

Dose/Concentration: Exposed group: 62.7 μg/m3

PM, 55.7 ppb NO2,; 

Control group: 14.3 μg/m3

 PM, 5.1 ppb NO2 

Time to Analysis: Exposed for 24, 48, 60 wk. 

Parameters measured immediately following 

exposure. 

Respiratory Tissue: Post 24 wk, the lung surface was 

light gray with some BC particle deposits. Post 48-60 

wk, however, the surface was scattered with particle 

deposits in addition to its light gray color. 

Airway Changes: After 60 wk, no remarkable changes 

seen in the epithelium. The structure of the airways 

remained normal. 

Cells: No proliferation or ectopic growth of goblet cells 

were noted. Mast cells increased in epithelial 

intercellular space. No mast cell degranulation was 

observed. Lysosomes increased in ciliated cells post 

48 wk. Clara cells were unaffected. 

Lymph Nodes: Deposition of carbon particles were 

noted in the trachea and bronchiole-associated lymph 

nodes post 24 wk. 

Alveolar Changes: No changes in morphology of 

broncho-alveolar junctions were noted. Anthracosis 

observed within alveolar walls and pleura post 24 wk 

and became progressively marked with increased 

exposure. No change in the number of alveolar holes 

between exposure and control groups were observed. 

Reference: Kato et 

al. (2003, 198882) 

Species: Rat 

Gender: Male 

Strain: SD 

Age: 7 wk 

Weight: 190-220 g 

Polystyrene latex suspension of 

latex beads (Japan Synthetic 

Rubber Co.), uncoated or coated 

with lecithin 

Particle Size: 240 nm 

Route: IT Instillation with nebulizer 

Dose/Concentration: 5 ml of 0.2% suspension 

administered over 20 min at flow rate of 0.25 

ml/min

Time to Analysis: Exposed for 20 min. 

Parameters measured 30 min following treatment. 

Alveolar Macrophages: Following treatment, AMs 

appeared undamaged. AMs ingested more uncoated 

than coated beads, but both were ingested. Ingestion 

of beads differed as coated beads were engulfed 

individually while uncoated beads were engulfed 

individually or in aggregates. 

Epithelial Cells: Type I cells incorporated coated 

beads within a layer of cytoplasm. Type II cells 

incorporated beads in lamellar bodies. Uncoated beads 

were not incorporated. 

Other: Neither type of beads were incorporated into 

endothelial cells, fibroblasts or interstitium of alveolar 

wall 

Monocytes: Only the coated beads were incorporated 

by the monocytes. They were found inside and outside 

phagosomes and lysosomes of monocytes. PMNs did 

not incorporate any beads. 

Reference: 

Kleinman et al. 

(2003, 053535) 

Species: Rat 

Gender: NR 

Strain: F344n-NIA 

Age: 22-24 m 

O3

CCL: O3 + Ammonium bisulfate 

(ABS) + Elemental Carbon (EC) 

CCH: O3 + ABS + EC 

Purified Air (control) 

Particle Size: CCL: 0.30 ± 2.5 

μm; CCH: 0.29 ± 2.3 μm 

Route: Nose-only Inhalation 

Dose/Concentration: O3: 0.2 ppm 

CCL: 50 μg/m3

 EC + 70 μg/m3 ABS + 0.2 ppm O3

CCH: 100 μg/m3

 EC + 140 μg/m3 ABS + 0.2 ppm 

O3

Time to Analysis: 4 h/days, 3 consecutive 

days/wk for 4 wk 

BALF Cells: CCL and CCH induced macrophage 

respiratory burst activity. The effect induced by O3 was 

not significant. 

BAL Inflammatory/Injury Markers: Total protein, 

mucus glycoprotein and albumin were somewhat 

elevated in all exposure groups but only reached 

statistically significance for CCL and protein (very high 

variability). CCL and CCH both depressed Fc receptor 

side binding. No effect for O3 was observed. 

DNA Replication: O3 caused a slight effect of 20-40% 

increase. CCL and CCH caused between 250 - 340% 

increase for interstitial and epithelial cells. CCL induced 

greater reactions than the high dose. 

Reference: 

Kleinman and 

Phalen (2006, 

088596) 

Species: Rat 

Gender: Male 

Strain: SD 

Age: 6 wk 

Weight: 200 g 

LO3: Low O3

HO3: High O3

LS: Low H2SO4

HS: High H2SO4

LOLS: Low O3 + Low H2SO4

LOHS: Low O3 + High H2SO4

HOLS: High O3 + low H2SO4

HOHS: High O3 + high H2SO4

Particle Size: LS = 0.23 μm ± 

2.3 

HS = 0.28 μm ± 2.1 

LOLS = 0.23 μm ± 2.3 

LOHS = 0.28 μm ± 2.1 

HOLS = 0.23 μm ± 2.3 

HOHS = 0.28 μm ± 2.1 

Route: Nose-only Inhalation 

Dose/Concentration: LO3 = 0.30 ppm 

HO3 = 0.61 ppm 

LS = 0.48 mg/m3

HS = 1.00 mg/m3

LOLS = 0.31 ppm + 0.41 mg/m3

LOHS = 0.31 ppm +1.04 mg/m3

HOLS = 0.60 ppm + 0.52 mg/m3

HOHS = 0.60 ppm + 0.86 mg/m3

Time to Analysis: Exposed for 4 h. Parameters 

measured 42 h post-exposure. 

Inflammatory Lesions in Lung Parenchyma: Neither 

Type 1 or 2 lung lesions were affected by sulfuric acid 

alone. HO3 doubled Type 1 lesions and increased Type 

2 lesions 25-fold. Additions of H2SO4 to O3 appeared to 

have a dose-dependent protective effect for both types 

of lesions. 

DNA Synthesis in Nasal, Tracheal and Lung Tissue:

Increased DNA synthesis was observed at all high O3

exposures but was not affected by coexposure to 

H2SO4. 

Macrophage FcR binding: No effects were observed 

(no data for LO3 and HO3). 

Macrophage Phagocytosis: All levels of exposure (no 

data for LO3 and HO3) decreased phagocytosis. 

December 2009 D-96 
Reference Pollutant Exposure Effects 

Reference: 

Kodavanti et al. 

(2005, 087946) 

Species: Rat 

Gender: Male 

Strain: WKY and 

SH/NCrlBR 

Age: 11-14 wk 

CAPs (EPA, NC) 

Measured components included 

Al, Be, Ba, Co, Cu, Zn, Pb, Mn, 

Ni, Ag, Ti, As. 

Particle Size: 1 day: 1.07-1.19 

μm; 2 days: 1.27-1.48 μm 

Route: Whole-body Inhalation 

Dose/Concentration: 1 day study: 1138-1765 

μg/m3

2 day study: 144-2758 μg/m3

Time to Analysis: 4 hr (SH only); 4 hr/day, 2 day 

(WKY and SH) 

Post-exposure: 1 day: 3 h except study #4, 18-20 

h; 2 day: 18-20 h 

Breathing Parameters: In a paired analysis of control 

SH and treated SH, treated SH showed an increase in 

expiratory and inspiratory time due to CAPs. The 

treated and control groups of WKY rats did not show 

significant differences. 

BALF Cells: In the 2 day study, WKY rats showed 

decreases in total cells; this decrease was associated 

with decreased macrophages. WKY showed an 

increase in neutrophils. 

BAL Inflammatory/Injury Markers: Total protein and 

albumin in WKY rats decreased whereas SH rats 

maintained the same approximate level. LDH activity 

lowered slightly in both strains. 

Cell Membrane Integrity: SH rats showed increased 

GGT (membrane bound enzyme) activity and plasma 

fibrinogen for 5/7 exposures but these increases did 

not appear to be dose-dependent. 

Cytokines: Levels were undetermined in SH rats. 

WKY showed slight increases in IL-6, TNF-α, and MIP2 but these increases were not statistically significant. 

Reference: Kooter 

et al. (2006, 097547) 

Species: Rat 

Gender: Male 

Strain: SH 

Age: 12-14 wk 

CAP-F = fine (Site I) 

CAP-UF = fine + ultrafine (Site 

II) 

(Netherlands) 

Some measured components:

Ammonium, nitrate, sulfate ions: 

56 ± 16% CAP-F mass, 17 ± 

6% CAP-UF mass 

Particle Size: 0.15<CAP-F<2.5 

0.65-0.75 μm 

CAP-UF<2.5 

0.58-1.41 μm 

Route: Nose-only Inhalation 

Dose/Concentration: CAP-F 399- 3613 μg/m3

CAP-UF 269-556 μg/m3

Time to Analysis: 6 h/day for 2 days consecutive, 

18 h 

BALF Cells: A decrease in absolute neutrophils as well 

as percentages of reticulocytes and percentages of 

neutrophils were observed with CAP-F. Increased 

percentages of lymphocytes were observed with CAPF. 

BALF Inflammatory/Injury Markers: Based on 

unchanged levels of LDH and ALP, no cytotoxicity was 

noted. No significant change in the levels of total cells 

were observed. MDA (malondialdehyde) decreased 

with CAP-UF. Ho-1 increased with CAP-UF and CAP-F.

Cytokines: CC16 decreased at 457μg/m3 of CAP-F 

and increased at 3613 μg/m3

 of CAP-F. 

Pathology. No changes were observed. 

Reference: Kumar 

et al. (2004, 096655) 

Species: Rat 

Gender: Male 

Strain: Wistar Kyoto 

Weight: 150 ± 20 g 

Fly Ash (Obra Thermal power 

Station, India) 

Particle Size: PM <5 μm (90%) 

Route: Whole-body Inhalation 

Dose/Concentration: 14.4 ± 1.77 mg/m3

(fluid bed generator) 

Time to Analysis: 4 h/day for 28 day. Parameters 

measured immediately following last exposure. 

Lung Weight: Lung body weight increased 25.58% 

relative to controls. Total body weight slightly 

decreased in the treated group. 

BALF Cells: Only eosinophils(%) increased 95% over 

controls. Congestion and focal infiltration of monocytes 

in alveolar area was seen. Fly ash laden macrophages 

in alveoli combined with hypertrophy of epithelial lining 

cells was observed. 

BAL Inflammatory/Injury Markers: LDH, GGT, ALP 

and lavagable protein increased by 140, 450, 160 and 

50%, respectively. 

Reference: Lei et al. 

(2004, 087999) 

Species: Rat 

Gender: Male 

Strain: SD 

Weight: 318 ± 8 g 

CAPs 

(Yaipei, Taiwan) 

Particle Size: 0.01- 2.5 μm 

Reference: Nose-only Inhalation 

Dose/Concentration: 371 ± 208 μg/m3

Time to Analysis: 6 h/day for 3 day, 

5 h post-exposure pulmonary function. 

2 day post-exposure for BALF collection 

Pulmonary hypertension induced 2 wk preexposure 

Respiratory Effects: Decreased respiratory frequency 

and increased tidal volume for both experimental and 

control groups were observed. However, only the 

experimental group levels were statistically significant. 

There was an increase in airway responsiveness 

(Penh/methacholine) for CAPs group when compared 

to the control. 

BALF Cells: A massive increase in total cell number 

and percent neutrophils was observed. There were no 

changes in percent macrophages, lymphocytes and 

eosinophils. 

BAL Inflammatory/Injury Markers: Total protein and 

LDH increased in the CAPs group. 

Cytokines: TNF-α and IL-6 were not affected. 

December 2009 D-97 
Reference Pollutant Exposure Effects 

Reference: Lei et al. 

(2004, 087884) 

Species: Rat 

Gender: Male 

Strain: SD 

Weight: 300-350 g 

CAPs from Asian dust storm 

(Taiwan) 

Measured Components: Si, Al, 

S, Ca, K, Mg, Fe, As, Ni, W, V, 

OC, EC, SO2, NO2, nitrate, 

sulfate 

Particle Size: 0.01- 2.5 μm 

Route: Nose-only Inhalation 

Dose/Concentration: 315.6 μg/m3 (Low) or 684.5 

μg/m3 (High) 

Time to Analysis: Low: Exposed for 6 h. 

Sacrificed 36 h post-exposure 

High: Exposed for 4.5 h. Sacrificed 36 h postexposure 

Pulmonary hypertension induced 2 wk preexposure. 

BALF Cells: PM induced dose-dependent increases in 

total cells and percentage of neutrophils. No change in 

macrophages, lymphocytes or eosinophils occurred. 

Basophils were highly variable. 

BALF Inflammatory/Injury Markers: Dose-dependent 

increases were observed for total protein and LDH. 

Cytokines: IL-6 increased dose-dependently. (control: 

33.5 ± 7.5, low 165.1 ± 117.2, 273.6 ± 62.8 pg/mL). 

Reference: Li et al. 

(2007, 155929) 

Species: Mouse 

Gender: Female 

Strain: BALB/c, 

C57BL/6 

Age: 9 wk 

Weight: NR 

DEP (2369-cc diesel engine 

manufactured by Isuzu Motor, 

operated at 1050 rpm, 80% load, 

commercial light oil) 

Particle Size: NR 

Route: Inhalation 

Dose/Concentration: DEP: 103.1 ± 9.2 μg/m3, 

CO: 3.5 ± 0.1 ppm, NO2: 2.2 ± 0.3 ppm, SO2: 

<0.01 ppm 

Time to Analysis: Protocol 1: Exposed 7h/day, 5 

days/wk. Sacrificed at day 0, week 1, 4, 8. Protocol 

2: DE alone or DE+NAC 7h/day, 1-5 days. 

Airway Hyperresponsiveness: Penh values 

increased in BALB/c mice compared to the control at 

day 0, but no significant changes occurred after this 

time. Penh values increased in C57BL/6 mice at 1 wk 

compared to the control but returned to control levels at 

8 wk. 

BALF: Compared to the other strain, the total number 

of cells and macrophages increased significantly at 1 

wk in C57BL/6 mice and at 8 wk in BALB/c mice. 

Neutrophils, lymphocytes, MCP-1, IL-12, IL-10, IL-4, IL13 increased significantly for both strains. No 

eosinophils were found. IL-1β and IFN-γ increased 

significantly in BALB/c mice compared to C57BL/6 

mice. 

HO-1 mRNA and Protein: HO-1 mRNA was more 

marked in BALB/c mice at 1 wk and C57BL/6 mice at 4 

and 8 wk. HO-1 protein percentage changes from the 

control were greater in BALB/c mice at 1 wk and 

C57BL/c mice at 8 wk. 

NAC: NAC inhibited the increased Penh values, total 

number of cells and macrophages in C57BL/6 mice at 

1 wk and neutrophils and lymphocytes in both strains. 

Reference: Liu et al. 

(2008, 156709) 

Species: Mouse 

Gender: Female 

Strain: BALB/c 

Age: 11 wk 

Weight: NR 

 DEP (5500-watt single-cylinder 

diesel engine generator 

(Yanmar, Model YDG 5500E), 

406 cc displacement air-cooled 

engine, Number 2 Diesel 

Certification Fuel, 40 weight 

motor oil) 

Particle Size: ~0.1 μm (MMAD) 

Route: Intranasal 

Dose/Concentration: Average particle 

concentration: 1.28 mg/m3

Time to Analysis: Four groups: saline+air control, 

saline+DEP, A. fumigatus+air, A.fumigatus+DEP. A. 

fumigatus exposure every 4 day for 6 doses. DEP 

exposure 5 h/day for 3 wk concurrent with A. 

fumigatus exposure. 

A.fumigatus+DEP increased IgE, the mean BAL 

eosinophil percentage, goblet cell hyperplasia, and 

eosinophilic and mononuclear cell inflammatory 

infiltrate around the airways and blood vessels 

compared to the A. fumigatus or DEP treatments. 

A.fumigatus+DEP also caused methylation at the IFN-γ

promoter sites CpG-53, CpG-45, and CpG-205. 

Reference: Lopes 

et al. (2009, 190430) 

Species: Mouse 

Gender: Male 

Strain: BALB/c 

Age: 6-8 wk 

Weight: NR

PM (high density traffic; winter 

2004; São Paulo, Brazil) (NO2, 

CO, SO2) 

Particle Size: 10 μm (diameter) 

Route: Open-Top Exposure Chamber 

Dose/Concentration: 33.86 ± 2.09 μg/m3

Time to Analysis: Some rats pretreated with 

papain. Exposed to UAP or filtered air 24 h/day, 7 

days/wk, 2 mo.

The papain+UAP treatment increased Lm values, 

collagen fibers, and decreased the density of elastin 

fibers over the papain+filtered air treatment. The 

papin+UAP treatment increased 8-isoprotane more 

than any other group. 

December 2009 D-98 
Reference Pollutant Exposure Effects 

Reference: 

Mangum et al. 

(2004, 097326) 

Species: Rat 

Gender: Female 

Strain: CDF 

(F344)/CrlBR 

Age: 7 wk 

TiO2 (DuPont) 

Particle Size: NR 

Route: Whole-body Inhalation 

Dose/Concentration: 10, 50 or 250 mg/m3

Time to Analysis: 6 h/day, 5 days/wk, 13 wk. 

Parameters measured 0, 4, 13, 26, 52 wk postexposure. 

OPN (osteopontin) Expression: At 0 wk, OPN mRNA 

expression exhibited a dose-dependent increase. Low 

dose induced a 2-fold increase while the high dose 

induced an almost 100 -fold increase. At 4 wk, the middose and high-dose elevated OPN mRNA levels. At 13 

wk, the high dose elevated OPN mRNA levels. No 

significant elevation with mid dose level was observed. 

At 26 wk, the mid and high dose induced elevated OPN 

mRNA levels. At 52 wk, rats in the low, mid and high 

dose groups all indicated elevated levels of OPN 

mRNA. Specifically, the low, mid and high doses 

induced a 3-fold increase, 7-fold increase and 400-fold 

increase, respectively. 

OPN Protein in BALF: Data was not reported at 0 and 

4 wk. At 13 wk, protein increased 9-fold (~800 pg/mL 

OPN) at mid dose and 100 -fold (~8000 pg/mL OPn) at 

high dose. At 26 wk, the mid and high dose groups 

remained elevated. At 52 wk, protein increased by 2.5 

fold in low dose, 7-fold in mid dose and 166-fold in high 

dose group. 

Histopathology: At 52 wk, slight OPN 

immunoreactivity was observed in control and low dose 

group (immunostaining mostly limited to intraalveolar 

MACS).Trichrome-stained lung sections from control 

and low dose groups showed no increase in collagen. 

Rats exposed to mid or high dose groups showed 

areas of lesions. 

Reference: Martin 

et al. (2007, 096366) 

Species: Mouse 

Gender: Male 

Strain: BALB/c 

Age: 1-2 mo 

UAP-BA: Urban Air particles 

(Buenos Airs, Argentina) 

Particle Size: <2.5 μm 

Route: Intranasal Installation 

Dose/Concentration: 0.17 mg/kg

Time to Analysis: 3×day, 3 days/wk, 2 days apart 

(1, 4, 7 day). Parameters measured 1 h postexposure. 

Particle Characteristics: 3 types, ultrafines <0.2 um 

(inorganics ND), bunched agglomerates of ultrafines 

and <40 um with aluminum silicates, ions and trace 

metals. 

BALF Cells: Increased amount of phagocytes in 

alveolar area, reducing airspace percentage (control 

52.9% ± 1.39, UAP-BA 24.7% ± 2.87). Increased 

number of PAS positive cells. 

Morphometry: Induced focal inflammatory lesions. 

Accumulation of refractile material in upper and lower 

respiratory tract. PM in phagocytes of bronchiolar 

lumen and alveolar space. No evidence of fibrosis 

and/or collagen changes. 

Reference: Mauad 

et al. (2008, 156743) 

Species: Mouse 

Gender: Male, 

Female 

Strain: BALB/c 

Age: 10 day 

Weight: Parental: 

21.4 ± 4.0 - 26.3 ± 

2.8 g; 15 day-old 

offspring: 7.8 ± 1.1 - 

9.0 ± 1.0 g; 90 dayold offspring: 20.3 ± 

2.3 - 27.4 ± 1.8 g 

PM (busy traffic street São 

Paulo, Brazil; Aug. 2005-April 

2006) (NO2, SO2, CO) 

Particle Size: 2.5, 10 μm 

(diameter) 

Route: Open-Top Chamber 

Dose/Concentration: PM2.5: filtered chamber- 2.9 

± 3.0 μg/m3

, nonfiltered chamber- 16.9 ± 8.3 

μg/m3

; Outdoor concentration: PM10- 36.3 ± 15.8 

μg/m3

, CO- 1.7 ± 0.7ppm, NO- 89.4 ± 31.9 μg/m3, 

SO2- 8.1 ± 4.8 μg/m3

Time to Analysis: Nonfiltered exposure 24 h/day 

for 4 mo. Mated at 120 days exposure. After birth, 

30 females and offspring transferred to filtered or 

nonfiltered chamber. Killed 15 or 90 day of age. 

Mild foci of macrophage accumulations containing 

black dots of carbon pigment occurred in the alveolar 

areas on 90 day-old mice. Surface-to-volume ratio 

decreased from 15 to 90 days of age and was higher in 

mice exposed to air pollution. PM exposure reduced 

inspiratory and expiratory volumes at higher levels of 

transpulmonary pressure. 

Reference: 

McDonald et al. 

(2004, 087459) 

Species: Mouse 

Strain: C57BL/6 

Age: 8-10 wk 

DEE: high load, No 2, No cat 

(620: 1 dilution) 

DEE-ER (Control): Emissions 

Reduced (high load, low sulfur 

ECD1) (same dilution) 

(Yanmar diesel generator, 406 

cc, 5500 watt load) 

Particle Size: DEE: 110 nm; 

DEE-ER: NR 

Route: Whole-body inhalation 

Dose/Concentration: DEE PM: 236 μg/m3

DEE-ER PM: 7 μg/m3

Time to Analysis: DEE: 6 h/day for 7 days. 

DEE-ER: 6 h/day for 7 days. RSV administered 

post-exposure for some: single, 4 days. Those not 

infected with RSV sacrificed immediately upon last 

exposure. 

Differences in Exposure Conditions: CO, PM, EC, 

OC, nitrate, alkyne, c2-c212 alkenes, phenanthrenes, 

total particle PAHs, total Oxy-PAHs, benzene, pyrene, 

benzo(ayrene, zinc were reduced by 90-100% in the 

emissions reduction case. Most other components 

were reduced by around 60%. 

DEE vs. DEE-ER Effects: DEE increased viral 

retention and lung histopathology. DEE-ER increases 

were not statistically significant. 

Cytokines: DEE increased TNF-α, IL-6, IFN-γ and HO1. DEE-ER responses were not statistically significant 

(significantly higher variability in DEE-ER controls vs. 

DEE controls). 

December 2009 D-99 
Reference Pollutant Exposure Effects 

Reference: 

McQueen et al. 

(2007, 096266) 

Species: Rat 

Gender: Male 

Strain: Wistar Kyoto 

Weight: 228-500 g 

DEP: SRM 2975 (NIST) 

Particle Size: NR 

Route: IT Instillation 

Dose/Concentration: 0.5 mL/rat of 1 mg/mL; 1-

2.2 mg/kg

Time to Analysis: 6 h. 

Pre-exposure: Vagotomy (sectioning of vagus 

nerve) or atropine, 1 mg/kg i.p. administered 30 

min prior, 2 and 4 h post. 

BALF Cells: A 9-fold increase in neutrophils with high 

individual variability in response was observed. 

Bilateral vagotomy prior to DEP reduced neutrophil 

increase to 3 fold. Vagotomy with saline instillation had 

no effect. Atropine reduced neutrophils to levels similar 

to saline response. No differences were observed 

between DEP response in anesthetized when 

compared to conscious animals. Macrophages, 

eosinophils and lymphocytes remain unchanged. 

Respiratory Response: RMV increased post DEP. 

Vagatomy reduced response by one-third. Atropine pretreatment did not have effect. 

Reference: 

Medeiros et al. 

(2004, 096012) 

Species: Mouse 

Gender: Male 

Strain: BALB/c 

Age: 60 days 

Weight: 20-30 g 

CP: Carbon particles 

PSA: ROFA (solid waste 

incinerator hospital Sao Paulo, 

Brazil) 

PSB: electric precipitator, steel 

plant, Brazil) 

PSA/PSB Characteristics: 

Generally, PSB had greater 

component concentrations than 

PSA: Br (100+x), Cr (3x), Fe 

(10+x), Mn (2x), Rb (60+x), Se 

(7x), Zn (4x). PMA>PMB: Ce 

(3x), Co (10+x), La (100x), Sb 

(15x), V (50x). 

Particle Size: CP: 1.7 ± 2.5 μm 

(78%<2.5 μm); PMA: 1.2 ± 2.2 

μm(98 %<2.5 μm); PMB: 1.2 ± 

2.2 μm (98%<2.5 μm) 

Reference: Intranasal Instillation 

Dose/Concentration: CP: 10 μg/mouse; 0.5 

mg/kg 

PSA: 0.1, 1 or 10 μg/mouse; 0.005, 0.05, 0.5 

mg/kg 

PSB: 0.1, 1 or 10 μg/mouse; 0.005, 0.05, 0.5 

mg/kg

Time to Analysis: Single, 24 h 

BALF Cells: No change in BAL cell count was seen. 

Quantitative cellular counts increased for perivascular 

area for both groups at all dose levels. Inflammatory 

cells in alveolar septum area only increased for PSA. 

Reference: Mutlu et 

al. (2006, 155994)

Species: Mouse 

Strain: C57BL/6 

Age: 6-8 wk 

Weight: 20-25 g 

PM10

Collected by baghouse from 

Dusseldorf, Germany 

Particle Size: NR 

Route: IT Instillation 

Dose/Concentration: 100 ng/mouse; 1 μg/mouse; 

10 μg/mouse; 100 μg/mouse

Time to Analysis: 1-7 days 

Alveolar Fluid Clearance: At 100 μg/mouse, 

decreased clearance peaked at 24 h and recovered at 

7 days. 

Histology: Evidence of mild lung injury at doses of 100 

μg/mouse or more was seen. 

BALF Cells: Significant increase in total cell number 

was observed. Neutrophils increased but this was not 

statistically significant. 

Wet/Dry Ratio: Exposure did not induce any effects. 

Na, K-ATPase: At 100 μg/mouse, decreased activity of 

Na, K-ATPase in basolateral membranes was 

observed. 

Reference: 

Nadziejko, et al. 

(2002, 087460) 

Species: Rat 

Gender: Male 

Strain: SH 

Age: 16 wk 

CAPs: produced at Tuxedo, NY 

laboratory using centrifugal 

aerosol concentrator 

FA: Fine Particle Sulfuric Acid 

Aerosol 

UFA: Ultra-Fine Particle Sulfuric 

Acid Aerosol 

Particle Size: CAPs: PM2.5; 

FA: 160 nm; UFA: 50-75 nm 

Route: Nose-only Inhalation 

Dose/Concentration: CAPS 80, 66 μg/m3; avg 73 

μg/m3

FA: 299, 280, 119, 203 μg/m3

; avg 225 μg/m3

UFA: 140, 565, 416, 750 μg/m3

; avg 468 μg/m3

Time to Analysis: 10 exposures of 4 h each, each 

exposure at least 1 wk apart. 

(2 exposures to CAPs, 4 to FA and 4 to UFA)

Respiratory Rate: CAPs decreased the respiratory 

rate as did FA at all dose levels. However, the FAinduced respiratory rate was not statistically significant 

unless the data was combined. UFA increased this rate 

significantly. 

Reference: Nemmar 

et al. (2007, 156800) 

Species: Rat 

Gender: Male 

Strain: Wistar Kyoto 

Age: 16 wk 

Weight: 424 ± 8g 

DEP: SRM 2975 

Particle Size: <1 μm 

Route: Intravenous Injection 

Dose/Concentration: 0.02, 0.1 or 0.5 mg/kg 

Time to Analysis: single, 24 h 

BALF Cells: Marked cellular influx at all dose levels 

Was observed. Macrophages increased at the high 

dose, but this was not statistically significant. PMN 

increased significantly at all dose levels. 

Wet/Dry Ratio: All dose levels induced increases. 

December 2009 D-100 
Reference Pollutant Exposure Effects 

Reference: Nemmar 

et al. (2003, 087931) 

Species: Hamster 

Gender: Male and 

Female 

Weight: 100-110 g 

PS: Polystyrene particles 

PSC: Polystyrene particles, Carboxylate modified 

PSA: Polystyrene particles, 

Amine modified 

Particle Size: PS, PSC, PSA60: 60 nm; PSA-400: 400 nm 

Route: IT Instillation 

Dose/Concentration: 5, 50 or 500 μg/animal; 

0.05, 0.5, 5 mg/kg

Time to Analysis: Single, 10 min post-exposure 

Rose Bengal administered to induce thrombosis, 

immediate study thereafter 

BALF Cells: Both PSA-60 and PSA-400 (PSA60>PSA-400) induced a massive influx of PMNs. PSA60 effect may exhibit some dose-dependency. 

BALF Inflammatory/Injury Markers: Small increases 

in total protein were seen at 500 μg level for both PSA60 and PSA-400. LDH was increased at all PSA-60 

levels but not for 500 ug PSA-400. Histamine increased 

for all PSA-60 levels and PSA-400 but due to high 

variability only the effect at 500 μg PSA-60 was 

statistically significant. 

Reference: Nemmar 

et al. (2003, 097487) 

Species: Hamster 

Gender: NR 

Weight: 100-110 g 

DEP: SRM 1650 

Particle Size: NR 

Route: IT Instillation 

Dose/Concentration: 50 μg/animal 

Time to Analysis: Single exposure, parameters 

measured 1, 3, 6 or 24 h post- exposure. 

BALF Cells: DEP led to a significant PMN flux at 1 h 

(13% of total cell number), 6 h (22%) and 24 h (37%). 

Histamine: Concentrations in BALF were consistently 

elevated starting at 1 h. Plasma histamine did not 

increase until 6 h. 

Pretreatment with Histamine Receptor Antagonist:

A major decrease in DEP induced PMN infiltration was 

seen. No effect on histamine in BALF or plasma was 

observed. 

Reference: Pereira 

et al. (2007, 156019) 

Species: Rat 

Gender: Male 

Strain: Wistar Kyoto 

Age: 3 m 

Ambient Particles 

(Porto Allegre, Brazil) 

Particle Size: <10μm 

Route: Whole-body Inhalation 

Dose/Concentration: P-6: 34, 22 or 225 μg/m3

P-20: 139 or 112 μg/m3

P-I: 99 μg/m3

Time to Analysis: P-6: single/continuous for 6 h 

P-20: single/continuous for 20h 

P-I: intermittent (5 h) periods per day for 4 days 

consecutively 

Parameters measured 0 or 24 h post-exposure 

BAL Inflammatory/Injury Markers: An increase in 

lipid peroxidation was statistically significant only for 

the 20 h continuously exposed group. Leukocytes also 

increased at P-20. No change at P-6. Total protein 

remained unaffected at all dose levels. 

Wet to Dry Ratio (0h): No effect was observed. 

Reference: 

Pinkerton et al. 

(2004, 087465) 

Species: Rat 

Gender: Female 

(pregnant), 

Offspring- NR 

Strain: SD 

Age: 10 days 

(pups), Pregnant 

females- 10-14 days 

of gestation 

Weight: NR 

 PM (Fe and soot from 

combustion of acetylene and 

ethylene in a laminar diffusion 

flame system) 

Particle Size: Median diameter: 

72-74 nm; size range: 10-50 nm 

Route: Inhalation 

Dose/Concentration: Mean mass concentration: 

243 ± 34 μg/m3

; Average Fe concentration: 96 

μg/m3

Time to Analysis: Exposed 10 days postnatal 

age, 6 h/day, 3 days (consecutive). 

A significant reduction of cell proliferation occurred only 

within the proximal alveolar region of exposed animals 

compared to controls. There were no significant 

differences between the groups for alveolar formation 

and separation within the proximal alveolar region. 

Reference: 

Pinkerton et al. 

(2002, 087645) 

Species: Rat 

Gender: Male, 

Female 

Strain: SD 

Age: 11-13 wk (adult 

male), 10-12 days 

(neonatal) 

Weight: NR

PM (Fe, Soot) (ethylene, iron 

pentacarbonyl, acetylene 

combined; Fe2O3; soot: 60% EC, 

40% OC) (CO, NOX) 

Particle Size: Fe (diameter) 40 

nm; Soot (primary particles, 

diameter) 20-40 nm

Route: Whole-body Inhalation 

Dose/Concentration: Adult males: Fe- 57, 90 

μg/m3

, Soot- 250 μg/m3, Fe+Soot- Fe: 45 μg/m3, 

Total PM: 250 μg/m3

; Neonates: Fe+Soot- Low: 

Fe- 30 μg/m3, Total PM: 250 μg/m3, High: Fe- 100 

μg/m3

, Total PM: 250 μg/m3 

Time to Analysis: Adult males exposed to Fe, 

soot, Fe+Soot, or filtered air. Exposed 6 h/d, 3 

days (consecutive). BAL, 2 h postexposure, lung 

tissue, 24 h postexposure. Neonatal rats exposed 

to Fe+Soot 10-12 day-old and 23-25 day-old. 

Fe: Only the high dose had significant effects. This 

dose increased total protein in the lavage fluid, 

decreased total antioxidant power, induced GST 

activity, and induced a non-significant, increasing trend 

of GSH and GSSG. IL-1β, intracellular ferritin, and NFκB increased. 

Fe+Soot, Soot: Fe+Soot significantly reduced the total 

antioxidant power in BALF and supernatant from lung 

tissue homogenate. Fe+Soot significantly increased 

GSSG, IL-1β, NF-κB, CYP1A1, and CYP2E1. CYP2B1 

increased but was not significant. Soot alone was not 

significant for anything. 

Neonates: The high-dose significantly decreased cell 

viability, increased LDH activity, and increased IL-1β

and ferritin. Both doses significantly increased GSSG, 

GRR, and GST, and decreased total antioxidant power. 

December 2009 D-101 
Reference Pollutant Exposure Effects 

Reference: PiresNeto et al. (2006, 

096734) 

Species: Mouse 

Gender: Male 

Strain: Swiss 

Age: 6 days 

Ambient Air: PM2.5, NO2 and CB 

(Sao Paulo, Brazil) 

Particle Size: PM2.5

Route: Whole-body Inhalation 

Dose/Concentration: PM2.5: 46.49 μg/m3

Control: 18.62 μg/m3

NO2: 59.52 μg/m3

Control: 37.08 μg/m3

CB: 12.52 μg/m3

 

Control: 0 μg/m3

Time to Analysis: 24 h/day, 7 days/wk for 5 mo 

(weaned at 21 days into exposure, mothers 

removed) 

Nasal Cavity: Increased total mucus and acidic mucus 

at proximal and medial areas of cavity. Nonsecretory 

epithelium declined. No significant changes in amount 

of neutral mucus, volume proportion of neutral mucus, 

volume proportion of total mucus, thickness of 

epithelium, volume proportion of nonsecretory 

epithelium or ratio between neutral and acidic mucus 

were observed. 

Types of Acidic Mucus Cells: Proximal and medium 

cells increased. Effects on distal cells were equivocal. 

Reference: 

Pourazar et al. 

(2005, 088305) 

Species: Human 

Gender: Male and 

Female (nonatopic & 

nonsmokers) 

Age: 21-28 yr 

DEP: generated from idling 

Volvo diesel engine 

DEP 300 μg/m3

 comprised of: 

NO2 1.6 ppm 

NO 4.5 ppm 

CO 7.5 ppm 

Hydrocarbons 4.3 ppm 

Formaldehyde 0.26 mg/m3

Suspended particulates 

4.3×106

/cm3

Particle Size: <10 μm 

Route: Whole-body Inhalation 

Dose/Concentration: DEP 300 μg/m3

Time to Analysis: Single exposure for 1 h. 

Parameters measured 6 h post exposure. 

Transcription Factors: Exposure induced increased 

cytoplasmic and nuclear immunoreactivity of phosphorylated p38 MAPK in bronchial epithelium. 

Increased nuclear translocation of phosphorylated p38 

and JNK, MAPK as well as increased nuclear 

phosphorylated tyrosine immunoreactivity were 

observed. No change in total or nuclear c-fos 

immunoreactivity was seen. Exposure induced 

increased nuclear translocation of phosphorylated JNK 

significantly associated with phosphorylation of nuclear 

c-jun and also resulted in an increase in nuclear p65. 

Cytokines: Expression of IL-8 was positively 

associated with nuclear phosphorylated p38 postexposure. 

Reference: Pradhan 

et al. (2005, 096128) 

Species: Rat 

Gender: Female 

Strain: Wistar Albino 

Weight: 120-180 g 

RSPM: Respirable Suspended 

PM 

(Lucknow, India) 

Quartz dust (positive control) 

Particle Size: < 5 μm 

Route: IT Instillation 

Dose/Concentration: 2.5, 5.0, or 10.0 mg/ 0.05 

ml; 20, 42, 83 mg/kg

Time to Analysis: 15 days. 

Relative Lung Weight: A dose-dependent increase in 

total lung weight of RSPM-instilled animals was 

observed. 

BALF Cells: Exposure induced a dose-dependent 

increase in total cells dose-dependent with the low and 

mid dose levels. PMNs increased massively at all dose 

levels with RSPM inducing less of an increase than 

Quartz. Exposure at low dose levels resulted in an 

influx of inflammatory cells (predominantly 

macrophages into lumen of alveolar ducts and alveoli). 

Reaction at the high dose was more intense than that 

seen in mid dose-exposed lungs. 

BAL Inflammatory/Injury Markers: A significant dosedependent increase in LDH and NO was observed, but 

the Quartz-induced increase was greater than the 

RSPM-induced increase. An increase in protein was 

significant at the mid dose level for RSPM and 

significant at the high dose level for both RSPM and 

Quartz. 

Lung Biochemistry: An increase in lipid peroxidation 

was dose-dependent. Superoxide dismutase (SOD) 

enzyme levels showed a dose-dependent decrease. 

Reference: Ramos 

et al. (2009, 190116) 

Species: Guinea 

Pig 

Gender: NR 

Strain: NR 

Age: NR 

Weight: 330-370 g

WS (Pine wood) (CO(<80ppm), 

CO2 (0.35%), O2 (20.1%), PM2.5, 

PM10) 

Particle Size: PM2.5, PM10

Route: Whole-body Inhalation 

Dose/Concentration: WS: 60 g, PM2.5: 363 ± 23 

μg/m3

, PM10: 502 ± 34 μg/m3 

Time to Analysis: Exposed 3 h, 5 days/wk for 1, 

2, 3, 4, 6, 7 mo.

WS significantly decreased body weight between 4 and 

7 m exposure. The concentration of blood 

carboxyhemoglobin increased. Recovered BALF cells 

were higher in WS-exposed pigs. Macrophages and 

neutrophils increased. Inflammation in the lungs was 

seen. Pulmonary arterial hypertension and 

emphysematous lesions were observed. Macrophage 

and lung tissue homogenate elastolysis increased. 

Collagenolysis increased. Generally, MMP-2, MMP-9, 

and MMP-1 increased. BAL macrophage apoptosis 

increased with time. 

December 2009 D-102 
Reference Pollutant Exposure Effects 

Reference: Rao et 

al. (2005, 095756) 

Species: Rat 

Strain: SD 

Weight: 175 g 

DEP: SRM 2975 

Particle Size: 0.5 μm 

Route: IT Instillation 

Dose/Concentration: 5, 35, 50 mg/kg bw

Time to Analysis: Sacrificed 1, 7, 30 days post 

single exposure. Cytokines measured after 24 h 

incubation (in vitro). 

BALF Cells: Macrophages unaffected. Increased 

PMNs at 1 day for all dose levels, sustained elevation 

at 7days for mid and high dose and at 30 days for all 

dose levels. 

BAL Inflammatory/Injury Markers: Increased albumin 

at 1 and 30 days at all dose levels. Increased LDH 

except at low dose at 7 days. 

Cytokines: The high dose induced a significant 

increase of mRNA expression for IL-1β, iNOS, MCP-1, 

and MIP-2 in BAL cells. MCP-1 mRNA sustained high 

levels at 7 days for mid and high dose and at 30 days 

for all dose levels. mRNA expression of IL-6, IL-10, 

TGF-β1, TNF-α were unaffected. However, IL-6 and 

MCP-1 proteins increased significantly in BALF at 1 

day for mid and high dose, returning to basal levels at 7 

days. MIP-2 increased for all dose levels at all time 

points. NO level unaffected. 

Reference: Reed et 

al. (2006, 156043) 

Species: Rat, 

Mouse 

Gender: Male and 

Female 

Strain: CDF 

(F344)/CrlBR (rat), 

SH (rat), A/J 

(mouse), and 

C57BL/6 (mouse) 

Age: 6-12 wk 

HWS (burned mix of hardwood 

in noncertified wood stove using 

a Pineridge model 27000, 

Heating and Energy Systems, 

Inc. Clackamas, OR) 

Measured Components: EC, 

OM, NO3, SO4, NH4, metals 

Particle Size: ~0.25 μm 

Route: Whole-body Inhalation 

Dose/Concentration: Low: 30 μg/m3 

Mid-low: 100 μg/m3

 

Mid-high: 300 μg/m3

 

High: 1000 μg/m3

 

Time to Analysis: 6 hr/day, 7 days/wk for 1wk or 6 

mo. Immediate post-exposure analysis. 

Organ Weights: Liver declined in rats of both genders 

at 1 wk and female rats at 6 m. Lung volume increased 

and lung weight decreased in female rats at 6 m. 

Spleen weight increased in female mice and rats at 1 

wk. Thymus weight decreased in male rats at 1 wk. 

Cells: Eosinophils decreased and lymphocytes 

increased in males at 6m. Neutrophils decreased at 6m 

in both genders. Minimal increases in alveolar 

macrophages and sparse brown-appearing 

macrophages in all species. 

Bacterial Clearance: Mice instilled with bacteria were 

mostly unaffected by exposure, except for a decline in 

histopathology summary score after 6m. 

Tumorigenesis: No values for exposed groups differed 

significantly from controls. There was no evidence of 

progressive exposure related trend. 

Reference: Reed et 

al. (2004, 055625) 

Species: Rat, 

Mouse 

Gender: Male and 

Female 

Strain: CDF 

(F344)/CrlBR (rat), 

A/J (mouse) 

Age: 12 wk 

DE: generated from two 2000 

model 5.9 L Cummins ISM turbo 

diesel engines 

Co-exposure to 8 gas and 8 

solid exhaust components 

measured 

Particle Size: 0.10 - 0.15 μm 

Route: Whole-body Inhalation 

Dose/Concentration: Low: 30 μg/m3 

Mid-low: 100 μg/m3

 

Mid-high: 300 μg/m3

 

High: 1000 μg/m3

 

Time to Analysis: 6 h/day, 7 days/wk for 1wk or 6 

mo. Analyzed 1 day post-exposure. 

Organ Weights: Kidney weight increased after 6m for 

both males and female rats at the high dose. Kidney 

and liver weight increased for female mice at all dose 

levels at 6 mo. Lung weight increased at high dose at 

6m for female mice and male rats. Spleen weight 

decreased in male mice at the low and mid-high levels. 

Cells: Minimal increases in alveolar macrophages and 

PM within the macrophages were seen. 

Cytokines: TNF-α decreased in female rats after 6m. 

Tumorigenesis: No significant effect was observed. 

Reference: Reed et 

al. (2008, 156903)

Species: Mouse 

Gender: Male, 

Female 

Strain: C57BL/6, 

A/J, BALB/c 

Age: NR 

Weight: NR 

GEE (2 1996 General Motors 

4.3-L V-6 engines; unleaded 

gasoline) 

Particle Size: 150 nm (MMAD) 

Route: Whole-body Inhalation 

Dose/Concentration: Control: 2.5 ± 2.9, Lowexposure: 6.6 ± 3.7, Mid-exposure: 30.3 ± 11.8, 

High-exposure: 59.1 ± 28.3, High filtered exposure: 

2.3 ± 2.6 μg/m3

Time to Analysis: Exposed 6 h/day, 7 days/wk, 3 

days-6 mo. 

Body and Organ Weight and Histopathology in A/J: 

Kidney weight decreased, but no effects pertaining to 

weight were significant. No visible inflammatory 

changes were seen. 

Lung Damage in A/J: No significant effect was seen, 

but hypomethylation was seen in females at 1wk, and 

methylation was reduced in all exposed female groups. 

Bacteria in Lungs of C57BL/6: Exposure did not 

affect the clearance of bacteria from the lung. 

Respiratory Allergic Response in BALB/c: Exposure 

had little effect, but serum total IgE increased 

significantly for the high-exposure group. Increasing 

trends were seen in OVA-specific serum IgE and IgG1, 

as well as neutrophils and eosinophils. 

December 2009 D-103 
Reference Pollutant Exposure Effects 

Reference: Reed et 

al. (2008, 156903) 

Species: Mouse 

Gender: Male, 

Female (only 

BALB/c) 

Strain: C57BL/6, 

A/J, BALB/c 

Age: NR 

Weight: NR

GEE (two 1996 General Motors 

4.3-L V-6 engines; regular, 

unleaded, non-oxygenated, nonreformulated gasoline blended to 

US average consumption for 

summer 2001 and winter 2001-

2002- Chevron-Phillips) 

Particle Size: 150 nm (MMAD) 

Route: Whole-body Inhalation 

Dose/Concentration: PM: Low- 6.6 ± 3.7 μg/m3, 

Medium- 30.3 ± 11.8 μg/m3, High- 59.1 ± 28.3 

μg/m3

Time to Analysis: A/J - exposed 6 h/days, 7 

days/wk, 3 days-6 mo. C57BL/6- 1wk exposure. 

Instillation of P. aeruginosa. Killed 18 h 

postinstillation. BALB/c- Conditioned to exposure 

chambers and mated. Pregnant females exposed 

GD 1 and throughout gestation. Offspring 

exposures continued until 4 wk-old. Half of 

offspring sensitized to OVA. Tested for airway 

reactivity by methacholine challenge 48 h postinstillation and euthanized.

The kidney weight of female A/J mice decreased at 6m 

and was strongly related to PM by the removal of 

emission PM. PM-containing macrophages increased 

by 6 mo. Hypomethylation occurred in females at 1 wk. 

The clearance of P. aeruginosa was unaffected by 

exposure. Serum total IgE significantly and dosedependently increased. OVA-specific IgE and IgG1 

gave slight exposure-related evidence but were not 

significant. 

Reference: Reed et 

al. (2008, 156903) 

Species: Rat 

Gender: Male, 

Female 

Strain: CDF 

(F344)/CrlBR, SHR

Age: NR 

Weight: NR

GEE (two 1996 General Motors 

4.3-L V-6 engines; regular, 

unleaded, non-oxygenated, nonreformulated Chevron-Phillips 

gasoline, U.S. average 

consumption for summer 2001 

and winter 2001-2002) 

Particle Size: 150 nm (MMAD) 

Route: Whole-body Inhalation 

Dose/Concentration: PM: Low- 6.6 ± 3.7 μg/m3, 

Medium- 30.3 ± 11.8 μg/m3

, High- 59.1 ± 28.3 

μg/m3

Time to Analysis: 6 h/day, 7 days/wk, 3 days-6 

mo. 

Organ Weight: At 6 mo. exposure, the heart weights of 

male and female rats increased and male rats’ seminal 

vesicle weight decreased. 

Histopathology: PM-containing macrophages 

increased by 6 mo. 

Lung DNA Damage: Hypermethylation occurred in 

medium- and high-exposure male rats at 6 mo. 

BAL: For both genders in the high-exposure group, 

LDH and MIP-2 significantly increased at 6 mo. ROS 

decreased at 1wk and 6 mo. Generally, the production 

of hydrogen peroxide and superoxide decreased in the 

high-exposure group and medium- and high-exposure 

groups, respectively. 

Removal of Emission PM: The removal of emission 

PM strongly linked PM to increased seminal vesicle 

weight, red blood cell counts, LDH, lipid peroxides, and 

methylation. 

Reference: 

Rengasamy et al. 

(2003, 156907) 

Species: Rat 

Gender: Male 

Strain: SD 

Weight: ~200 g 

DEP: SRM1650 

CB Elftex-12 furnace black, 

Cabot, Boston, MA 

Particle Size: NR 

Route: IT Instillation 

Dose/Concentration: 5, 15, or 35 μg/kg

Time to Analysis: Single; 1, 3, 5, 7 days post 

exposure 

CYP1A1: DEP at all doses significantly increased 

CYP1A1 protein, was maximal at 1 day, and 

normalized at 5 days. CB had no effect. 

CYP2B1: DEP and CB at 15 and 35 mg/kg inhibited 

activity at 1 day. 

Protein level significantly decreased at 1 day with 5, 15 

and 35 mg/kg DEP and at 15 and 35 mg/kg CB. A time 

dependent decrease was shown at 35 mg/kg for both 

DEP and CB. 

Reference: Renwick 

et al. (2004, 056067) 

Species: Rat 

Gender: Male 

Strain: Wistar Kyoto 

Weight: 370-470 g 

FCB: Fine Carbon Black (Huber 

990) 

UCB: Ultrafine Carbon Black 

(Printex 90, Degussa) 

FTO: Fine Titanium Dioxide 

(Tioxide) 

UTO: Ultrafine Titanium dioxide 

(Degussa) 

Particle Size: FCB: 260 nm; 

UCB: 14 nm; FTO: 250 nm; 

UTO: 29 nm 

Route: IT Instillation 

Dose/Concentration: 125 or 500 μg/rat 

Time to Analysis: Single, 24 h 

BALF Cells: UTO and UCB induced a large dosedependent increase in percent neutrophils (only 

statistically significant at 500 μg for UTO). 

BAL Inflammatory/Injury Markers: UTO and UCB 

also increased total protein content only at the 500 μg 

dose. UCB induced LDH release at 125 and 500 μg, 

UTO and CB at 500 μg. UTO and UCB induced large 

dose-dependent increases in GGT activity (only 

statistically significant at 500 μg for UTO). 

Phagocytosis: All 4 particles decreased but only at the 

500 μg level. 

Chemotaxis: Only UTO and UCB at 500 μg/l 

increased chemotactic migration. 

December 2009 D-104 
Reference Pollutant Exposure Effects 

Reference: Rhoden 

et al. (2004, 087969) 

Species: Rat 

Gender: Male 

Strain: SD 

Weight: 250-300 g 

CAPs 

(Boston, MA) 

Particle Size: CAPS: 0.1-2.5 μm 

Route: Whole-body Inhalation 

Dose/Concentration: 1060 ± 300 μg/m3

Time to Analysis: Single exposure for 5 h. 

Analyzed 24 h post-exposure. 

(CAPS-NAC = CAPS with 50 mg/kg bw NAS (Nacetylcysteine) pretreatment) 

Particle Characteristics: Major components did not 

appear to show any correlation to total particle mass. 

Included Na, Mg, Al, Si, S, Cl, K, Ca, Ti, V, Cr, Mn, Fe, 

Ni, Cu, Zn, Br, Ba, Pb. Metals Al, Si and Fe (somewhat 

less for Pb, Cu, K) correlated with TBARS. 

BALF Cells: CAPS increased PMN 4 fold. NAS 

treatment reduced this increase to control levels. 

BAL Inflammatory/Injury Markers: LDH and total 

protein not affected. Histology confirms slight 

inflammation with CAPS and no inflammation with 

CAPs-NAC.

Oxidative Stress: CAPS increased TBARS and 

oxidized protein by 2+ fold. NAS fully prevented the 

increase in TBARS and partially prevented an increase 

in protein carbonyl. 

Tissue Damage: Wet/dry ratio increased with CAPS 

but significantly decreased with NAC. 

Reference: Rhoden 

et al. (2008, 190475) 

Species: Rat 

Gender: Male 

Strain: SD 

Age: NR 

Weight: 300 g

Urban Air Particles (UAP) (SRM 

1649) 

Particle Size: NR 

Route: IT Instillation 

Dose/Concentration: 1mg in 100 μL saline 

Time to Analysis: Instilled with UAP. CL 

analysis:15 min post-exposure. BAL 

measurements: 4 h post-exposure. 

Some rats pre-treated with MnTBAP 2 h prior to 

UAP exposure.

UAP significantly increased the total cell number, PMN, 

MPO activity, and protein levels. MnTBAP prevented 

UAP-induced lung inflammation. UAP increased 

oxidants in lung CL, which was prevented by MnTBAP.

Reference: Rivero 

et al. (2005, 088653) 

Species: Rat 

Gender: Male 

Strain: Wistar Kyoto 

Age: 3 mo. 

Weight: 250 g 

Ambient air (Sao Paulo, Brazil) 

Particle Size: <2.5 μm 

Route: IT Instillation 

Dose/Concentration: 100 or 500 μg/rat; 0.4 or 2 

mg/kg

Time to Analysis: Single, 24 h 

Histopathology: At both doses, acute alveolar 

inflammation was observed and was more pronounced 

in the 500 μg group. 

Lung Morphometry: Lumen wall ratio values show a 

dose-dependent increase in peribronchial as well as 

intra-acinar pulmonary arterioles. No effect in 

myocardial arterioles were observed. 

Tissue Damage: Lung wet/dry ratios were unaffected. 

Reference: Roberts 

et al. (2004, 198903) 

Species: Rat 

Gender: Male 

Strain: SD 

Age: 60-90 days 

Weight: 300-350 g 

ROFA: SRI (cyclone power 

plant) 

Particle Size: NR 

Route: IT Instillation 

Dose/Concentration: 0.5 mg/rat; 1.67 mg/kg

Time to Analysis: Single, 6 and 24 h 

Technology: Laser capture microdissection of airway 

cells were used to analyze results. 

Protein: pERK1/2: ERK1/2 ratio increased by 60% at 

6 h and 80% at 24 h. NF-κB activity increased at 6 h 

but was not statistically significant. 

Reference: Saber et 

al. (2005, 097865) 

Species: Mouse 

Gender: Female 

Strain: TNF(-/-) (B6, 

129S-Tnftm1Gk1), 

C57/BL 

Age: 9-10 wk

DEP: SRM 2975 

CB: Printex 90 (Degussa) 

Particle Size: DEP: 215 nm; 

CB: 90 nm 

Route: Nose-only Inhalation 

Dose/Concentration: DEP: 20 mg/m3; CB: 20 

mg/m3

Time to Analysis: 90 min/day for 4 days 

consecutively, 1 h

BALF Cells: Neutrophils increased significantly to 15% 

when compared to control (4%) with DEP exposure. No 

response difference was observed between TNF (+/+) 

and TNF(-/-). CB did not induce any changes in 

neutrophil numbers. 

Cytokines: IL-6 increased 2-3 fold in DEP and CB 

exposure in both normal and knockout mice. IL-1β was 

unaffected. 

mRNA: In TNF (+/+) mice, DEP and CB increased 

expression of TNF mRNA 2- fold. IL-6 mRNA 

expression was high in DEP-exposed knockout mice 

when compared to normal mice. 

DNA: DNA strand breaks increased in both strains. 

Knockout mice showed a higher response to CB and 

DEP exposure. For normal mice, only CB induced a 

statistically significant effect. 

December 2009 D-105 
Reference Pollutant Exposure Effects 

Reference: Schins 

et al. (2004, 

054173) 

Species: Rat 

Gender: Female 

Strain: Wistar Kyoto 

Weight: 350-550 g 

Soluble fractions 

PMC: PM10-2.5

PMF: PM2.5 

-B: Borken, Germany (rural) 

-D: Duisburg, Germany (industrialized) 

Particle Size: PM10-2.5, PM2.5

Route: IT Instillation 

Dose/Concentration: 0.32 ± 0.01 mg/rat; 0.91± 

0.58 mg/kg

Time to Analysis: Single, 18 h 

BALF cells: Both PMC showed a massive increase in 

neutrophils. PMC-B induced the greatest increase 

followed by PMC-D. Both PMF did not induce a 

significant increase. 

BAL Inflammatory/Injury Markers: PMC from both 

sites induced markedly higher endotoxin concentration 

vs PMF as follows in decreasing order: PMC-B, PMCD, PMF-B, PMF-D, control. Glutathione decreased only 

for PMC-B. LDH and total protein were unaffected. 

Cytokines: TNF-α and IL-8 increased with PMC from 

both sites. PMF induced a slight increase in IL-8 but did 

not induce an increase in TNF-α. 

Radical Formation: Formation of hydroxyl radicals 

increased with exposure. Relative intensity was: PMCD, PMF-D, PMC-B, PMF-B, and control. 

Reference: 

Seagrave et al. 

(2005, 088000) 

Species: Rat 

Gender: Male 

Strain: F344/DCrl 

BR 

Age: 11 ± 1 wk 

PM from 3 sources: 

NT: New Technology bus, Detroit 

Diesel 50G, exhaust oxidation 

catalyst, 216 miles, 2002 model - 

in use 

NE: Normal emitter bus, Detroit 

Diesel 50G, no catalyst, 134259 

miles, 1997 model - in use 

HE: High Emitter bus, Cummins 

L10G, no catalyst, >250, 000 

miles, 1992, retired 

Fuel composition very similar for 

3 vehicles: methane (96-96.8%), 

ethane (1.6-1.9%), carbon 

dioxide (0.9-1.1%), nitrogen (0.6-

0.8%), traces of other gases 

Particle Size: NR 

Route: IT Instillation 

Dose/Concentration: 0.25-2.2 mg/rat in 0.5mL 

saline 

Time to Analysis: Single, 24 h 

Engine Specific Emission data: HE had significantly 

higher PM and SVOC recovered emission rates than 

NE and NT. 

Organic mass in PM: The following PM sources are 

listed in decreasing order of percent of total mass: HE, 

NE, NT. 

Total PAH: The following PM sources are listed in 

decreasing order of total mass: HE, NT, Control, NE. 

Nitro PAH: The following PM sources are also listed in 

decreasing order of total mass: NE, HE, Control, NT. 

Authors note confounding technical issues (mostly 

technique related) with mostly mild effects. 

BAL Inflammatory/Injury Markers: LDH showed 

dose-dependent increases with HE inducing higher 

increases than NT and NE. Total protein exhibited 

dose-dependent increases with HE, NT and the 

positive control SRM2975 inducing higher levels than 

NE. 

Potency Factors Cytotoxicity and Inflammation: HE 

was significantly more potent than NT and NE, with NT 

also showing significant potency. 

Lung Toxicity: The results were highly variable but the 

general toxicity levels in increasing order is the 

following: NE, NT, HE, Normal gasoline, diesels, and 

high gasolines, though individual factors may differ 

greatly. 

Reference: 

Seagrave et al. 

(2006, 091291) 

Species: Rat 

Gender: Male 

Strain: F344/Crl BR, 

Age: 11 ± 1 wk 

PM2.5 sources: BHM: Birmingham, Alabama; urban 

JST: Jefferson Street, Atlanta, 

Georgia; urban 

PNS: Pensacola, Florida; urban/ 

residential 

CTR: Centreville, Alabama; rural 

“smoke” = downwind of forest 

fires/burns (NR) 

Particle Size: PM2.5

Route: IT Instillation 

Dose/Concentration: 0.75, 1.5, 3 mg/rat 

Time to Analysis: Single, 24 h 

BALF PMN: In general, the winter samples induced 

greater increases in potency than the summer samples 

except for PNS. For the winter samples, the samples 

that induced the greatest increases, in descending 

order, are: JST, BHM, CTR, PNS and Smoke. For the 

summer, the samples that induced increases, in 

descending order, are: BHM, JST, PNS, and CTR. 

BALF Macrophages: For the winter, the BHM and JST 

samples significantly increased potency whereas the 

PNS sample induced significantly negative potency. 

For the summer, only the BHM sample significantly 

induced potency. 

BALF Lymphocytes: Only the JST-W and BHM-W 

significantly increased potency. The BHM-S, CTR-S 

and PNS-S also significantly increased potency. 

Histopathology: All the winter and summer samples, 

excepting PNS, significantly induced inflammation. 

Lung weight/body Weight Ratio: In general, for all 

end points, JST-S was significantly less potent than 

JST-W. The summer samples of BHM and CTR were 

also generally more potent than their winter 

counterparts. 

December 2009 D-106 
Reference Pollutant Exposure Effects 

Reference: 

Seagrave et al. 

(2005, 088000) 

Species: Rat 

Gender: Male, 

Female 

Strain: CDF(F344)/CrlBR 

Age: 10-12 wk 

DE: 

(Two 6 cyl Cummins ISB turb0) 

HWS = hardwood smoke 

(mixed black/white oak, 

uncertified conventional wood 

stove) 

DE: 

EC = 557 μg/m3

OC = 269 μg/m3

NO = 45 ppm 

NO2 = 4 ppm 

CO = 30 ppm 

THV = 2 ppm 

HWS: 

EC = 43 μg/m3

OC = 908 μg/m3

NO or NO2 = 0 ppm 

CO = 13 ppm 

THV = 3 ppm 

Particle Size: DE: 0.14 ± 1.8 

μm; HWS: 0.36 ± 2.1 μm 

Route: Whole-body Inhalation 

Dose/Concentration: 30, 100, 300, 1000 μg/m3

TPM 

Time to Analysis: 6 h/day, 7 days/wk for 6 mo. 1 

day post-exposure 

Particle Characteristics: Major differences K: 

HWS>>DE; Ca DE>>HWS; Zn: DE>>HWS. 

BALF Cells: No effects were observed except for an 

increase in macrophages at 30 μg/m3

 for HWS males 

exposed to HWS. 

Cytokines: IL-1β was unaffected by DE or HWS. MIP2 decreased for both genders at 1000 HWS. TNF-α

decreased in females with DE exposure. No TNF-α

effects for HWS were observed. 

BAL Inflammatory/Injury Markers: LDH was 

unaffected by DE. Exposure to HWS induced an 

increase for males only at 100 and 300 but not at 1000 

μg/m3

. Protein was unaffected by DE. HWS exposure 

showed male-only effects at 100 and 300 μg/m3

 but not 

at 1000. AP was unaffected by DE or HWS except for 

slight decline induced by HWS at 1000 μg/m3

 for both 

genders. 

Other: β-glucose was unaffected by DE. HWSexposed females showed decreased β-glucose at 100 

and 300 but not at 1000 μg/m3

. 

BALF GSH to (GSH+GSSG): No effects for DE were 

observed. HWS significantly decreased the ratio in both 

males and females at 1000 μg/m3

. The effect for 

females was greater than the male effect. 

Reference: 

Seagrave et al. 

(2008, 191990) 

Species: Rat 

Gender: Male 

Strain: SD 

Age: 10-12 wk 

Weight: 250-300 g

GEE (2 1996 General Motors 

4.3-L V6 gasoline engines; 

conventional Chevron Phillips 

gasoline, U.S. average 

composition) (CO, NO, NO2, 

SO2, THC) (PM2.5 compositionEC, OC, SO4, NH4, NO3) 

Simulated downwind coal 

emission atmospheres (SDCAs) 

(fly ash, gas-phase pollutants, 

sulfate aerosols, NO, NO2, SO2) 

Paved Road Dust (RD) (Los 

Angeles, CA; New York City, NY; 

Atlanta, GA) 

Particle Size: GEE: MMAD- 150 

nm, RD: 2.6 ± 1.7 μm, SDCA: 

0.1-1.0 μm 

Route: Nose-only Inhalation 

Dose/Concentration: GEE: 60 μg/m3, SDCAs: 

317-1072 μg/m3

, RD: 306-954 μg/m3; GEE: CO104 ppm, NO- 16.7 ppm, NO2- 1.1 ppm, SO2- 

1.0ppm, THC- 12 ppm; SDCAs: CO- <1 ppm, NO0.19-0.62 ppm, NO2- 0.10-0.37 ppm, SO2- 0.07-

0.24 ppm, THC- <1 ppm 

Time to Analysis: 6 h exposure, immediately 

post-exposure 

GEE produced CL in the lungs, heart, and liver. RD 

produced a significant effect in the heart at the low 

dose. SDCAs had no effect on CL. GEE did not affect 

the amount of macrophages or PMN. SDCAs increased 

macrophages. The RD low dose increased 

macrophages and PMN. SDCAs increased Penh 

values and tidal volumes. 

Reference: Singh et 

al. (2004, 087472) 

Species: Mouse 

Gender: Female 

Strain: CD-1 

Age: 6-8 wk 

A-DEP (4cyl light duty 2.7l Isuzu 

diesel at 6 kg/m) 

DEP: SRM 2975 

Particle Size: A-DEP >50 μm 

Route: Oropharyngeal Aspiration 

Dose/Concentration: 25 or 100 μg/mouse 

Time to Analysis: single, 4 h 

(18 h post-exposure measurements taken but NR 

due to similar results) 

Particle Characteristics: DEP had 60% EC vs 9% in 

A-DEP. A-DEP had 50% OC vs 5% in DEP. 

Phenanthrene and Fluoranthene fractions were much 

more prevalent in PAH from DEP than A-DEP. 

BALF Cells: PMNs significantly increased dosedependently with DEP and remained elevated at 18h. 

Endotoxin induced the greatest increases of PMNs. 

Macrophages increased with A-DEP and were 

unaffected by DEP. 

Cytokines: Endotoxin induced massive responses for 

IL-6, MIP-2 and TNF-α but no response from IL-5. ADEP increased all 4 cytokines but only at the 100 μg 

dose level. Similarly, DEP only increased IL-6 at the 

100 μg dose level.

BAL Inflammatory/Injury Markers: Microalbumin 

increased for both pollutants except DEP induced 

increases only at 100 μg. Endotoxin increased microalbumin. NAG increased with 100 μg A-DEP. 

December 2009 D-107 
Reference Pollutant Exposure Effects 

Reference: Smith et 

al. (2003, 042107) 

Species: Rat 

Gender: Male 

Strain: SD 

Age: 11-12 wk 

CAPs 

(Fresno, CA) 

Particle Size: <2.5 μm 

Route: Whole-body Inhalation 

Dose/Concentration: 6 exp in 2 sets of 3: 

Fall1 = 847 μg/m3

Fall2 = 260 μg/m3

Fall3 = 369 μg/m3

Winter1 = 815 μg/m3

Winter2 = 190 μg/m3

Winter3 = 371 μg/m3

Time to Analysis: 4 h/days for 3 consecutive 

days. Parameters measured immediately following 

last exposure. 

Particle Characteristics: Nitrate showed the highest 

variability near 10 fold, followed by Si, S and EC. OC 

concentration was relatively consistent. Metals 

otherwise appeared proportionate to the 

concentrations. 

BALF Cells: Total cells increased at wk1. Percent of 

macrophages reduced in wk2 with CAPs. Number of 

neutrophils increased with CAPs, but only achieved 

statistical significance during wk1 of the fall and winter. 

Lymphocytes increased but were not statistically 

significant. 

BAL cell permeability: Upon CAPs exposure, the 

proportion of nonviable cells were increased up to 

242% when compared to controls. The fall of wk2 

induced the highest significant increases followed by 

fall wk1, fall wk3, and winter wk3. 

Reference: Smith et 

al. (2006, 110864) 

Species: Rat 

Gender: Male 

Strain: SD 

Age: 8 wk 

Weight: 260-270 g 

CFA: Coal Fly Ash (400 MW, 

Wasatch Plateau, Utah) 

(aerodynamic separation) 

Particle Size: 0.4-2.5 μm 

Route: Nose-only Inhalation 

Dose/Concentration: 1400 μg/m3 PM2.5 including 

600 μg/m3

 PM1

Time to Analysis: 4 h/days for 3 consecutive 

days. Parameters measured 18 or 36 h postexposure. 

BALF Cells: Percent and total number of neutrophils in 

BALF and blood increased significantly at both 18 and 

36 h. Percent of macrophages decreased slightly while 

number of macrophages increased in bronchiolealveolar duct regions at both time periods. 

Cytokines: MIP-2 and transferrin increased at 18 h. IL1β increased at 36 h. 

Other: Gamma glutamyl transferase decreased at 

36 h. Lung antioxidant increased at 18 h. 

Reference: Song et 

al. (2008, 156093) 

Species: Mouse, 

Gender: Female 

Strain: BALB/c 

Age: 5-6 wk 

DEP collected from a 4JB1-type, 

light-duty (2740 cc), four-cylinder 

diesel engine operated using 

standard diesel fuel at speeds of 

1500 rpm under a load of 10 

torque. 

Particle Size: 0.4 μm (mean 

diameter) 

Route: Intranasal Instillation (days 1-5), Wholebody Inhalation (days 6-8) 

Dose/Concentration: 0.6 mg/mL in 50 μL of saline 

(days 1-5), 6mg/m3 for 1 h/day for 3 days (days 6-

8). 

Time to Analysis: Enhanced Pause (Penh), 

measured on day 9. BAL and lung tissues 

collected on day 10. 

Airway Hyperresponsiveness: Intranasal exposure 

plus aerosolized DEP caused a significant increase in 

methacholine-induced Penh over the control. 

BAL Analysis: There was no significant increase in 

IFN-γ in the BAL fluid following DEP treatment but 

there was a significant increase in IL-4 levels compared 

to the control. (IL-4 increase could indicate that DEP 

modulates Th-2 cytokines in the mouse model). DEP 

also induced an increase in total neutrophils and 

lymphocytes in the BAL when compared to the control. 

The nitrite concentration in BAL (indicating NO 

generation) was significantly greater in the DEP 

exposed group than the control. 

Histology: Peribronchial and perivascular infiltrates 

were more common in the group exposed to DEP than 

the control. 

Ym1 and Ym2 Expression: (see explanation in 

comments section) Ym1 and Ym2 transcripts were 

upregulated in response to DEP exposure in mice. 

Reference: 

Steerenberg et al. 

(2006, 088249) 

Species: Rat 

Strain: Crl/WKY 

Age: 6-8 wk 

Ambient air samples 

PMC, PMF: 

-I: Rome, Italy 

-N: Oslo, Norway 

-PL: Lodz, Poland 

-NL: Amsterdam, Netherlands 

Measured Components: Li, Be, 

B, Na, Mg, Al, K, Ca, Ti, V, Cr, 

Mn, Fe, Co, Ni, Cu, Zn, As, Se, 

Sr, Mo, Cd, Sn, Sb, Ba, Ce, Nd, 

Sm, Au, Hg, Tl, Pb, Bi, U, Si, 

Endotoxins, Cl, NO-, SO4 

Particle Size: PMC: 2.35-8.5 

μm; PMF: 0.12-2.35 μm 

Route: IT Instillation 

Dose/Concentration: 1 and 2.5 mg/animal

Time to Analysis: Single, 24 h 

Particle Characteristics: Concentrations of metals 

were highest in Rome. Amsterdam was noted for high 

Mg and V. Lodz was noted for high Pb, Zn, PAH. More 

of PMC was composed of Fe, Mn, Al, Cr, Cu. More of 

PMF, on the other hand, was composed of Zn, Pb, Ni, 

V. 

BALF Cells: PMNs increased.

Cytokines: MIP-2 increased dose-dependently. TNF-α

also increased. 

BAL Inflammatory/Injury Markers: CC16 decreased 

substantially. Crustal material (endotoxin, Na, Cl and 

metals but not Ti, As, Cd, Zn, V, Ni, Se) was positively 

associated with short term CC16. Albumin increased. 

December 2009 D-108 
Reference Pollutant Exposure Effects 

Reference: Stinn et 

al. (2005, 088307) 

Species: Rat 

Gender: Male and 

Female 

Strain: Crl: (WIU BR 

Age: 40 days 

DE (generated from 1.6 L VW 

diesel under USFTP 72) 

CO: 10, 37 ppm 

CO2: 2170, 6540 ppm 

NO: 7.0, 22.8 ppm 

NOX: 8.6, 28.3 ppm 

SO2: 0.83, 3.09 ppm 

NH4: ND 

Measured Major Components:

NO, SO2, 1-nitropyrene, Zi. 50% 

by DE weight is EC. 

Particle Size: 0.19-0.21 μm 

(MMAD) 

Route: Nose-only Inhalation 

Dose/Concentration: 3 and 10 mg/m3

Time to Analysis: 6 h/day, 7 days/wk for 24 mo; 6 

mo. post-exposure 

Body Weight: Mean weight increased substantially 

during the first few weeks in all groups. Food 

consumption decreased in 1-24 mo but was recovered 

in 24-30 mo. Body weight decreased at 23 mo in all 

categories, but recovered except in high dose males at 

30 mo. 

Organ Weight: Absolute weight of lungs, larynx and 

trachea increased from 0 to 12 to 24 mo and stayed 

elevated at 30 mo: Low<Hi, male ~ female. 

Pulmonary Parameters: Respiratory frequency, tidal 

volume, and minute volume were unaffected in any 

group measured between 3 and 24 mo. 

EC increased dose-dependently in exposure groups. 

No male/female difference was observed, but 

increases were greater at 24 mo than at 18 mo. 

BALF Cells: PMNs and lymphocytes showed dose and 

time-dependent effects at 18 and 24 mo (no data at 30 

mo). Lymphocytes increased 50 fold in high dose 

males at 24 mo. Peripheral monocytes and neutrophils 

increased 3 fold in DE groups at the end of the study. 

Particle-filled macrophages in alveolar lumen and 

interstitium increased at 12, 24, 30 mo in both genders 

at all dose levels. 

BAL Inflammatory/Injury Markers: LDH increased in 

dose and time-dependent manner. 

Nasal Cavity Histopathology: All effects were 

resolved at 30 mo. Nasal cavity hyperplasia increased 

at the high dose at 12 and 24 mo in both genders. 

Squamous metaplasia of respiratory epithelium 

increased in high dose females (12, 24 mo). 

Larynx Histopathology: No effects were observed. 

Lung Histopathology: Alveolar region hyperplasia of 

alveolar epithelium increased at 12, 24, 30 mo in both 

genders at all dose levels except for 12 mo low dose 

males and females. Above lung histopathology was not 

time-dependent, though perhaps some small dosedependence was observed. The following 

histopathology findings showed strong dose- and timedependent increases that occurred in both genders 

(24-30 mo): goblet cell hyperplasia of bronchial 

epithelia, cuboidal/columnar hyperplasia of alveolar 

epithelium, chronic active inflammation and septal 

fibrosis. 

Tumorigenicity: Lung tumors were more prevalent in 

females than males and appeared to be dosedependent. The major 3 types of tumors are the 

following:: bronchio-alveolar adenoma, bronchioloalveolar adenoma and benign keratinizing cystic cell 

tumors. Enhanced effects in females versus males may 

be the result of enhanced metabolism (body volume 

versus body weight) and increased respiratory 

volume/bw for females. 

December 2009 D-109 
Reference Pollutant Exposure Effects 

Reference: 

Sureshkumar et al. 

(2005, 088306) 

Species: Mouse 

Gender: Male 

Strain: Swiss 

Age: 10-12 wk 

Weight: 20-25 g 

GE: Gasoline Exhaust 

(Honda generator EBK 1200, 

four stroke one cyl) 

Including: SO2 = 0.11 mg/m3

NOX = 0.49 mg/m3

CO = 18.7 ppm 

Particle Size: GE 

>4 μm = 34.1% 

3-4 μm = 15.8% 

2-3 μm = 15.8% 

1.5-2 μm = 10.6% 

0.5-1.5 μm = 5.3% 

<0.5 μm = 18.4% 

Route: Nose-only Inhalation 

Dose/Concentration: 0.635 mg/m3

Time to Analysis: 15 min/day 7, 14 or 21 days; ; 

<1 h post-exposure 

BALF Cells: Neutrophils (%) increased at 7, 14 and 21 

days (stable). Total cell count, macrophages and 

eosinophils were unaffected. Leukocytes and 

lymphocytes increased, though not significantly. 

Cytokines: GE caused time-dependent increases in 

TNF-α and IL-6. IL-10 and IL-1β were unaffected. 

BAL Inflammatory/Injury Markers: y-GGT, ALP and 

LDH increased after 2 wk of GE exposure and stayed 

stable at 21 days. Total protein slightly increased on 14 

and 21 days, though these increases were not 

statistically significant. 

Histopathology: Minor changes at 7 days, mild edema 

in alveolar region at 14 days and sloughing of epithelial 

cells in bronchiolar region and focal accumulation of 

inflammatory cells in alveolar region at 21 days were 

observed in a time-dependent manner. 

Reference: 

Tesfaigzi et al. 

(2002, 025575) 

Species: Rat 

Gender: NR 

Strain: BrownNorway 

Age: 7-8 wk 

Weight: 310-330 g 

WS (wood stove- Vogelzang 

Boxwood Stove, Model BX-42E, 

wood- Pinus edulis) (CO, NO, 

NOX, total hydrocarbon) 

Particle Size: Smaller size 

fraction: 0.405-0.496 μm, larger 

size fraction: 6.7-11.7 μm

Route: Whole-body Inhalation 

Dose/Concentration: Target concentration (low, 

high exposure): 1, 10 mg/m3

; CO- 15-106.4 ppm, 

NO- 2.2-18.9 ppm, NOX- 2.4-19.7 ppm, total 

hydrocarbon- 3.5-13.8 ppm 

Time to Analysis: 3 h/day, 5 days/wk, 4 or 12 wk. 

Respiratory Function: Total pulmonary resistance 

increased for exposure groups and was significant for 

the low-exposure group. In exposed groups, forced 

expiratory flows and quasistatic compliance were lower 

and dynamic lung compliance higher, the latter being 

significant for the high-exposure group. For the highexposure group, vital capacity slightly decreased, 

residual volume slightly increased, and CO-diffusing 

capacity had a slight, significant decrease. 

BALF Cells: Macrophages decreased significantly in 

the high-exposure group. Particle-laden macrophages 

increased with concentration. Lymphocytes and 

neutrophils slightly increased in the high-exposure 

group. 

Cytokines: LDH increased slightly and protein levels 

decreased slightly in the high-exposure group. 

Cytokines were below detectable levels. 

Histopathology: WS caused minimal to mild chronic 

inflammation in the epiglottis of the larynx. PAS-positive 

cells increased in the 30 day high-exposure group. AMs 

increased with time and concentration. Particle-laden 

macrophages were seen after 90 days. AB- and PASpositive epithelial cells increased for the 90 day low 

exposure group. 

Reference: Tin-TinWin-Shwe et al. 

(2006, 088415) 

Species: Mouse 

Gender: Male 

Strain: BALB/c 

Age: 7wk 

CB14: Printex 90 (Degussa) 

CB90: Flammruss 101 

(Degussa) 

Particle Size: CB14: 14 nm 

CB95: 95 nm 

Route: IT Instillation 

Dose/Concentration: 25, 125, 625 μg/mouse; 

approx. 1, 5, 25 mg/kg

Time to Analysis: 1/wk for 4wk. 4 h postexposure 

Body Weight, Thymus, Spleen, Splenic Cell Count:

No effects were observed. 

BALF Cells: Increased total cell numbers were 

observed for 125, 625 μg CB14 (dose-dependent) and 

625 μg CB95. Total cell count was twice as high for 

CB14 at 125 and 625 μg compared to CB95. AM 

numbers exhibited a dose-dependent response for both 

CB14 and CB95 for all doses except 125 μg. Lymphocyte numbers increased at 125 and 625 μg for CB14 

and 625 μg for CB95. PMN numbers increased at 125 

and 625 μg for CB14 and CB95, but the response was 

greater with CB14. PMN numbers were proportional to 

dose surface area for both PM sizes. 

BAL Cytokines: CB14 and CB95 induced dosedependent increases in IL-1β. TNF-α increased at 125 

and 625 μg dose in CB14 with the 125 dose inducing a 

slightly greater increase. CB14 and CB95 induced 

CCL-3 increases 125 and 625 μg. 

Chemokine mRNA in lung and lymph nodes: CCL-3 

mRNA increased for CB14 but not CB95 4 h following 

the last exposure. CCL-2 was unchanged. 

Mediastinal lymph nodes: The number of CB-laden 

phagocytes increased in a dose-dependent manner for 

CB14 and CB95. CB14 had higher numbers at all 

doses compared to CB95. 

December 2009 D-110 
Reference Pollutant Exposure Effects 

Reference: Tong et 

al. (2006, 097699) 

Species: Mouse 

Gender: Male 

Strain: KP600 CD-1 

Weight: 22-26 g 

PM2.5 (collected from stacked 

filter air sampler in Shanghai, 

China) 

Fe: FeSO4

Zn: ZnSO4

PMF: PM2.5 + FeSO4

PMFZ: PM2.5 + FeSO4 + ZnSO4

Major Measured Components:

Fe 26 ppm, Zn 9 ppm, S 61 ppm 

Particle Size: PM2.5

Route: IT Instillation 

Dose/Concentration: PM: 25 mg/mL, 1mg/mouse 

Fe: 15mg/mL, 0.6 mg/mouse 

Zn: 15mg/mL, 0.6 mg/mouse 

PMF: PM 25 mg/mL + Fe 15 mg/mL, 

1.6 mg/mouse 

PMFZ: PM 25 mg/mL + Fe 15 mg/mL, 

1.6 mg/mouse 

Time to Analysis: Instilled twice at 0 and 24 h. 

Parameters measured 24 h following last exposure 

(at 48 h). 

Synchrotron X-ray imaging: PMFZ showed the 

greatest increase in alveolar changes. Fe induced 

more hemorrhagic changes, whereas Zn induced more 

nonuniformity of lung texture. This suggests that Zn 

induces PBMC in a dose-dependent manner which 

releases IL-1, IL-6, TNF-α, and IFN-y. 

Histopathology: PMFZ induced the most severe 

changes including serious inflammation/pus in bronchia 

and bronchial epidermal cell hyperplasia. For Fe or 

PMF hemorrhagic changes predominated but were less 

severe than PMFZ. 

Reference: 

Upadhyay et al. 

(2008, 159345) 

Species: Rat 

Gender: Male 

Strain: SH

Age: 6 mo. 

Weight: NR

Ultrafine Carbon Particles 

(UFCP) 

Particle Size: Size- 31 ± 0.3 nm, 

MMAD- 46 nm, Surface area 

concentration- 0.139 m2

particles/m3

, Mass specific 

surface area- 807 m2/g 

Route: Whole-body Inhalation 

Dose/Concentration: 172 μg/m3

Time to Analysis: 24 h exposure. 4 days 

recovery. Sacrificed 1st or 3rd day of recovery. 

Pulmonary Inflammation: UFCP did not cause 

pulmonary inflammation. 

Pulmonary and Cardiac Tissue: HO-1, ET-1, ETA, 

ETB, TF, PAI-1 significantly increased in the lung on 

the 3rd recovery day. HO-1 was repressed in the heart, 

but the other markers had slight, nonsignificant 

increases.

Reference: 

Wallenborn et al. 

(2007, 156144) 

Species: Rat 

Gender: Male 

Strain: WKY, SH,, 

and stroke-prone SH 

(SHRSP) 

Age: 12-15 wk 

PM: precipitator unit power plant 

residual oil combustion 

Particle Size: PM: 3.76 μm 

(bulk) ± 2.15 

Route: IT Instillation 

Dose/Concentration: WKY vs SHRSP: 1.11, 3.33, 

8.33 mg/kg 

SH vs SHRSP: 3.33, 8.33 mg/kg

Time to Analysis: Single, 24 h 

Note: 4 h post-exposure study done on WKY vs 

SHRSP but not published. 

BALF Cells: A dose-dependent increase in total cells 

and neutrophils was observed. Equal response for all 3 

strains except for SH, for both concentrations was 

observed. 

BAL inflammation/Injury Markers: LDH exhibited a 

dose-dependent increase in equal response for all 3 

strains. WKY had higher baseline levels of NAG activity 

but, upon PM exposure, SHRSP induced higher 

increases than WKY. GGT exhibited a dose-dependent 

response for all 3 strains. SHRSP showed the highest 

increase followed by WKY and SH. Protein levels 

increased at the high dose level with SHRSP exhibiting 

the highest increases followed by SH and WKY. 

Albumin levels were inconsistent between experiments.

Oxidative Stress - Lung: (WKY vs SHRSP only): SOD 

decreased following increased exposure levels with 

SHRSP levels generally higher than WKY. Ferritin 

levels declined only in SHRSP. 

GPx: No action but SHRSP levels were similar to SHR 

and, in the WKY vs SHRSP experiment, SHRSP 

exhibited higher activity level than WKY. 

Ferritin: Equivocal results were observed. Levels 

decreased at the high dose for WKY and SHRSP but 

increased at medium doses for SH and SHRSP. 

ICDH: Levels increased for WKY and decreased for 

SHRSP. 

Reference: 

Wallenborn et al. 

(2008, 191171) 

Species: Rat 

Gender: Male 

Strain: Wistar Kyoto 

Age: 13 wk 

Weight: NR

Zinc Sulfate (ZnSO4, 

aerosolized) 

Particle Size: NR 

Route: Nose-only Inhalation 

Dose/Concentration: 9.0 ± 2.1 μg/m3, 35 ± 8.1 

μg/m3

, 123.2 ± 29.6 μg/m3

Time to Analysis: Exposed 5 h/days, 3 days/wk, 

16 wk. Half of the rats used for plasma/serum 

analysis, other half for isolation of cardiac 

mitochondria.

A trend toward increased BALF protein was seen. No 

pulmonary-related effects were seen. 

December 2009 D-111 
Reference Pollutant Exposure Effects 

Reference: 

Wegesser and Last 

(2008, 190506) 

Species: Mouse 

Gender: Male 

Strain: BALB/c 

Age: 8-10 wk 

Ambient PM2.5-10

Collected from San Joaquin 

Valley, CA 

Particle Size: PM10-2.5

Route: IT Instillation 

Dose/Concentration: 25-50 μg/mouse

Time to Analysis: 3, 6, 18, 24, 48, 72 h post IT 

instillation. 

BALF Cells: Increased amount of viable cells found in 

PM-exposed mice with dose-response relationship 

between dose of PM and number of total cells 

recovered in BALF. At 6 h, increased numbers of 

macrophages at both 25 and 50 μg/mouse. Increased 

percentage of neutrophils observed with 50 μg/mouse 

PM only. Furthermore, both macrophages and 

neutrophils increased with longer time period from 

instillation, peaking at 24 h. At 50 μg/mouse, MIP-2 

concentrations increased, peaking at 3 h, though not 

statistically significant and returned to basal levels by 

6 h. Positive correlation observed between MIP-2 

concentration and increased neutrophil counts. No 

correlation found between MIP-2 and macrophages. 

Reference: 

Whitekus et al. 

(2002, 157142) 

Species: Mouse 

Gender: Female 

Strain: BALB/c 

Age: 6-8 wk 

Weight: NR 

DEP (light-duty, four-cylinder 

engine- 4JB1 type, Isuzu 

Automobile, Japan; standard 

diesel fuel) (extracts) 

Particle Size: 0.5-4 μm

Route: Inhalation 

Dose/Concentration: 200, 600, 2000 μg/m3

Time to Analysis: Exposed 1 h/day,10 days. 

Animals receiving OVA had 20 min OVA exposure 

after DEP exposure. 

DEP+OVA dose-dependently increased IgE and IgG1, 

being more effective than the OVA-alone treatment. 

This effect was significantly suppressed by thiol 

antioxidants NAC or BUC. DEP+OVA increased 

carbonyl protein and lipid peroxide over OVA. NAC or 

BUC suppressed lipid peroxide and protein oxidation. 

No general markers for inflammation were observed. 

Reference: Wichers 

et al. (2004, 055636) 

Species: Rat 

Gender: Male 

Strain: SH 

Age: 75 days 

PM (HP-12): inside wall of stack 

of Boston, MA power plant 

burning # 6 oil. 

Particle Size: PM: 3.76 μm ± 

2.15 

Route: IT Instillation 

Dose/Concentration: 0.83, 3.33 or 8.33 mg/kg

Time to Analysis: single, 6 h for Whole-body 

plethysmographs (WBP) and repeated daily for 4-7 

days, 

96 or 192 h post-exposure 

non-WBP animals: single, 

24, 96, 192 h post-exposure 

Tidal Volume: A dose-dependent decrease in tidal 

volume (45 % at high dose) was sustained for 1 day 

with very slow recovery over 7 days. 

Breathing Frequency: Dose-dependent increase (100 

% at high dose) with recovery at 7 days was observed. 

Minute Ventilation: Small dose-dependent increases 

were observed with a return to normal ventilation in 2 

days. 

Penh (enhanced pause): Equivocal results in all 

groups were observed (due to major control variation). 

BALF Cells: Dose-dependent increases in total cells at 

24 h, with declined, but still elevated, levels at 192 h. 

Neutrophils increased significantly (10 fold) at 24 h in 

the mid and high dose groups and showed declined, 

but still elevated, levels at 192 h. Macrophages slowly 

increased in a dose-dependent manner at 192 h. 

BAL Inflammatory/Injury Markers: Protein and 

albumin increased at 24 h, returned to relative basal 

level at 192 h at the mid and high dose levels. NAG 

exhibited dose-dependent increases at 24 h and 

sustained these levels through 192 h. 

Reference: Wichers 

et al. (2006, 103806) 

Species: Rat 

Gender: Male 

Strain: SH 

Age: 71-73 days 

Weight: 255-278 g 

PM (HP-12): inside wall of stack 

of Boston, MA power plant 

burning # 6 oil. 

Particle Size: 1.95 μm ± 3.49 

Route: Whole-body Inhalation 

Dose/Concentration: 13 mg/m3 

Time to Analysis: Phase I: 1st day, filtered air, 

2nd day, 6 h of PM 

Phase II: 1st day filtered air, 4 days of 6 h PM each

Immediate post-exposure 

Body/ Lung Weight: No effects on Phase I rats were 

observed. HP-12 exposure increased body weight, left 

lung, right intercostal, and right diaphragmatic lobes in 

Phase II rats. However, results appeared due to normal 

growth in juvenile rats over 4 days. 

Lung lobe to Body Weight Ratio: No effects at 1 or 4 

days were observed. 

Deposition calculations: V and Co were used to 

estimate deposition rates (good correlation between 

two metals at R2 = 0.94). Total HP-12 deposition using 

Co was 26 and 99 μg (for 1 day and 4 day 

experiments) and using V was 31 and 116 μg. 

Modeling information estimated HP-12 deposition at 

43% in conducting airways and 57% in alveolar region. 

Breathing parameters: No changes were observed for 

1 or 4 days studies except for a possible decrease in 

frequency for the 1 day study. 

December 2009 D-112 
Reference Pollutant Exposure Effects 

Reference: Witten 

et al. (2005, 087485) 

Species: Rat 

Gender: Female 

Strain: F344 

Age: 8 wk 

Weight: ~175 g 

DEP (heavy-duty Cummins N14 

research engine operated at 

75% throttle) 

Particle Size: 7.234-294.27 nm 

Route: Nose-only Inhalation 

Dose/Concentration: Low- 35.3 ± 4.9 μg/m3, 

High- 632.9 ± 47.61 μg/m3 

Time to Analysis: Exposed 4 h/day, 5 days/wk, 

3 wk. Pretreated with saline or capsaicin. 

There were no differences for substance P. The lowexposure group had significantly less NK1. DEP 

reduced NEP activity. Plasma extraversion dosedependently increased and was greatest in capsaicin 

animals. Respiratory permeability dose-dependently 

increased. IL-1β was significantly higher for the lowexposure group. IL-12 was significantly lower in the 

capsaicin high-exposure group. TNF-α increased in the 

high-exposure group and capsaicin low-exposure 

group. High exposure induced particle-laden AMs in the 

lungs, perivascular cuffing consisting of mononuclear 

cells, alveolar edema and increased mast cell number. 

Neutrophil and eosinophil influx was not seen. 

Reference: Wong et 

al. (2003, 097707) 

Species: Rat 

Gender: Female 

Strain: F344/NH 

Age: ~4 wk 

Weight: ~175 g 

DEP (Cummins N14 research 

engine at 75% throttle) (EC34.93-601.67 μg/m3

, OC- 1.90-

11.25 μg/m3

, Sulfates 0.94-17.96 

μg/m3

, Na- 4.07-4.78 ng/m3, Mg0.60-0.86 ng/m3

, Ca- 5.05-10.66 

ng/m3

, Fe- 3.17-6.44, Cr- 0.68-

1.31 ng/m3

, Mn- 0.11-0.22 ng/m3, 

Pb- 0.97-1.24 ng/m3

) 

Particle Size: 7.5-294.3 nm 

Route: Nose-only Inhalation 

Dose/Concentration: Low- 35.3 ± 4.9 μg/m3, 

High- 669.3 ± 47.6 μg/m3 

Time to Analysis: Exposed 4 h/day, 5 days/wk, 

 3 wk. Pretreated with saline or capsaicin. 

DEP dose-dependently increased plasma extraversion, 

which was further increased by capsaicin. In the highexposure group, particle-laden AMs (which were 

reduced by capsaicin), inflammatory cell margination, 

perivascular cuffing with subsequent mononuclear cell 

migration and dispersal, increased mast cells, and 

decreased substance P were all seen. NK-1R was 

downregulated in the low-exposure group and 

upregulated in the capsaicin-pretreated high-exposure 

group. NEP decreased significantly for both groups. 

Reference: Wu et 

al. (2003, 199749) 

Species: Rat 

Gender: Male 

Strain: SD 

Age: 60 days 

Zn2+

Particle Size: NA 

Route: IT Instillation 

Dose/Concentration: 50 μm/rat 

Time to Analysis: Single, 24 h 

Cells: Decreased number of airway epithelial cells 

shown with PTEN protein immunostaining. 

Macrophages were unaffected. 

Reference: 

Yamamoto et al. 

(2006, 096671) 

Species: Mouse 

Gender: Male 

Strain: BALB/c 

Age: 7 wk 

Weight: 23 g 

CB14: Printex 90 (Degussa) 

CB95: Flammruss 101 

(Degussa) 

LTA: Lipoteichoic acid 

14CL: CB14 + LTA 

95CL: CB95 + LTA 

CB14 measured Components: C 

96.79%, HR 0.19%, N0.13%, S 

0.11%, Ash 0.05%, O 2.74% 

CB95 measured Components: C 

97.98%, HR 0.15%, N 0.28%, S 

0.46%, Ash 0%, O 1.14% 

Particle Size: CB14: 14 nm; 

CB95: 90 nm 

Route: IT Instillation 

Dose/Concentration: CB14: 0, 25, 125, 625 

μg/mouse 

CB95: 0, 25, 125, 625 μg/mouse 

LTA: 10 or 50 μg/mouse 

14CL: 125 μg CB14 + 10 or 50 μg LTA 

95CL: 125 μg CB95 + 10 or 50 μg LTA 

Time to Analysis: Single, 4 and 24 h 

BALF Cells: CB95 induced dose-dependent increases 

of PMN. CB14 induced an increase in PMNs but the 

increases were not dose-dependent. LTA massively 

increased PMN. LTA induced dose-dependent 

increases in total cells, especially at high dose at 24 h. 

LTA had massive synergistic effect with CB14 and 

CB95 for total cells and PMNs. Total cell count and 

PMN levels were highest in 14CL with levels at 24 h 

higher than at 4 h. Macrophage data were inconsistent. 

Cytokines: CB95 induced dose-dependent increases 

in IL-6, TNF-α, CCL2 and CCL3. CB14 induced dosedependent increase in CCL2 and CCL3. Exposure 

induced increases of IL-6 at the high dose only. Slight 

effect on TNF-α was observed. LTA induced dosedependent increases of IL-6, TNF-α and CCL3. 14CL 

massively induced IL-6 and CCL2. No combination of 

CB and LTA affected TNF-α or CCL3. 

mRNA Expression: LTA, 14CL and 95CL increased 

TLR2 mRNA expression with 95CL and 14CL inducing 

higher increases than LTA. No effect on TLR4 mRNA 

expression was observed. 

December 2009 D-113 
Reference Pollutant Exposure Effects 

Reference: 

Yanagisawa et al. 

(2003, 087487) 

Species: Mouse 

Gender: Male 

Strain: ICR 

Age: 6 wk 

Weight: 29-33 g 

DEP: 

(4JB1 light duty 4cyc 2, 74 liter 

Isuzu engine) 

LPS 

DEP-OC: organic compounds 

DL: DEP + LPS 

DOL: DEP-OC + LPS 

Particle Size: 0.4 μm 

Route: IT Instillation 

Dose/Concentration: DEP/DEP-OC: 125 

μg/mouse 

LPS: 75 μg/mouse 

Time to Analysis: Single, 24 h 

BALF Cells: DEP and DEP-OC increased neutrophils 

but the increases were not statistically significant. LPS 

increased neutrophils significantly. DL and DOL 

massively increased neutrophils at greater levels than 

LPS alone. Macrophages were unaffected. 

Cytokines: LPS increased IL-1β, MIP-1α, MCP-1 and 

KC. DEP and DEP-OC had no effect. DL induced 

further increases. DOL decreased cytokines compared 

to LPS alone. DEP-OC increased IL-1β and MIP-1α

mRNA expression slightly. DEP had no effect. LPS 

significantly increased IL-1β and MIP-1α mRNA 

expression. DL increased expressions while DOL did 

not. 

Pulmonary Edema: LPS, DEP and DEP-OC increased 

edema. DL further increased this effect. DOL had no 

effect compared to LPS alone. 

Histology: DL elevated neutrophil inflammation 

interstitial edema and alveolar hemorrhages. DOL 

induced neutrophilic inflammation without the alveolar 

hemorrhages. 

mRNA Expression of TLRs: DEP-OC, DL, DOL and 

LPS increased TLR2. DEP had no effect. All particles 

increased TLR4 mRNA expression. 

Reference: 

Yokohira et al. 

(2007, 097976) 

Species: Rat 

Gender: Male 

Strain: F344/DuCrj 

Age: 10 wk 

DQ-12: Quartz dust (Douche 

Montan) 

HT: Hydrotalcite (Kyoward 500, 

PL-1686, KYOWA) 

POF: Potassium Octatitanate 

fiber (TISMO, Otsuka) 

PdO: Palladium Oxide 

CB: Carbon Black (Mitsubishi 

Kasei) 

Particle Size: DQ12 <7 μm 

HT: 7.8 ± 1.5 μm 

POF: <50 um length; <2 μm 

width 

PdO: 0.54 ± 1.11 μm 

CB: 28 nm 

Route: IT Instillation 

Dose/Concentration: 4 mg/rat in 0.2 ml saline 

Time to Analysis: Single, 1 and 28 days 

Lung Weight/Body Weight Ratio: DQ-12, HT and 

POF induced increases after 1 day. After 28 days, all 

samples induced increases in lung weight. 

BALF Cells: Neutrophils increased significantly in 

walls and alveolar spaces in all groups on 1 day except 

at HT. At 28 days, this increase was maintained only in 

walls with severe and moderate elevations, except for 

DQ-12. 

Histopathology: DQ-12 caused pulmonary edema 

both at 1 and 28 days. PdO and CB induced edema at 

28 days. Fibrosis was observed after 28 days with the 

most significant increase, in decreasing order, induced 

by DQ-12,PdO, POF, HT, CB, and the control. 

Histiocyte infiltration was observed after 1 day for DQ12, POF and PdO. At 28 days, infiltration was observed 

for DQ-12, HT, POF and PdO. Restructuring of alveolar 

walls and microgranulation was observed for all 5 

particles but only at 28 days with DQ 12, PdO, HT, 

POF, CB and control. 

Immunohistochemistry: BrdU: At 1 day all 5 particles 

elevated in both area and number. Activity declined 

after 28 days but was still higher than the control. 

iNOS: At 1day DQ-12, POF and PdO induced 

increases. At 28 days, DQ-12 and HT induced increases. 

MMP-3: DQ-12 induced increases at both 1 and 28 

 days and PdO at 28 days. 

Toxicity scoring: The levels of toxicity are, in 

decreasing order, as follows: DQ-12, HT/PdO/POF, and 

CB. 

December 2009 D-114 
Reference Pollutant Exposure Effects 

Reference: Zhao et 

al. (2006, 100996) 

Species: Rat 

Strain: SD 

Age: NR 

Weight: 200 g 

DEP: SRM 2975 

DEPE: SRM 1975 

Particle Size: NR 

Route: IT Instillation 

Dose/Concentration: 35 mg/kg 

Time to Analysis: Single, 1 day 

AG group coexposed 30 pre and 3, 6, 9 h post 

DEP/DEPE 

iNOS Expression in AMs: Both DEP and DEPE 

increased 12 and 6 fold respectively. NO and 

peroxynitrite levels increased accordingly. AG had no 

effect on iNOS expression but AG attenuated NO for 

both DEP and DEPE but peroxynitrite only for DEPE. 

DEP induced much higher levels of oxidants than 

DEPE. Unlike DEPE, DEP was unaffected by AG. 

Role of iNOS in Lung Injury: DEP and DEPE induced 

inflammation (PMN), cellular toxicity (LDH) and lung 

injury (protein). AG significantly attenuated the DEPE 

response but no effect was observed on the DEP 

responses. 

Cytokines: IL-12 levels were induced by both DEPE 

and DEP, with DEPE inducing higher increases than 

DEP, and both were significantly attenuated by AG. 

DEP and DEPE induced similar increases in IL-10 

levels. AG increased DEP effect 3 fold and attenuated 

DEPE to control. 

CYP Enzymes: DEP and DEPE induced increases in 

CYP1A1 level and activity. AG attenuated CYP1A1 

activity for both DEP and DEPE. CYP2B1 level and 

activity were slightly decreased by DEP and DEPE. AG 

had no effect. 

Cytosol Phase II Enzymes: DEPE had no effect; AG 

treatment increased catalase activity. DEP reduced 

catalase and GST activities. AG had no effect. Neither 

DEP, DEPE nor AG affected QR quinone reductase. 

Reference: Zhou et 

al. (2003, 087940) 

Species: Rat 

Gender: Male 

Strain: SD 

Age: 10-12 wk 

UFe: Ultrafine Fe particles 

Particle Size: 72 nm 

Route: Whole-body Inhalation 

Dose/Concentration: 57 or 90 μg/m3

Time to Analysis: 6 h/days for 3 days, parameters 

measured within 2 h post-exposure. 

BALF Cells: No significant changes observed in total 

cell number, cell viability or cell differentials. 

Cytokines: Only at the high dose was an increase in 

IL-1β observed. No effect on TNF-α or NF-κB-DNA 

binding activity was observed.

BAL Inflammatory/Injury Markers: At the high dose, 

total protein increased. No significant changes were 

observed in LDH. 

Intracellular Ferritin: The high dose induced 

increases. No significant differences were observed 

between the low dose and control. 

Oxidative stress: Antioxidant level by FRAP value 

decreased at the high dose. GST (glutathione-Stranferase) activity increased at the high dose. No 

effect on intracellular GSH and GSSG (glutathione 

disulfide) was observed. 

December 2009 D-115 
Table D-4. Effects related to immunity and allergy. 

Study Pollutant Exposure Effects 

Reference: 

Apicella et al. 

(2006, 096586)

Species: Mouse 

Strain: BALB/c 

Cell Line: 112D5 

hybridoma 

Primary 

Macrophages: 

Peritoneal 

Poly OVA (Ovalbumin on polystyrene 

beads) Soluble OVA 

Particle Size: NR 

Route: Cell Culture 

Dose/Concentration: PolyOVA and Soluble OVA: 

0.2,1.0 or 5.0 μg/mL

Time to Analysis: 48 h 

IL-6: Stimulation with PolyOVA higher than 

stimulation with soluble OVA 

TNF-α: Stimulation with PolyOVA higher than 

stimulation with soluble OVA. 

IL-10: No modifications in levels after PolyOVA 

or soluble OVA stimulation. 

Viability of Peritoneal Macrophages:

Stimulation with PolyOVA led to 33% decrease 

in viability. Stimulation with soluble OVA led to 

24% in viability. 

Effects of PolyOVA Stimulated Macrophages:

Culture supernatants from PolyOVA stimulated 

macrophages had a percentage increase of 

asymmetric IgG; however, the addition of rmIL-6 

at identical concentrations did not induce a 

significant increase. It also decreased the 

proliferation of 112D5 hybridoma. 

Reference: 

Arantes-Costa et 

al. (2008, 187137) 

Species: Mouse 

Gender: Male 

Strain: BALB/c 

Age: 6 wk 

Weight: NR

ROFA (solid waste incinerator powered 

by combustible oil; São Paulo, Brazil) 

Particle Size: NR 

Route: Intranasal Instillation 

Dose/Concentration: 60 μg ROFA in 50 μL saline 

Time to Analysis: OVA sensitized days 1 and 14. 

OVA-challenged days 22, 24, 26, and 28. ROFA 

exposed 1-3 h after OVA challenge or saline. 

Pulmonary responsiveness measured day 30 then 

sacrificed. Lungs removed, fixed for 48 h. 

ROFA increased pulmonary responsiveness and 

decreased ciliated cells in nonsensitized mice, 

which were both further amplified in the 

presence of OVA. ROFA did not affect 

eosinophils, macrophages, chronic 

inflammation, or neutral or acidic mucus. 

Reference: Archer 

et al. (2004, 

088097) 

Species: Mouse 

Strain: BALB/c 

DO11.10+/+ 

transgenic - ova 

specific receptor 

for OVA peptide 

323-339 

Age: 4 wk 

PM = SRM 1648 (NIST) 

TiO2

Particle Size: 

SRM1648: avg 1.4 μm

TiO2: avg 0.3 μg (sic) 

Route: Intranasal instillation 

Dose/Concentration: 500 μg/30 μl sterile saline, 

initial 0-750 μg range finding 

Time to Analysis: Ova challenge at 68 h, Methacholine aerosolization/AR at 72 h 

Airway responsiveness (WBP): AR induced by 

Ova/Mch challenge was significantly and dosedependently increased at doses of 

SRM1648 ≥500 μg . TiO2/Ova exposure was not 

significantly different from saline. PM associated 

endotoxin did not contribute to enhanced AR. 

Lung inflammation/pathology: No increases 

in BAL macrophages or eosinophils and no 

histological alterations after PM exposure. Both 

TiO2 and PM increased pulmonary neutrophils, 

indicating particles alone were responsible for 

this increase and that the inflammatory 

response could occur independently of AR. 

Reference: Barrett 

et al. (2006, 

155677) 

Species: Mouse 

Gender: Male 

Strain: BALB/c 

Age: 8-10 wk 

HWS (black/white oak) 

CO 

Total Vapor Hydrocarbon (TVH) 

Particle Size: 0.25 ± 3.3, 0.35 ± 2.5, 

0.35 ± 2.0, 0.36 ± 2.1 μm (MMAD±GSD) 

Route: Whole-body Inhalation 

Dose/Concentration: HWS: 30, 100 300, 

1000 μg/m3

CO: 0.7, 1.6, 4.0, 13 ppm 

TVH: 0.3, 0.6, 1.3, 3.1 ppm 

Time to Analysis: Pretreatment: ip 10 μg OVA and 

2 mg aluminum hydroxide post-OVA. OVA aerosol 

challenge on day 14, followed by 3 days of HWS. 

Pre-OVA received aerosol OVA challenge on day 

14, then 3 days of HWS on days 26-28 and an 

immediate (second) OVA challenge HWS 6 h/day 

for 3 days. Sacrificed 18 h post-exposure. 

Allergic Inflammation: A statistically significant 

increase in eosinophils was observed at 

300 μg/m3

 HWS following OVA challenge as 

compared to OVA alone. No changes in 

macrophages, neurophils and lymphocytes were 

observed. Post-OVA HWS did not significantly 

alter BAL cytokine or serum antibody levels, but 

linear trend analyses indicated decreases in IL2, IL-4, and IFN-γ in the absence of OVA, as 

well as a statistically significant upward trend in 

OVA-specific IgE when HWS exposure followed 

OVA challenge. HWS exposure pre-OVA (prior 

to second OVA challenge) resulted in a 

decrease in IL-13 (statistically significant at the 

high dose but no evidence of an exposuredependent response), an increase in OVA IgG1 

(trend significant) and no change in IL-2, IL-4, 

IL-5, IFN-γ, OVA IgE, total IgE or OVA IgG2a. 

 

December 2009 D-116 
Study Pollutant Exposure Effects 

Reference: 

Burchiel et al. 

(2005, 088090) 

Species: Mouse 

Gender: Female 

Strain: A/J 

Age: 12-14 wk 

HWS (black/white oak) 

HWS particle Mass 

BC 

OC 

CO 

Total Vapor Hydrocarbon 

29 other minor components PAH and 

metals 

Particle Size: 0.3 ± 3, 0.4 ± 2, 0.4 ± 2, 

0.4 ± 2 μm (MMAD ± GSD) 

Route: Inhalation 

Dose/Concentration: HWS: 30, 100, 300, 

1000 μg/m3

BC: 3, 12, 25, 43 μg/m3

OC: 40, 107, 281, 908 μg/m3

CO: 1, 2, 4, 13 ppm 

TVH: ND, 1, 1, 3 ppm 

Time to Analysis: 6 h/day for 6 mo. 

Proliferative Responses: HWS increased 

splenic T cell proliferation at 100 μg/m3

 with a 

dose dependent decrease at 300 and 

1000 μg/m3 exposures (p<0.05) HWS exposure 

did not affect T (CD3), helper T cell (Th, CD4), 

cytotoxic T cell (CTL, CD8), macrophage (Mac1), natural killer cell (NK, CD16) cell markers or 

B cell proliferative response to LPS. 

Reference: 

Burchiel et al. 

(2004, 055557) 

Species: Mouse 

Strain: AJ 

Age: 10-12 wk 

DE generated alternatively from two 

2000 Cummins ISB Turbo Diesel 5.9 L 

engines using no 2 (chevron) oil and 

15w/40 oil (Rotella T, Shell) run according to USEPA Dynamometer 

Schedule for Heavy-Duty Diesel Engines

18 PAHs quantified at exposure levels 

(text mentions 65) 

Particle Size: NR 

Route: Inhalation 

Dose/Concentration: 30, 100, 300, 1000 mg/m3

diesel PM 

Time to Analysis: 6 h/day, 7 days/wk for 6 mo. 

Proliferative Responses: DE depressed 

splenic T cell proliferation at all exposure levels 

but was not dose-dependent and most 

pronounced at the 30 μg/m3

 level. (p<0.05 at all 

levels) Splenic B cell proliferation was increased 

at the 30 μg/m3 level, but not at the other 

exposure levels. Little, if any, PAH was found in 

DE, and the majority of PAH tested in vitro 

enhanced T cell proliferation (below), so PAH is 

likely not responsible for the 

immunosuppressive effect of DE on murine 

spleen cell responses. 

Reference: Chan 

et al. (2006, 

097468) 

Species: 

Mouse 

Strain: DO11.10, 

BALB/c, Nrf 2-/-

Cell Types: 

Primary bone 

marrow dendritic 

cells and dendritic 

cell line (BC1), T 

cells (BMDC) 

DEP: DE particles 

DEP methanol extract: 

Particle Size: NR 

Route: Cell Culture 

Dose/Concentration: DEP: 10 μg/mL 

LPS: 5 ng/mL 

Time to Analysis: 24 h 

Dendritic Cell Maturation: Organic DEP 

chemicals interfered in the expression of several 

DC maturation markers. Both DEP and DEP 

extracts were found to inhibit CD86 expression 

and IL-12 production in LPS-exposed DCs, and 

intact particles were not as effective as DEP 

extract. DEP extract treatment of BC1 cells 

reduced their ability to stimulate co-cultured 

antigen-specific T cells, leading to decreased 

IFN- y and increased IL-10 without affecting IL-4 

or IL-13. DEP extract also induced oxidative 

stress and interfered with DC activation by 

several other Toll-like receptor agonists as well 

as the NF-kB cascade. Inhibition of IL-12 

production by DEP extract was shown to be 

mediated by pro-oxidative chemicals that 

engage the Nrf2 pathway. Taken together the 

inhibition of both IL-12 and IFN- y indicates a 

suppression of the Th1 pathway and provides a 

novel explanation for the adjuvant effect of 

DEPs on allergic inflammation.

December 2009 D-117 
Study Pollutant Exposure Effects 

Reference: 

Ciencewicki et al. 

(2007, 096557) 

Species: Mouse 

Gender: Female 

Strain: BALB/c 

Age: 10-12 wk 

Weight: 17-20 g 

DE: generated from a 30-kW (40 hp), 4-

cylinder Deutz BF4M1008 diesel engine 

Influenza A/Bangkok/1/79 (H3N2 

serotype) from Dr. Melinda Beck of the 

University of North Carolina, Chapel Hill 

O2, CO, NO2, NO, SO2

O2: 20.9- 20.5% (Lo, Hi) 

CO: 0.9-5.4 ppm 

NO2: 0.25-1.13 ppm 

NO: 2.5-10.8 ppm 

SO2: 0.06-0.32 ppm 

H3N2: NR 

Particle Size: NR 

Route: Inhalation; Oropharyngeal aspiration (virus) 

Dose/Concentration: DE: 529 or 2070 μg/m3

Time to Analysis: 4 h/day for 5 days. Virus 

exposure immediately after last DE exposure. 

Analyzed 18 h post infection. 

DE exposure on susceptibility to Influenza 

Infection: Mice exposed to 0.5 mg/m3

 had 

significantly greater levels of HA mRNA 

compared to air-exposed mice. HA levels not 

significantly altered in mice exposed to 2.0 

mg/m3

. 

DE Exposure on the Influenza-induced 

Inflammatory Response: f IL-6 mRNA levels 

were significantly greater when exposed to 0.5 

mg/m3

 of DE prior to infection compared to air 

exposure. Significantly increased amount of IL-6 

protein observed in exposed mice. Exposure to 

DE in absence of influenza infection had no 

significant effect on IL-6 mRNA or protein levels. 

DE Exposure on Pulmonary Injury: Infection 

with the influenza virus increases levels of PMN 

in BAL fluid. Exposure to either dose of DE prior 

to infection showed no significant effect on PMN 

levels Exposure to DE alone had no effect on 

PMNs in BAL fluid. Neither exposure to DE nor 

infection with influenza significantly increased 

BAL fluid protein levels when compared to noninfected, air-exposed. 

Other Markers of Injury, NAG and MIA were 

not statistically affected by DE or influenza 

exposure. 

DE Exposure on the Influenza Induced 

Interferon Response: No significant change in 

TFN-α mRNA levels at either dose of DE, 

although mice exposed to 0.5 mg/m3

 of DE prior 

to infection had significantly greater levels of 

IFN-B MRNA compared to air controls. No effect 

on any of the IFNs observed in uninfected mice 

exposed to DE. 

DE Exposure on Surfactant Protein 

Expression: Influenza virus infection alone 

significantly increased expression of SP-A in airexposed. Exposure to 0.5 mg/m3

 of DE prior to 

infection had significant decreases in levels of 

SP-A mRNA in the lungs, this effect was not 

observed in 2.0 mg/m3

 DE exposed. Decrease 

seen in expression of SP-A protein in lungs of 

mice exposed to 0.5 mg/m3 DE prior to infection. 

Levels of SP-D mRNA and protein were 

significantly decreased in lungs of mice exposed 

to 0.5 mg/m3

 of DE prior to infection compared 

with mice exposed to air or 2.0 mg/m3

 DE prior 

to infection. Exposure to 0.5 mg/m3

 of DE prior 

to infection with influenza decreased levels of 

SP-D, especially in airways. Mice exposed to 

2.0 mg/m3

 DE prior to infection showed no 

significant difference. 

Reference: Day et 

al. (2008, 190204) 

Species: Mouse 

Gender: Male 

Strain: BALB/c 

Age: 8-10 wk 

Weight: NR

GEE (General Motors 1996 model 4.3-L 

V6 engine; regular unleaded fuel) (CO, 

NO, NO2, SO2, NH3) 

Particle Size: NR 

Route: Whole-body Inhalation 

Dose/Concentration: Low(L)- 6.6 ± 3.7 PM/m3, 

Medium(M)- 30.3 ± 11.8 PM/m3, High(H)- 59.1 ± 

28.3 PM/m3

, High-Filtered(HF) 

Time to Analysis: Pre-OVA protocol: OVA or saline 

sensitized 7 days. OVA challenge day 14. GEE or 

air exposed 6 h/day on days 26-28. Immediately 

after exposure on day 28 challenged with OVA. 

Tested for MCh-induced changes 24 h postexposure then sacrificed. Post-OVA protocol: OVA 

or saline sensitized 7 days. OVA challenge day 14. 

GEE or air exposed days 15-17. Tested for MChinduced changes 24 h post-exposure then 

sacrificed. 

Pre-OVA: In nonsensitized mice, neutrophils 

and IgE decreased in the H group. IL-2 

increased in the HF group and was dosedependent. Eosinophils dose-dependently 

decreased. OVA-specific IgE increased in the H 

group, and OVA-specific IgG2a dosedependently increased. In OVA-sensitized mice, 

OVA-specific IgG1 increased in the M group. 

Airway hyperresponsiveness was lower in the M 

and HF groups. 

Post-OVA: In nonsensitized mice, neutrophils 

dose-dependently decreased, IL-4 decreased in 

the M group, IL-5 decreased in the HF group, 

and IFN-γ decreased at all exposures. In OVAsensitized mice, IL-13 dose-dependently 

decreased.

December 2009 D-118 
Study Pollutant Exposure Effects 

Reference: de 

Haar et al. (2005, 

097872) 

Species: Mouse 

Gender: Female 

Strain: 

BALB/cANNCrl 

Age: 6-8 wk 

Weight: NR 

CBP: Carbon black particles in 

phosphate buffered saline, 1: 10 & 1: 

100 dilutions (Brunschwich Chemicals, 

Amsterdam, The Netherlands) 

OVA: Ovalbumin 

Particle Size: CBP: 30-50 nm 

Route: Intranasal Droplet 

Dose/Concentration: CBP± OVA 200, 20, 2 μg 

(3.3, 0.33, 0.033 mg/ml) 

OVA only: 20 μg (0.5 mg/ml) 

Time to Analysis: Droplet applied on days 0, 1, 2. 

Sacrificed on day 4 or challenged with OVA droplet 

on days 25, 26, & 27. Sacrificed on day 28 

Acute Airway Damage and Inflammation:

Only day 4 had LDH increased in the 200 μg 

CBP+OVA group. The 200 μg CBP+OVA group 

induced significantly higher numbers of BAL 

cells compared to OVA control. Total protein and 

TNF-a levels were increased only in 200 μg 

CBP+OVA group. RAS, parameter for 

phagocytosis, 200 μg and 20 μg CBP+OVA had 

higher levels than OVA controls. 

Adjuvant Activity on PBLN: Total lymphocytes 

in PBLN significantly increased 4-5 fold in the 

200 μg CBP+OVA exposed. 20 μg and 2 μg 

exposures did not increase the number of PBLN 

cells compared to OVA control. All CBP+OVA 

concentrations induced higher levels of IL-4, IL5, IL-10, and IL-13, with 200 μg concentration 

having 10-200 times higher levels. IFN-y 

cytokine was increased in the 200 μg dose. 

IgE Production: In CBP+OVA, IgE were 

significantly increased. 

PBLN and Lung Lymphocytes after OVA 

Challenge: PBLN cell numbers increased in 

OVA and CBP+OVA sensitized mice. CD4 and 

CD8 populations increased in both groups. 

PBLN levels in CBP+OVA and challenged with 

PBS were higher than mice treated with OVA 

and challenged with PBS, both groups cytokine 

production was low, only IL-5 levels were 

significant in the CBP+OVA/PBS group. Higher 

lung lymphocyte numbers were caused by 

higher numbers of CD4 and CD19. Production 

of IL-5 and IL-10 was four to five times higher 

than in OVA treated mice. 

OVA Challenge Induces Asthma like Airway 

Inflammation in CBP+OVA Sensitized Mice:

Total number of cells in BAL increased 10 fold in 

CBP+OVA mice challenged with OVA. 

Eosinophils exhibited highest increase in CBP+ 

OVA/OVA group. Perivascular and peribronchial 

infiltrates and goblet cell hyperplasia in 

CBP+OVA/OVA was confirmed by histological 

examination. Antigen specific inflammation 

induced in CBP+OVA mice. 

December 2009 D-119 
Study Pollutant Exposure Effects 

Reference: de 

Haar (2006, 

144746) 

Species: Mouse 

Gender: Female 

Strain: 

BALB/cANNCr 

Age: 6-8 wk 

Weight: NR 

CBP: fine (F) and ultrafine (UF) carbon 

black particles (Ken Donaldson Group) 

TiO2: fine and ultrafine 

OVA: Ovalbumin 

Particle Size: F CBP: 260.0 nm 

UF CBP: 14.0 nm 

F TiO2: 250.0 nm 

UF TiO2: 29.0 nm 

Route: Intranasal Droplet 

Dose/Concentration: CBP: 200 μg (3.3 mg/mL) 

TiO2: 200 μg (3.3 mg/mL) 

OVA: 10 μg 

CBP+OVA: 200 +10 μg 

Time to Analysis: Days 0,1,2: Exposed to OVA or 

CBP+OVA. Sacrificed on day 8 & analyzed after 2 

h, or continued to second group. 

Second group: days 25, 26, 27 given OVA 

challenge day 28: sacrificed , analyzed 24 h post 

sacrifice 

Ultrafine Particles Induce Lung 

Inflammation: UF TiO2 and CBP induced a 

local inflammatory response in the airways and 

showed higher levels of LDH and total protein 

as compared to mice exposed to the F particles. 

Cytokine levels were much higher in groups 

exposed to ultrafine particles. Histologic 

analysis of the airways showed that exposure to 

ultrafine TiO2 or CBP leads to peribronchial and 

perivascular inflammatory infiltrates (mostly 

neutrophils). Exposure to OVA alone, or 

combined with fine TiO2 and fine CBP had no 

effects on lung histology. 

Ultrafine Stimulate Local Immune 

Responses: TiO2 and CBP particles stimulated 

the local immune response against co 

administered OVA antigen. Fine TiO2 particles 

induced a low but significant increase in PBLN 

cell number. Both types of ultrafine particles 

elicited higher levels of Th-2 associated 

cytokines, with UF CBP stimulating a greater 

response. IFN-y production was low, but 

significantly higher than OVA exposures. 

Ultrafine TiO2 Increase OVA-specific IgE and 

IgG1 Levels: Levels of OVA specific IgE were 

significantly increased in animals exposed to the 

UF TiO2+ OVA compared to F TiO2 or OVA-only. 

Average IgE level in mice exposed to ultrafine 

CBP+OVA was not a significant increase. OVAspecific IgG2a not detected in any groups. 

Ultrafine Particles Stimulate Allergic Airway 

Sensitization Against OVA: At day 28, the 

PBLN cell numbers were significantly higher in 

both ultrafine and combination with OVA. 

Production of OVA specific IL-4, IL-5, IL-10 and 

IL-13 by PBLN cells was significantly increased 

in both ultrafine TiO2 and CBP. IFN-y levels were 

significantly increased in ultrafine CBP+OVA 

treated animals. F TiO2 had low, but significant, 

increases in IL-4 and IFN-y compared to OVA 

only. Allergic airway inflammation and Influxes of 

eosinophils, neutrophils and lymphocytes were 

only found in both groups exposed to ultrafine 

particles. 

Reference: de 

Haar (2008, 

187128) 

Species: Mouse 

Gender: Female 

Strain: BALB/c, 

CD80/CD86-

deficient, DO11.10 

Age: 6-8 wk 

Weight: NR

Ultrafine Carbon Black (UFCB) 

(Brunschwich Chemicals; Amsterdam, 

The Netherlands) 

Particle Size: Diameter: 30-50 nm 

Route: Intranasal Exposure 

Dose/Concentration: 20 μg/mL 

Time to Analysis: Exposed days 1, 2, 3. OVA 

challenge days 25, 26, 27. Spleens and lymph 

nodes from DO11.10 mice pooled and CD4+ T-cells 

isolated. Solution injected into tail veins of BALB/c 

mice day 0. CTLA4-Ig ip injected days 0, 2. PBLN 

cell suspensions plated, restimulated with OVA 4 

day. 

UFCB+OVA induced proliferation of CD4+ Tcells, increased cytokine production. 

UFCB+OVA did not induce any effects in 

CD80/CD86-deficient mice. UFCB-induced 

airway inflammation is dose-dependent. 

Reference: de 

Haar et al. (2008, 

187128) 

Species: Mouse 

Cell Line: Myeloid 

dendritic cells 

(mDCs)

Ultrafine Carbon Black (UFCB) 

(Brunschwich Chemicals; Amsterdam, 

The Netherlands) 

Particle Size: Diameter: 30-50 nm 

Route: Cell Culture 

Dose/Concentration: 25 μg/mL 

Time to Analysis: 18 h 

UFCB+OVA increased mDCs in the 

peribronchial lymph nodes, and their 

expressions of CD80, CD86, and MHC-11. 

December 2009 D-120 
Study Pollutant Exposure Effects 

Reference: Dong 

et al. (2005, 

088079) 

Species: Rat 

Gender: Male 

Strain: BrownNorway 

(BN/CrlBR) 

Age: NR 

Weight: 200-225 g 

DEP: SRM 2975 (NIST, Gaithersburg, 

MD) 

OVA: Ovalbumin 

Particle Size: 0.5 μm (MMAD) 

Route: Inhalation 

Dose/Concentration: DEP: 20.6 ± 2.7 mg/m3

OVA 40.5 ± 6.3 mg/m3

Time to Analysis: 4 h/dayfor 5 days + OVA 30 

min/day1 x wk on days 8,15 & 29. Sacrificed on 

days 9 or 30. 

Lung Inflammation/Injury: Both the BAL 

proteins and inflammatory cell counts for DEP 

exposure alone were not different from those of 

the air exposed control, suggesting that DEP 

exposure did not cause lung injury at 9 or 30 

days post-exposure. OVA exposure caused 

significant increases in neutrophils, 

lymphocytes, eosinophils, albumin and LDH 

activity in the lung after two exposures. DEP did 

show a strong effect on OVA-induced 

inflammatory responses. 

Alveolar Macrophage (AM) function: OVA 

exposure resulted in an increase in NO levels in 

the acellular BAL fluid and AM conditioned 

media. This increase was significantly 

attenuated in rats exposed to DEP. DEP 

exposure had no significant effect on the 

production of IL-10 or IL-12 by AM recovered 

from rats 9 and 30 days post exposure. In 

contrast, OVA sensitization elevated both IL-10 

and IL-12 secretion by AM at both time points. 

Lymphocyte population and cytokine 

production: DEP exposure was found to 

increase the numbers of total lymphocytes, T 

cells and their CD4+ and CD8+ subsets in 

LDLN. OVA exposure also significantly 

increased these cell counts on days 9 and 30. 

DEP+OVA exposure showed a significant 

reduction in total lymphocytes, T cells, CD4+ 

and CD8+ subsets on day 30. Levels of IL-4 and 

IFN-y in lymphocyte conditioned media were 

below detection limit of the ELISA kits. 

Intracellular GSH levels in AM and 

Lymphocytes: DEP exposure alone slightly 

decrease GSH levels in AM, but markedly 

reduced GSH concentration in lymphocytes on 

days 9 and 30. OVA exposure significantly 

decreased intracellular GSH in both cell types. 

Combined exposure showed AM and 

lymphocytes to have depleted intracellular GSH. 

OVA specific IgE and IgG levels in serum: In 

all samples collected on day 9, both serum IgG 

and IgE levels were under the detection limits. 

On day 30, no measureable IgE levels were 

found. The OVA exposure, however, resulted in 

elevated IgE levels, and was enhanced in rats 

preexposed to DEP. IgE and IgG levels for 

DEP+OVA was tw0 times higher than OVA alone 

indicating that DEP has an adjuvant effect on 

the production of IgG and IgE. 

Effects of DEP and OVA on Lung iNOS 

expression: AM from various exposure groups 

did not stain for iNOS. 1 rat at day 9 from the 

combined DEP+OVA group showed a slightly 

positive iNOS staining. On day 30, 2 of 5 rats 

from combined exposure group and 1 from the 

OVA group showed a positive airway staining. 

December 2009 D-121 
Study Pollutant Exposure Effects 

Reference: Dong 

et al. (2005, 

088083) 

Species: Rat 

Gender: Male 

Strain: BrownNorway 

(BN/CrlBR) 

Age: NR 

Weight: 200-225 g 

DEP: SRM 2975 Diesel Exhaust 

Particles (NIST) 

OVA: Ovalbumin 

Particle Size: 0.5 μm (MMAD) 

Route: Nose-only Inhalation 

Dose/Concentration: DEP 22.7 ± 2.5 mg/m3

OVA 42.3 ± 5.7 mg/m3

Time to Analysis: Day 1, 8, 15: OVA exposure 30 

min/day

Days 24-28: DEP exposure 4 h/day 

Day 29: OVA challenge 

Day 30: Whole-body plethysmography 

Day 31: Sacrifice 

Effect of DEP on OVA Induced Allergic 

Responses: DEP exposure had a synergistic 

effect with OVA on inducing airway hyperresponsiveness (AHR) in rats. DEP alone had 

no effect on IgG production. Levels of OVAspecific IgG and IgE increased in OVA+DEP 

exposure. This indicates that DEP pre-exposure 

augments the immune response of rats to OVA 

in the production of allergen specific IgG and 

IgE. 

Effect of DEP on OVA Induced Cell 

Differentiation: Neither DEP, OVA nor the 

combination induced elevated levels of LDH 

activity or albumin content, indicating that the 

exposure protocols did not cause significant 

lung injury. DEP alone induced moderate but 

significant increase of neutrophil numbers. OVA 

exposure induced a greater infiltration of 

neutrophils than DEP, and infiltration of 

eosinophils and lymphocytes. OVA-induced 

eosinophil count markedly increased with DEP 

exposure. Total lymphocytes, T cells, and their 

CD4+ and CD8+ subsets in LDLN from rats 

sensitized and challenged by OVA were 

significantly higher than those of air-exposed 

non sensitized rats. DEP+OVA exposure 

resulted in substantial increase in T cells 

compared to OVA alone. 

Effect of DEP on OVA-induced Oxidant 

Generation and GSH Depletion: Exposure to 

DEP or OVA alone had no effect on ROS 

production by AM. Substantial elevation seen in 

ROS for the DEP+OVA exposed group. Both 

OVA and DEP exposures resulted in an 

increased presence of NO in the acellular BAL 

fluid and in AM conditioned media; OVA+DEP 

exposure further increased these levels. The 

ATII cells from OVA exposed rats exhibited a 

higher percentage of cells that produce NO and 

superoxide than air exposed, non sensitized 

rats. DEP and OVA exposure resulted in a significant increase in the percentage of cells that 

produce NO and superoxide over the control. 

iNOS Expression: Immunohistological analysis 

in lung tissues showed no AM staining in any 

group. Airway epithelium was found to be 

positive in all 5 rats from the DEP+OVA group 

and 3 of 5 rats from single exposure of DEP or 

OVA and 2 of 5 in air only exposed rats. iNOS 

expression was significantly higher in ATII cells 

isolated from rats exposed to combined DEP 

and OVA . 

GSH levels in AM and lymphocytes: Levels 

were slightly lowered by DEP or OVA exposure, 

though not statistically significant. DEP+OVA 

showed a significant reduction in GSH levels. 

December 2009 D-122 
Study Pollutant Exposure Effects 

Reference: Drela 

et al. (2006, 

096352) 

Species: Mouse 

Gender: Male 

Strain: BALB/c 

Age: 6 wk 

Weight: NR 

ASM: Air suspended PM from Upper 

Silesia (Poland) 

1μg of ASM: 

Pb (1.136 ng) 

Cu (0.004 μg) 

Co (0.072 ng) 

Mn (0.406 ng) 

Fe (0.016 μg) 

Cd (0.154 ng) 

Cr (0.418 ng) 

Ni (0.238 ng) 

Particle Size: 0.3-10 μm 

Route: Intraperitoneal Injection 

Dose/Concentration: 170 mg/kg 

Time to Analysis: Single, 72 h 

CD28 Expression on Thymocytes at Different 

Stages of Development: ASM exposure 

accelerated thymocyte maturation but did not 

alter the expression of CD28 on peripheral CD4 

and CD8 T cells isolated from lymph nodes. A 

slight but not statistically significant decrease in 

the expression of CD28 on spleen T cells from 

ASM animals was observed. 

Distribution of CD28(low) and CD28(high):

Acute exposure to ASM resulted in the increase 

of CD28(low) and decrease of CD28 (high) 

thymocyte percentages in the total thymocyte 

population. The percentages of CD28 low and 

high thymocytes did not differ between intact 

and PBS controls. Acute ASM exposure resulted 

in the increase of the percentage of CD28(low) 

and the decrease of CD28(high) thymocytes in 

the CD3 low subset. The percentage of CD28 

low and high positive thymocytes did not differ in 

CD3 high thymocyte subset. 

Natural Regulatory CD4+ CD25+ T Cells in 

the Thymus: The development of thymic 

natural regulatory cells was unaffected by ASM. 

Proliferation of Splenocytes and Lymph 

Node Lymphocytes: Decreased proliferative 

responses were evident in splenocytes from 

ASM-exposed animals when cells were 

stimulated with low but not high levels of antiCD3 mAb. In contrast, lymph node lymphocytes 

from ASM treated mice had increased 

proliferative responses independent of anti-CD3 

concentration. Both CD4+ and CD8+ T cells 

from ASM treated mice proliferated more 

vigorously than from controls. Almost all CD8+ T 

cells from ASM mice were induced to proliferate. 

Reference: Dybing 

et al. (2004, 

097545) 

Species: Mouse 

Gender: NR 

Strain: BALB/cA 

Age: NR 

Weight: NR 

UP: Urban ambient particles collected in 

5 different sites (Amsterdam, Lodz, Oslo, 

Rome, Dutch seaside) during four-wk 

periods in spring, summer, winter 

seasons from March 2001 to March 

2004. 

DEP as reference std: SRM 1650 (NIST) 

OVA: Ovalbumin (Sigma Chemical, St. 

Louis, MO) 

Particle Size: UP: PM10 and PM2.5

Route: Injection in hind foot pad 

Dose/Concentration: UP: 100- 200 μg

DEP: 50 μg 

OVA: 50 μg 

Time to Analysis: 

Day 0: 1 exposure to OVA alone, OVA w/particles, 

particles alone. 

Day 6: Lymph nodes harvested 

Day 21: 1 OVA w/o particles exposure 

Day 26: Antibody assay 

Allergy Screening: All samples were 

immunostimulatory in the popliteal lymph node 

assay; activity was weak in the absence of OVA 

but statistically significant when injected with 

OVA, indicating an adjuvant effect. Particle 

adjuvancy was further demonstrated via 

significant enhancement of OVA-specific 

antibody responses. All ambient particle 

fractions from all seasons increased IgG1. 

Except for a few coarse samples, all fractions 

significantly increased IgE. All fine fractions and 

some coarse fractions significantly increased 

IgG2a, indicating that most particles could exert 

both Th1 and Th2 adjuvancy. In general, fine 

particles demonstrated stronger adjuvant activity 

than coarse in a pair-wise comparison of coarse 

and fine particles from the same location. 

Reference: 

Dybing, et al. 

(2004, 097545) 

Species Rat 

Cell Lines: Type 2 

cells, AM 

UP: Urban ambient particles collected in 

5 different sites (Amsterdam, Lodz, Oslo, 

Rome, Dutch seaside) during four-wk 

periods in spring, summer, winter 

seasons from March 2001 to March 

2004. 

DEP: SRM 2975 (NIST) 

OVA: Ovalbumin (Sigma Chemical, St. 

Louis, MO) 

Particle Size: PM10 and PM2.5

Route: Cell Culture 

Dose/Concentration: 0-50 μg/ml 

Time to Analysis: 20 h 

Inflammation: The coarse fractions were more 

potent than the fine fractions. Among the 

samples, the overall effects of the coarse 

fractions on the cells were dependent on the site 

of collection. High MIP-2 levels were found 

using particles from some spring collections. 

Coarse particles collected in summer 

demonstrated the highest potency, and samples 

collected during winter proved to be less potent 

but seasonal variation was not obvious for all 

sites. Only minor responses were observed 

using fine fractions from urban sites. 

December 2009 D-123 
Study Pollutant Exposure Effects 

Reference: Farraj 

et al. (2006, 

141730) 

Species: Mouse 

Gender: Male 

Strain: BALB/c 

Age: 6 wk 

Weight: NR 

DEP: SRM 2975 NIST 

OVA: Ovalbumin 

Anti-p75: Rabbit anti-mouse p75 

neurotrophin receptor polyclonal 

antibody (Chemicon, Temecula, CA) 

Anti-trkA: anti-mouse trkA NGF receptor 

antibody (Santa Cruz, Santa Cruz, CA) 

Particle Size: DEP: 1.47 μm (MMAD), 

2.75 GSD 

Route: Nose-only Inhalation

Dose/Concentration: DEP: 1.78 to 2.18 mg/m3

Anti-p75: 50 μl 

Anti-trkA: 50 μl 

OVA injection: 20 μg 

MCH: 0, 16, 32, 64 mg/ml 

Time to Analysis: On day 0: ip injection of 20 μg 

OVA 

Day 14: intranasal instillation of 50 μl anti-p75 or 

anti-trkA, 1 h after 1st exposure challenged with 

OVA aerosol for 1 h followed by a h exposure to 

DEP 

24 h after DEP exposure: MCH challenge 

Airways Responsiveness: No significant 

differences in avg baseline Penh values of any 

treatment groups. 

Vehicle sensitized mice: exposure to DEP, antip75 or anti-trkA had no effect on MCH-induced 

Penh values. 

OVA-sensitized DEP-exposed: seen increase of 

Penh values. Administration of anti-p75 or antitrkA to OVA sensitized mice reversed DEP 

induced Penh increases. 

Lung Function in Ventilated Mice: Compared 

to vehicle sensitized mice, central airway 

resistance (Rn) increased 62% in OVA 

sensitized mice was not a significant increase. 

OVA-sensitized DEP-exposed mice, anti-p75 

decreased central airway resistance (Rn ) and 

anti-trkA did not significantly alter Rn. though Rn 

response for anti-p75 was significantly less than 

anti-trkA response, Constant phase model 

parameter of tissue elastance not significantly 

affected by any treatments or by increasing 

MCH dose, indicating development of significant 

regional ventilation inhomogeneity during 

bronchoconstriction. 

Airway Pathology: OVA-sensitized mice had 

small increases in intraepithelial mucus 

compared to vehicle-sensitized mice. DEP 

exposure did not enhance severity of OVAinduced airway pathology. Anti-p75 or anti-trkA 

administration did not influence airway 

morphology. 

BAL Cells: Vehicle-sensitized DEP-exposed 

mice had significantly enhanced macrophage 

numbers by 92% compared to air-exposed, 

vehicle-sensitized mice. Anti-p75 or Anti-trkA 

administration significantly suppressed DEPinduced macrophage increase to levels similar 

to air-exposed, vehicle-sensitized group. DEP 

co exposure significantly decreased number of 

macrophages in OVA-sensitized mice to control 

levels. Anti-trkA or anti-p75 had no effect in 

OVA-sensitized, DEP-exposed. Eosinophil 

number greater in OVA-sensitized DEP-exposed 

mice than in vehicle-sensitized air-exposed 

mice. No significant effects of DEP exposure on 

neutrophils from vehicle- or OVA-sensitized 

mice. 

Cytokines: IL4: OVA-sensitized DEP-exposed 

had five-fold increase over vehicle-sensitized, 

air-exposed mice and anti-trkA or anti-p75 

significantly inhibited the DEP-induced increase. 

IL5, IL13: OVA-sensitized DEP-exposed had no 

significant change. Anti-p75 or anti-trkA 

administration had no significant effect. 

Serum IgE: OVA sensitized mice had a 10 fold 

increase in IgE levels for air and DEP exposed 

mice. Anti-p75, anti-trkA treatment did not cause 

significant effects on IgE levels. 

December 2009 D-124 
Study Pollutant Exposure Effects 

Reference: Farraj 

et al. (2006, 

088469) 

Species: Mouse 

Gender: Male 

Strains: C57/Bl6 

Age: 6 wk 

DEP: SRM 2975 collected from dieselpowered industrial forklift filter (NIST) 

OVA: Ovalbumin 

Anti-p75: Rabbit anti-mouse p75 

neurotrophin receptor polyclonal 

antibody 

Particle Size: 1.47 (MMAD), 2.75 (GSD)

Route: Nose-only Inhalation 

Dose/Concentration: DEP: 0.87 mg/m3

MCH: 0, 16, 32, 64 mg/ml 

OVA: 20 μg ip 

Anti-p75: 50 μl 

Time to Analysis: Day 0: OVA in gel vehicle, ip 

Day 14: anti-p75 exposure, intranasal instillation 

1 h post anti-p75 exposure, OVA aerosol challenge 

for1 h 

1 h post OVA challenge: DEP exposure for 5 h 

48 h post DEP exposure: MCH challenge 

Airway Responsiveness: No significant 

differences in average Penh values among any 

vehicle control groups. No significant differences 

in treatment groups in OVA-sensitized mice at 

baseline 0, 16, or 32 mg/mL of MCH. At 64 

mg/mL MCH, OVA-sensitized, DEP-exposed 

mice had a 22% increase in Penh compared to 

vehicle mice, and a 68% increase compared to 

vehicle-sensitized, air-exposed mice. Instillation 

of anti-p75 inhibited the DEP induced increased 

Penh. 

BALF Cells: DEP exposure in vehiclesensitized mice significantly increased 

macrophages by 161% compared to airexposed, vehicle-sensitized mice, while OVAsensitized mice had 69% increase. Anti-p75 

administration significantly suppressed DEPinduced macrophage increase in vehiclesensitized mice. No significant effects of DEP 

exposure or anti-p75 treatment in OVA-allergic 

mice. 

OVA-sensitized air-exposed mice had a several 

hundred fold increase in the number of 

eosinophils. No significant effects of DEP 

exposure or anti-p75 treatment on eosinophils 

from OVA-sensitized mice. OVA-exposure or 

DEP-exposure had no significant effects on 

neutrophil or lymphocyte number . 

Cytokines: No significant effects of DEP alone 

or with OVA on IL-4, IL-5, or IL-13. 

Serum IgE: OVA sensitization in the presence 

or absence of DEP or anti-p75 caused at least a 

3 fold increase in IgE levels. No significant 

effects of DEP or anti-p75 treatment on IgE 

levels. 

Reference: 

Finkelman et al. 

(2004, 096572) 

Species: Mouse 

Gender: Female 

Strains: BALB/c, 

C57BL/6 

Age: 2-4 mo 

DEP: 4JB1 type; Isuzu Automobile, 

Tokyo, Japan 

Particle Size: 2 μm (MMAD) 

Route: Group1: 1 ip injection of 2 mg of DEP. 

Group 2: daily ip injections of 2 mg of DEP 

Dose/Concentration: 2 mg 

Time to Analysis: 2-96 h 

Serum Cytokines: Mice in group 1 

demonstrated an increase in serum IL-6 

production but no increase in IL-4 or IL-2 

production. IFN-γ levels were decreased in 

group 2. TNF production was not affected.

Spleen Cytokines: When injected before LPS, 

DEP had little effect on the LPS-induced TNF-α

and IL-6 response, but resulted in a minor 

suppression of INF-γ and IL-10. DEP LPSinduced increase in INF-γ mRNA responses in 

spleen cells. DEP caused a dose related 

suppression of LPS stimulated INF-γ. DEP had 

little or no effect on the percentage of NK or 

NKT cells in the spleen and inhibited LPSinduced IFN-γ production by NK and NKT. DEP 

failed to inhibit the IFN-γ response by anti-CD3 

mAb-activated NKT cells. Oxidant activity was 

not responsible for DEP inhibition of LPSinduced IFN-γ production. 

Reference: 

Fujimaki and 

Kurokawa (2004, 

096575) 

Species: Mouse 

Gender: Male 

Strains: BALB/c 

Age: 4 wk 

Cell Types: 

Cervical lymphnode (CLN) cells 

DE ± particles: Comparison of exposure 

to DE including particles and exposure to 

particle-filtered DE 

DE: 12.09 ± 0.15 NOX, 1.99 ± 0.02 NO2, 

10.02 ± 0.12 NO, 0.18 ± 0.002 SO2 and 

1769.2 ± 13.2 CO2 (all in ppm). 

DE gas: 11.93 ± 0.13 NOX, 2.93 ± 0.06 

NO2, 8.91 ± 0.09 NO, 0.11 ± 0.003 SO2

and 1838.8 ± 15.3 CO2 (all in ppm) 

Particle Size: 0.4 μm (MMAD) 

Route: Whole-body Inhalation 

Dose/Concentration: Exposure to: 0, 1.0 mg/m3 or 

1.0 mg/m3 DE gas only (0.04 mg/m3 PM) 

Time to Analysis: Exposure for 12 h daily for 5 wk. 

Days 14 and 35 challenge with sugi basic protein 

(SBP), a cedar pollen allergen, intranasally. 

Evaluation is 24 and 48 h after final SBP injection. 

CLN Response: Exposure to DE or DE gas did 

not affect B1 lymphocyte subpopulations of 

CLN. Culture supernatants of CLN cells from DE 

exposed/SBP immunized mice showed 

significant increase in MCP-1 at 24 and 48 h. 

Exposure to DE or DE gas significantly 

increased the amount of TARC and MIP-1α in 

CLN cells from SBP-immunized mice at 48 h. 

December 2009 D-125 
Study Pollutant Exposure Effects 

Reference: 

Fujimaki et al. 

(2005, 156456) 

Species: Mouse 

Gender: Male 

Strains: C57BL/6 

Age: 4 wk 

DE generated by 4 cyl 2.74 l Isuzu diesel

DE gas = DE filtered to remove particles 

Composition of Diesel Exhaust: DE 

DEP: 1.01 mg/m3

 

1796 ppm CO2

12.09 ppm NOX 

0.18 ppm SO2

Composition of filtered DE Gas: DEP: 

0.04 mg/m3

 

1839ppm CO2

11.93ppm NOX 

0.11 ppm SO2

Sugi Basic Protein (SBP)- allergen 

Particle Size: 0.4 μm (average 

diameter) 

Route: Whole-body Inhalation 

Dose/Concentration: 1.0 mg DEP/m3 or 1.0 mg 

DEP/m3 DE gas 

Time to Analysis: 12 h daily, 5 wk. All mice were 

injected IP with 100 μg SBP before exposure to gas 

or DE and again received 50 μg SBP intranasally 

on days 14 and 35. Evaluation is 1 day after final 

SBP-immunization (mice are euthanized and CLN 

and blood samples are collected) 

CLN: Exposure to DE and gas led to a decrease 

in total number of CLN cells and percentage of 

CD4+ and TCR-Β levels. Cell proliferation 

response to SBP was higher in gas-exposed 

mice than in the control group. The production 

of MCP-1 increased in CLN cells when 

stimulated with SBP (in vitro) but the difference 

was not significant at 24 and 48 h. SBPstimulated cells in gas-exposed mice showed 

greatly enhanced MIP-1α production at 24 and 

48 h. Exposure to gas increased the amount of 

TARC in the culture supernatants of CLN cells. 

Plasma: Exposure to DE or gas significantly 

decreased the anti-SBP IgG1 antibody titers and 

increased the anti-SBP IgG2a antibody titers in 

mouse plasma. 

Reference: 

Fujimoto et al. 

(2005, 096556) 

Species: Mouse 

Gender: Female 

1st day of 

pregnancy) 

Strains: Slc: IRC 

DEP: generated by a 2369-cc diesel 

engine operated at 1050 rpm and 80% 

load with commercial light oil 

Particle Size: 0.4 μm (MMAD) 

Route: Whole-body Inhalation 

Dose/Concentration: 0.3, 1.0 and 3.0 mg DEP/m3 

(Groups 1,2,3) 

Time to Analysis: Exposure began at 2 days 

postcoitum and was continued until 13 days 

postcoitum. Exposure time was 12 h daily for 7 

days/wk. Pregnant females were sacrificed 14 days 

postcoitum. 

mRNA Expression in Placentas: In groups 

exposed to DE, the expression of CYP1A1 

mRNA decreased to undetectable levels during 

placental absorption and INF-γ was increased. 

Levels of CYP1A1 mRNA in normal placentas 

from DE-exposed mice were unchanged. mRNA 

levels of inflammatory cytokines IL-2, IL-5, IL12α, IL-12Β and GM-CSF increased in 

placentas of mice exposed to DE. 

Reference: Gao et 

al. (2004, 087950) 

Species: Human 

Cell Line: Lung 

fibroblasts infected 

with Mycoplasma 

fermentans 

ROFA: collected near a power plant in 

FL burning low sulfur # 6 oil. 

(PM from Dusseldorf, volcanic ash for 

Mt. St. Helens, PM from Utah used to 

compare against ROFA in one 

experiment) 

NiSO4, CuSO4, VOSO4, Na3VO4

Particle Size: NR 

Route: Cell Culture; seeded into 6-well plates (3-

4.5×105

 cells/3 mL/well) or 24-well plates (0.6-

1×105

 cells/1.0 mL/well) 

Dose/Concentration: PM: 3, 10, 20, 40, 50 μg/ml 

Metallic salts: 2, 20, 200 μM 

Time to Analysis: 24, 48h 

Cytokines: ROFA exposure in combination with 

Mycoplasma fermentans infection synergistically 

amplifies the induction of IL-6 production in 

human lung fibroblasts (HLF). PM from the other 

sources has little synergistic effect on IL-6 

release. Exposing HLF cells to,M. fermentans 

derived macrophage activating lipopeptide-2 

(MALP-2) and ROFA has the same synergistic 

effect as M. fermentans infection and ROFA. 

MALP-2 and ROFA extract have a similar 

synergistic effect that requires more time to 

appear. ROFA contains high levels of V, Ni, Fe 

and Cu. Exposure of HLF to NiSO4 alone and 

NiSO4 with MALP-2 produced 10 and 50 fold 

increases, respectively, in IL-6 production. 

Exposure of HLF to CuSO4, VOSO4 and 

Na3VO4, with and without the presence of 

MALP-2, did not produce as dramatic results as 

seen with Ni. The action of NiSO4 and MALP-2 

on IL-6 production was found to be dose 

dependent. 

December 2009 D-126 
Study Pollutant Exposure Effects 

Reference: Gavett 

et al. (2003, 

053153) 

Species: Mouse 

Gender: Female 

Strain: BALB/c 

Age: 7wk 

PM2.5 from the German cities of Hettstedt 

or Zerbst 

PM Composition: samples from Hettstedt 

have several-fold higher levels of Zn, 

Mg, Pb, Cu and Cd than samples from 

Zerbst. 

Particle Size: PM2.5 

Route: Oropharyngeal Aspirations 

Dose/Concentration: 50-100 μg 

Time to Analysis: Single, 18 h. 

Sensitization Model: Mice were exposed to 50 μg 

PM 2 h before being sensitized with 10 μg OVA, 

repeated two days later. On day 14 all mice were 

challenged with 20 μg OVA. 

Parameters measured on days 2 and 7 after final 

exposure to OVA. 

Challenge Model: Mice were sensitized IP with 20 

μg OVA or adjuvant only. 14 days later mice were 

exposed to 100 μg PM2.5 followed 2 h later by 20 μg 

OVA. Parameters measured on days 2 and 7 after 

final exposure to OVA. 

BAL Analysis: Hettstedt PM significantly 

increased BAL protein and NAG levels. Zerbst 

PM did not. Mice exposed to Zerbst had lower 

levels of LDH than control groups. Hettstedt 

exposed mice had increased levels of IL-1Β, IL6 and MIP-2 in comparison to control and to 

mice exposed to Zerbst PM. PM2.5 at a dose of 

100 μg was not found to be toxic, therefore used 

for subsequent studies. 

Airway Responsiveness (PenH): In allergic 

mice tested immediately after exposure, 

Hettstedt PM increased PenH 190% compared 

to baseline, Zerbst increased PenH by 120% 

and the Control increased by 44%.:.: Two days 

after OVA challenge, no differences in nonallergic mice from either group. In allergic mice, 

Hettstedt PM still caused a significant response 

to Mch responsiveness, Zerbst none. No effects 

on day seven. 

IgE Levels: Serum collected on day 2 showed 

antigen-specific IgE was increased by Hettstedt 

PM2.5 in both the sensitization and challenge 

phases when compared to the control and 

exposure to Zerbst. Day 7 serum indicated no 

effect. 

BALF Cells: In non-allergic mice both Hettstedt 

and Zerbst PM increased neutrophil numbers 

(3-fold; not statistically significant) and in allergic 

mice, only Hettstedt PM significantly increased 

neutrophil count. Eosinophil numbers were 

increased only in allergic mice exposed to 

Hettstedt PM. Lymphocyte numbers were not 

different among groups. 

BAL Injury Markers: At 2 days after both 

Hettstedt and Zerbst PM administered in allergic 

mice caused significant increases in protein, 

LDH and NAG compared to the non-allergic 

groups. Both PMs caused an increase in LDH in 

allergic mice compared to the allergic control, 

but only Hettstedt caused an increase NAG in 

allergic mice compared to control. At 7 days no 

effect. 

BAL Cytokines: Allergic mice had increased 

levels of IL-4, IL-5 and IL-13 compared to nonallergic mice (at 2 days after). IL-5 was 

significantly increased by exposure to either PM 

in allergic mice compared to non-allergic mice. 

Exposure to either PM caused an increase in 

TNF-α and IFN-γ (by 6-8 fold) in allergic mice, 

there was also an increase in these 

inflammatory cytokines in the non-allergic group 

but was not statistically significant. No 

significant effects were observed in animals that 

underwent the sensitization protocol alone for 

any measurement or endpoint. 

Reference: Gowdy 

et al. (2008, 

097226) 

Species: Mouse 

Gender: Female 

Strain: BALB/c 

Age: ~12-14 wk 

Weight: 17-20 g

DEP (30kW (40hp) 4-cylinder Deutz 

BF4M1008 diesel engine, steady state, 

20% full load) (Low dose: 21% O2, 0.4wt 

ratio OC/EC; High dose: 20.7% O2, 

0.4wt ratio OC/EC) (CO, NOX, SO2) 

Particle Size: Diameter: ~240 nm 

Route: Inhalation 

Dose/Concentration: Low- 514 ± 3 μg/m3, High2026 ± 38 μg/m3

Time to Analysis: 4 h/day, 1 or 5 days 

(consecutive). Necropsied immediately or 18 h 

postexposure. 

BAL Analysis: Neutrophils and lung injury 

dose-dependently increased. ICAM-1 increased 

immediately after both exposures and after 18h 

postexposure in the low dose. 

Cytokines: After 1 day exposure, IFN-γ and 

TNF-α increased immediately at both doses and 

the high dose, respectively. Immediately after 5 

days exposure TNF-α and IFN-γ increased at 

both concentrations and IL-6 increased at the 

low dose. At 18 h postexposure IL-6 and IFN-γ

increased at both doses, TNF-α and IL-13 

increased at the low dose, and MIP-2 dosedependently increased. 

CCSP, Surfactants: CCSP decreased. SP-A 

and SP-D decreases were only significant after 

5 days exposure, 18 h post-exposure. 

December 2009 D-127 
Study Pollutant Exposure Effects 

Reference: 

Hamada et al. 

(2007, 091235) 

Species: Mouse 

Gender: Female 

(Pregnant close to 

partruition) 

Strain: BALB/c 

ROFA (obtained from a precipitator until 

of a local power plant) 

Composition of ROFA (in μg/mL): 341.2 

Ni, 323.4 V, 232.2 Zn, 18.3 Co, 15.8 Mn, 

8.4 Ca, 6.7 Cu, 6.1 Sr, 5.0 mg, 0.9 Sb, 

and 0.6 Cd. 

Particle Size: NR 

Route: Nebulized ROFA leachate 

Dose/Concentration: 50 mg/mL dilution 

Time to Analysis: Pregnant mice exposed to 

nebulized ROFA leachate for 30 min/day at days 

14, 16 and 18 of pregnancy. 

Newborns received a single injection (ip) of OVA (5 

μg)+ alum (1mg) at day 0 followed by exposure to: 

1. aerosolized OVA days 12, 13 and 14 (2-wk old 

protocol) 

OR 

2. aerosolized OVA days 32, 33 and 34 (5 wk old 

protocol) 

Analysis 48 h after final allergen exposure 

Susceptibility to Asthma: Exposure of mother 

to PBS aerosols during pregnancy did not result 

in prominent asthma features in young. The 

offspring of the ROFA mothers revealed 

increasing AHR and elevated numbers of 

eosinophils in the BAL fluid. Similar results were 

seen in both the 2-wk and 5-wk old groups. 

IgE Levels: Histopathology revealed prominent 

inflammation in the lungs of the ROFA neonates 

and increased allergen-specific IgE and IgG1 

levels in the 5-wk group. 

Maternal Influence: Breast milk was not shown 

to be responsible for the increased susceptibility 

to allergy seen in offspring. 

IL-4 and IFN-γ: IL-4 and IFN-γ levels in 

maternal mice showed no difference between 

PBS exposed or ROFA exposed mice. Cultured 

spleen cells from mice born of ROFA-exposed 

mothers showed either increased or similar 

levels of IL-4 and decreased production of IFN-γ

causing an increase in the ratio of IL-4/IFN-γ

indicating greater susceptibility to develop Th2- 

allergic response. 

Eosinophils: Exposure of mothers to Ni levels 

similar to those found in ROFA had no 

appreciable effect on BAL eosinophil. 

Reference: Hao et 

al. (2003, 096565) 

Species: Mouse 

Gender: Female 

Strain: BALB/c 

Age: 6-7 wk 

DEP (4-cylinder diesel engine under a 

10-torque load) 

Particle Size: NR 

Route: Nebulization 

Dose/Concentration: 2 mg DEP m3 

Time to Analysis: Mild Sensitization- Mice receive 

IP OVA alum and are challenge with aerosolized 

OVA with and without DEPs. Mice sacrificed d19. 

Postchallenge Model- DEPs are delivered to mice 

sensitized by IP OVA and alum. Mice sacrificed 

d23. 

Transgenic Mice: Mice exposed to nebulized saline 

or DEPs for 1 h daily for 3 days. Mice sacrificed 

day 5. 

Mild Sensitization: Exposure of previously OVA 

sensitized mice to aerosolized DEP and OVA did 

not affect OVA-specific IgE production, BAL 

eosinophilia or methacholine-induced AHR. 

Aerosolized particles induced inflammation and 

increased MBP deposition and MBP positive 

eosinophils in the mucosa. 

IL-5 Transgenic: Exposure to aerosolized DEP 

did not change BAL cytokine levels, but did 

increase AHR and BAL cell count. 

Classic Sensitization, Post-Challenge: Did 

not lead to a discernable increase in OVAinduced AHR. DEP treatment was associated 

with increased airway inflammation and mucin 

production in larger and intermediary airways. 

Reference: 

Harkema et al. 

(2004, 056842) 

Species: Rat 

Gender: Male 

Strain: F344, BN 

Age: 10-12 wk 

Weight: NR

CAPs (Detroit; July-Sept. 2000; Harvard 

Ambient Fine Particle Concentrator) 

Particle Size: 2.5 μm (diameter) 

Route: Inhalation; IT Instillation.

Dose/Concentration: 4 day concentration: 676 ± 

288 μg/m3

, 5 day concentration: 313 ± 119 μg/m3, 

July concentration: 16-185 μg/m3

, September 

concentration: 81-755 μg/m3

; IT Instillation- 200 μL 

(soluble and insoluble) 

Time to Analysis: 10 h/day 1, 4, 5 day 

(consecutive); F344 rats sensitized to endotoxin, 

BN rats to OVA. Both groups killed 24 h postexposure.

The retention of PM in the airways was 

enhanced by allergic sensitization. Recovery of 

anthropogenic trace elements was greatest for 

CAPs-exposed rats. Temporal increases in 

these elements were associated with eosinophil 

influx, BAL protein content and increased airway 

mucosubstances. A mild pulmonary neutrophilic 

inflammation was observed in rats instilled with 

the insoluble fraction but instillation of total, 

soluble or insoluble PM2.5 in allergic rats did not 

result in differential effects. 

December 2009 D-128 
Study Pollutant Exposure Effects 

Reference: Harrod 

et al. (2003, 

097046) 

Species: Mouse 

Gender: NR 

Strains: C57BL/6 

Age: 8-10 wk 

DEE: Diesel Engine Emissions 

generated from a 5.9-liter turbo diesel 

engine fueled by Number 2 fuel. 

DEE Composition: 

NOX: 2.0-43.3 ppm 

CO: 0.94-29.0 ppm 

SO2: 8.3-364.9 ppb 

Particle Size: 0.1-0.2 μm (MMAD) 

Route: Whole-body Inhalation 

RSV: IT administration 

Dose/Concentration: DEE: 38.8 μg/m3 (low level)

or 10027 μg/m3

 (high level) 

RSV: 100 μl 

Time to Analysis: 6 h/day, 7 days 

After the final 6 h exposure period mice were 

infected with RSV. 

Parameters measured 4 days post infection 

Viral Gene Expression: For air+RSV, RSV-F 

gene expression was not apparent but RSV-G 

gene expression was detectable at very low 

levels. In DEE+RSV (for high and low levels), 

RSV-F and -G were markedly elevated. ß-Actin 

mRNA levels not changed in DEE-exposed 

compared to air-treated. DEE+RSV for high and 

low levels show 10- to 20- fold induction of RSVG mRNA levels as compared to air+RSV. 

BALF Cells: Uninfected low-level DEE did not 

induce statistically significant increase in cell 

numbers as compared to air+RSV. High level 

DEE+RSV caused increase as compared to 

air+RSV. Uninfected high-level DEE had 

increase as compared to uninfected air group. 

For all groups, alveolar macrophages were 

predominant cell type and no substantial 

changes in infiltrating cell populations by 

exposure to DEE were noted. 

Lung Inflammation & Airway Epithelial 

Morphology: Lung sections from air- or DEEexposed, uninfected did not exhibit any 

observable change. Low level DEE + RSV had 

increased inflammatory cell infiltration in 

peribronchial regions and loss of normal 

cuboidal appearance of Clara cells as compared 

to air+RSV. High level DEE+RSV had more 

apparent lung-inflammation, especially 

surrounding bronchi and bronchioles, and 

increased appearance of pseudo-stratified, 

columnar epithelial cell morphology and 

apparent airway epithelial cell sloughing as 

compared to low level DEE+RSV, indicating 

dose-related increase in lung histopathology to 

RSV infection by prior DEE exposure.

Cytokines: TNF-α and IFN-γ were significantly 

increased in RSV-infected mice exposed to low 

or high level DEE and not increased in RSVinfected mice exposed to air. TNF-α levels 

elevated to similar levels for low and high level 

DEE+RSV. IFN-γ exhibited more dose-related 

increase with higher levels in high level 

DEE+RSV versus low level DEE+RSV. 

Mucous Cell Metaplasia: DEE exposure in 

uninfected was not altered. Mucous metaplasia 

was increased in epithelium of RSV-infected 

mice when exposed to DEE in a dosedependent manner. Following high level 

DEE+RSV, mucous staining of airway epithelial 

cells in more distal airways was occasionally 

observed. 

CCSP Production in Airway Epithelium: DEE 

alone did not have an effect CCSP-producing 

cells, or Clara cells, decreased in Low DEE + 

RSV and further decreased in high level 

DEE+RSV in large and terminal airways. 

Surfactant Protein B: proSP-B staining post 

RSV alone shows now discernible decrease 

when compared to uninfected. Staining levels in 

alveolar lung regions decreased when exposed 

to low level DEE+RSV, and further decreased in 

high level DEE+ RSV. Staining in airway 

epithelium following high level DEE+RSV 

diminished when compared to RSV alone or low 

level DEE+RSV. 

SP-A: In alveolar type II cells and airway 

epithelial cells for untreated and air +RSV, no 

discernible changes in levels. Prior exposure to 

low or high level DEE decreased SP-A staining 

in alveolar type II cells and airways epithelial 

cells during RSV infection. 

December 2009 D-129 
Study Pollutant Exposure Effects 

Reference: Harrod 

et al. (2005, 

088144) 

Species: Mouse 

Gender: Male 

Strain: C57B1/6 

Age: 10-12 wk 

Weight: NR

DEE (2 2000 model 5.9-1 Cummins ISB 

turbo diesel engines, No. 2 certification 

diesel fuel) 

Particle Size: NR 

Route: Inhalation

Dose/Concentration: Low- 30 μg/m3 PM, MidLow- 100 μg/m3

 PM, Mid-High- 300 μg/m3 PM, 

High- 1000 μg/m3

 PM 

Time to Analysis: 6 h/d, 7 days/wk, 1 wk or 6 mo. 

1 wk exposure repeated on separate occasion. 

Immediately after exposure, mice anesthetized, IT 

instilled with Pseudomonas aeruginosa. 

Bacterial Clearance: Lung bacterial clearance 

was decreased at all levels after 1wk exposure 

and was concentration-dependent 18h 

postinfection. Bacterial clearance was not 

affected at 6m and bacterial counts were higher. 

Inflammation, Particle Deposition: Lung 

inflammation and histopathology were increased 

in all exposure groups postinfection. All 

exposure groups possessed particle-laden 

macrophages. Higher doses had a 

concentration-dependent increase. 

Ciliated, Clara Cells, TTF-1: Generally, ciliated 

cells decreased with exposure dose, were more 

discernible in inflamed airways, and higher 

doses caused effects in small distal airways. 

Clara cells decreased equally at all exposures 

and were most notable in the distal airway 

epithelium. TTF-1 decreased postinfection. 

Reference: 

Heidenfelder et al. 

(2009, 190026) 

Species: Rat 

Gender: Male 

Strain: BrownNorway 

Age: 10-12 wk 

Weight: NR

CAPs (Grand Rapids, MI; July) 

Particle Size: Diameter: 0.1-2.5 μm 

Route: Whole-body Inhalation 

Dose/Concentration: CAPs: 493 ± 391 μg/m3; OC: 

244 ± 144 μg/m3, EC: 10 ± 4 μg/m3, Sulfate: 79 ± 

131 μg/m3, Nitrate: 39 ± 67 μg/m3, Ammonium: 39 ± 

59 μg/m3

; Urban dust (Fe, Al, Ca, Si): 18 ± 6 μg/m3 

Time to Analysis: Sensitized to OVA 3 day. 

Challenged with OVA or saline 2wk later for 3 day. 

Exposed to CAPs 8h/d, 13d. OVA or saline 

challenge 9 day after first challenge. Sacrificed 24 h 

after last CAPs exposure.

CAPs enhanced the effects of OVA by causing 

differential expression in genes primarily 

involved in inflammation and airway remodeling. 

CAPs exposure alone had no effect on gene 

expression. CAPs+OVA also increased IgE, 

mucin glycoprotein, and BALF total protein, and 

caused a more severe bronchopneumonia, 

increased mucus cell metaplasia/hyperplasia 

and mucosubstances. 

Reference: 

Hiramatsu et al. 

(2003, 155846) 

Species: Mouse 

Gender: Female 

Strains: BALB/c 

and C57BL/6 

Age: 8 wk 

Weight: 17-22 g 

DE -DE (generated by diesel engine and 

diluted with filtered clean air) 

Particle Size: NR 

Route: Inhalation

Dose/Concentration: Low -0.1 mg/m3

High - 3 mg/m3

Time to Analysis: 7 h/day, 5 days/wk, 1 or 3 mo 

Lung Histopathology: DEP-laden 

macrophages accumulated in the alveoli and 

peribronchial tissues in a dose- and durationdependent manner in both strains. Lymphocytes 

and neutrophils increased in both strains, but 

were greatest in the BALB/c mice. 

BALF and Mac-1 Positive Cells: BALT 

formation in DEP-laden AMs was seen at the 

high dose group and was greater in the BALB/c 

mice. Mac-1 positive cells, a marker for 

phagocytic activation of the AMs, was observed 

in the high dose groups of both strains at 1 and 

3 mo, and in the low dose group at 1 mo. in 

BALB/c mice. 

Cytokine and iNOS mRNA expression:1 

month of exposure increased TNF-α, IL-12p40, 

IL-4 and IL-10 mRNA in a dose-dependent 

manner. IL-1Β and iNOS decreased in a dosedependent manner. IFN-γ mRNA expression 

increased in BALB/c mice and decreased in 

C57BL/6 mice. Similar results were seen at 3 

mo, except IL-4 and IFN-γ mRNA expression 

decreased in the BALB/c mice. In C57BL/6 

mice, IL-4 and IL-10 mRNA increased at the low 

dose but decreased at the high dose. NF-κB 

activation occurred after 1 wk and 1 month DE 

exposure and was more prevalent in BALB/c 

mice. 

December 2009 D-130 
Study Pollutant Exposure Effects 

Reference: 

Hiramatsu, (2005, 

088285) 

Species: Mouse 

Gender: Female 

Strains: BALB/c 

Age: 8 wk 

Weight: 17-22 g 

DE (generated by diesel engine and 

diluted with filtered clean air.) 

Mycobacterial Infection -M.tuberculosis 

(ATCC35812) Kurono strain 

Particle Size: NR 

Route: Inhalation

Dose/Concentration: Low - 0.1 mg/m3 

High - 3 mg/m3

Mycobaterial infection: 5 mL (nebulized) of a 106 

colony-forming units (CFU) suspension 

Time to Analysis: 7 h/day, 5 day/wk, 1, 2 or 6 mo. 

Subset infected on last day of DE exposure. CFU 

evaluation 7 wk postinfection. 

Histopathological Observations: DEP-laden 

AMs and DEPs in the alveoli and peribronchial 

tissues increased in a time-dependent manner. 

DE-exposed mice had a greater number of 

mycobacterial lesions, which were 

disseminated. Lesions in the control mice had 

clear borders and consisted of epithelial cells 

and lymphocytes. Tubercle bacilli and DEPs 

coexisted in AMs. BALT was seen around DEPs 

in the 2 and 6-month exposure groups. 

Inflammation cells increased in a timedependent manner with respect to DE exposure.

Granulomatous Lesions in Lungs: 6-month 

DE-exposed mice had a significantly higher 

amount of gross lesions than the 6-month 

control mice. 

Mycobacterial Burden: CFU in lungs were 

increased in DE-exposed animals but only the 6 

month exposure resulted in statistically 

significant increases (a ~4-fold increase over 

control). CFU in spleen were not significantly 

altered by DE exposure. 

Cytokines and iNOS mRNA Expression: 

Infected DE-exposed mice had time-dependent 

increases of TNF-α, IL-1Β, IL-12p40, IFN-γ and 

iNOS mRNAs compared to the infected control 

mice. IL-12 mRNA expression decreased in 

infected 6-month DE-exposed mice. 

Reference: 

Ichinose, T. et al. 

(2003, 041525) 

Species: Mouse 

Gender: NR 

Strains: 

BALB/cAnN, ICR, 

C3H/HeN 

Age: 6 wk 

Weight: NR 

DE: DE generated by 3059cc 4-cylinder 

diesel engine 

Der f: Crude extract of D. farinae 

Particle Size: 0.4 μm (MMAD) 

Route: Inhalation 

Dose/Concentration: 1. Air 

2. DE only: 3.0 mg/m3

3. Air + Der f: 1 mg Der f 

4. DE 3.0 mg/m3

 + 1 mg Der f 

Time to Analysis: DE: 12 h/day, 7 days/wk, 8 wk 

Der f: 2 wk intervals, 6 wk 

Analyzed 3 days after last instillation 

Light Microscopic Observations: DE 

exposure caused the proliferation of nonciliated 

cells and epithelial cell hypertrophy. Sootcontaining macrophages were found in the 

alveolar tissue spaces. Accumulated 

lymphocytes were present in the peribronchiolar 

lymphoid tissue. Inflammatory cells and sootcontaining macrophages were found in the 

submucosal layer and the vessel interstitium of 

mice treated with DE+Der f in all strains. 

DE+Der f treated C3H/He mice had 

desquamated goblet cells. 

Eosinophil Infiltration: DE treated C3H/He 

mice had a slight eosinophil infiltration in the 

submucosal layer. DE+Der f treated mice in all 

strains had a slight to moderate eosinophil 

infiltration. 

Lymphocyte Accumulation: Lymphocytes 

significantly increased in all strains under the 

DE treatment as compared to the air+saline 

treatment, and further increased under the 

DE+Der f treatment. 

Goblet Cell Proliferation: Little proliferation 

was seen in all strains under the DE treatment. 

DE+Der f caused a significant increase in 

proliferation compared to air+Der f in ICR mice, 

but a significant decrease in C3H/He mice. 

Local Cytokine and Chemokine Expression 

in Lung Tissue Supernatant: DE+saline 

significantly increased MIP-1α in all strains. 

MCP-1 also increased but not significantly. 

DE+Der f increased IL-5, RANTES, eotaxin, 

MCP-1 and MIP-1α in all strains as compared 

with air+saline and air+Der f. IL-5 decreased in 

C3H/He mice treated with DE+Der f compared 

to air+Der f. IL-3 decreased in ICR and C3H/He 

mice compared to air+saline. 

Der f-specific Immunoglobulin Production in 

Plasma: Increased production of IgG1 was 

statistically significant in ICR and C3H/He mice 

treated with DE+Der f as compared to air+Der f. 

IgE was low in all strains. 

December 2009 D-131 
Study Pollutant Exposure Effects 

Reference: 

Ichinose et al. 

(2004, 180367) 

Species: Mouse 

Gender: NR 

Strains: BALB/c, 

ICR and C3H/He 

Age: 5 wk 

Weight: NR 

DEP: 2740cc 4-cylinder engine 

D. farinae: crude extract 

Particle Size: 0.4 μm (MMAD) 

Route: IT Instillation 

Dose/Concentration: 1. D. farinae: 1 μm in PBS 

2. D. farinae + DEP: 1 μg in PBS + 50 μg mg DEP 

Time to Analysis: 4 times at 2 wk intervals. Mice 

examined 3 wk after last instillation 

Histological Changes: Mice in all three strains 

treated with DEP+D. farinae had a significant 

recruitment of eosinophils, more proliferation of 

goblet cells, and more eotaxin positive 

macrophages in the alveoli than mice treated 

with D. farinae alone. 

Local Cytokine Expression in Lung Tissue 

Supernatant: DEP+D. farinae induced 

significant elevation of IL-5 in ICR and C3H/He 

mice as compared to D. farinae alone. 

Production levels of IL-4 and RANTES did not 

correlate with the manifestations of allergic 

airway inflammation induced by the D. farinae 

treatment with or without DEP. 

Cytokine Expression in Plasma: IL-5 in 

C3H/He mice treated with DEP+D. farinae was 

significantly higher than D. farinae alone. 

RANTES was unaffected by the DEP treatment 

in all strains. 

D. farinae-specific Immunoglobulin 

Production in Plasma: The adjuvant effect of 

DEP on IgG1 production was observed in all 

three strains, with C3H/H3 being statistically 

significant. The production levels of IgG1 

correlated with the manifestations of 

eosinophilic airway inflammation by both 

treatments. No adjuvant effect on IgE production 

was observed. 

Reference: Inoue 

et al. (2007, 

096724) 

Species: Mouse 

Gender: Male 

Strain: ICR 

Age: 6 wk 

Weight: 29-33 g 

PM-OC: Urban PM, collected for 1 

month during early summer, 2001 in 

Urawa city Saitama, Japan 

LPS 

Particle Size: <2.0 μm 

Route: IT Instillation 

Dose/Concentration: Vehicle group: PBS 

PM-OC group: 4 mg/kg of PM-OC 

LPS group: 2.5 mg/kg of LPS 

PM-OC+LPS group: combined administration of 

PM-OC +LPS 

Time to Analysis: Single, 24 h 

Effects of PM-OC on LPS Related Lung 

Inflammation: PM-OC alone did not 

significantly increase the infiltration of 

neutrophils, but LPS challenge showed a 

marked increase in the number of neutrophils 

compared with vehicle. Administration of LPS 

combined with PM-OC significantly increased 

the infiltration of neutrophils compared with LPS 

administration alone. 

Effects of PM-OC on Histological Changes in 

the Lung: Combined treatment with PM-OC 

and LPS resulted in enhanced neutrophilic 

inflammation. 

Effects of PM-OC on Pulmonary Edema 

Related to LPS: LPS group compared with 

vehicle group had a significant increase in lung 

water. The combined administration of PM-OC 

and LPS resulted in further increase in the lung 

water compared with LPS administration alone, 

however it was not statistically significant. 

Effects of PM-OC on Protein Expression IL1Β, MIP-1α, MCP-1 and KC: The 

concentrations of these molecules were below 

the detection limits in the PM-OC group. LPS 

treatment significantly increased the protein 

levels of these molecules compared with the 

vehicle treatment. In the PM-OC + LPS group all 

concentrations, particularly KC, were smaller 

than in the LPS group. 

December 2009 D-132 
Study Pollutant Exposure Effects 

Reference: Inoue 

et al. (2006, 

090951) 

Species: Mouse 

Gender: Male 

Strain: ICR 

Age: 6 wk 

Weight: 29-33 g 

Carbon black (14 nm PrinteX 90; PrinteX 

25; Degussa, Dusseldorf, Germany) 

Particle Size: 14 nm - 300 m2/g 

56 nm - 45 m2

/g 

Route: IT Instillation 

Dose/Concentration: Vehicle group: PBS at pH7.4

LPS group: 2.5 mg/kg of LPS in vehicle 

Nanoparticle groups: 4 mg/kg carbon black 

nanoparticles (14 nm or 56 nm) in vehicle 

LPS + nanoparticle group: combined administration 

of carbon black and LPS in vehicle 

Time to Analysis: Single, 24 h 

Effects of Nanoparticles: Nanoparticles alone 

increased number of total cells and neutrophils, 

but not statistically significant. LPS exposure 

significantly increased numbers for both groups. 

Nanoparticles and/or LPS enhance pulmonary 

edema. 

Histology: Treatment with LPS+14 nm 

nanoparticles markedly enhanced neutrophil 

sequestration into the lung parenchyma 

compared to LPS alone. LPS+56 nm 

nanoparticles did not. 

Cytokines: IL-1Β level significantly greater for 

both LPS+ nanoparticles groups. TNF-α was not 

significantly altered among the experimental 

groups. 

Chemokines: Challenge with 14 nm 

nanoparticles alone elevated the levels of all 

chemokines without significance except for KC. 

LPS alone and with both nanoparticle groups 

caused significant increases in all chemokines. . 

Formations of 8-OHdG in Lung: LPS plus 

nanoparticles resulted in intensive expression 8-

OHdG, strongest in LPS+14 nm nanoparticle 

Plasma Coagulatory Changes: PT - no 

change for any group. APTT - some change with 

LPS and LPS + nanoparticle groups, fibrinogen 

level significantly elevated after LPS and for 

LPS+14 nm nanoparticle. APC decrease with 

LPS (significant) and LPS + nanoparticle 

groups. vWF increase with LPS (significant) and 

LPS+14 nm (significant). 

Reference: Inoue 

et al. (2004, 

087984) 

Species: Mouse 

Gender: Male 

Strain: ICR 

Age: 6 wk 

Weight: 29-33 g 

DEPs [4JB-1 type light-duty, fourcylinder, 2.74 liter Isuzu diesel engine 

(Isuzu Automobile Co., Tokyo Japan)] 

Washed DEP and DEP-OC - extracted 

with dichloromethane 

Particle Size: NR

Route: IT instillation 

Dose/Concentration: Vehicle group: PBS; 

Washed DEP group: 4mg/kg of DEP; DEP-OC 

group: 4mg/kg of DEP-OC;LPS group: 2.5mg/kg of 

LPS; Washed DEP+LPS group: combined 

administration of washed DEP +LPS; DEP-OC+ 

LPS group: combined administration of DEP-OC + 

LPS 

Time to Analysis: 4 h 

COX-1 mRNA: Slightly elevated in both washed 

DEP and DEP-OC groups, but slightly 

decreased in other groups compared to vehicle 

group. 

COX-2 mRNA: Slightly increased with DEP-OC, 

increased with LPS, washed DEP + LPS and 

DEP-OC + LPS groups compared to vehicle. 

COX-2 in the DEP-OC + LPS decreased when 

compared to the LPS only group. 

Pulmonary Edema: Washed DEP + LPS group 

showed a synergistic enhancement of 

pulmonary edema and local expression of 

proinflammatory chemokines (MCP-1, MIP-1α, KC, IL-1Β). 

Reference: Inoue 

et al. (2006, 

096720) 

Species: Mouse 

Gender: Male 

Strain: ICR 

Age: 6-7 wk 

Weight: 29-33 g 

Carbon black (PrinteX 90; PrinteX 25; 

Degussa, Dusseldorf, Germany) 

Particle Size: 14 nm - 300 m2/g 

56 nm - 45 m2

/g 

Route: IT instillation 

Dose/Concentration: Vehicle group: PBS 

Ovalbumin (OVA) group: 1mg OVA; Nanoparticle 

groups: 50 mg carbon black nanoparticles (14 nm 

or 56 nm);;OVA + nanoparticle group: combined 

administration of nanoparticles and OVA 

Time to Analysis: Vehicle group - weekly for 6wk 

OVA group - biweekly for 6 wk 

Nanoparticle groups - weekly for 6 wk 

OVA+Nanoparticle group (same protocol as OVA 

and Nanoparticle) studied 24 h after last 

administration 

Nanoparticles: Exposure to carbon 

nanoparticles resulted in the lung expression of 

TARC, GM-CSF and MIP-1α. The levels were 

higher in the 14 nm group compared to the 

 56 nm group. 

OVA: In the presence of OVA, nanoparticles 

enhanced levels of TARC, GM-CSF, MIP-1α, IL2 and IL-10, with the effects seen more 

prominently in the 14 nm particles + OVA group. 

December 2009 D-133 
Study Pollutant Exposure Effects 

Reference: Inoue 

et al. (2005, 

088625) 

Species: Mouse 

Gender: Male 

Strain: ICR 

Age: 6-7wk 

Weight: 29-33 g 

Carbon black (PrinteX 90; PrinteX 25; 

Degussa, Dusseldorf, Germany) 

Particle Size: 14 nm - 300 m2/g 

56 nm - 45 m2

/g 

Route: IT Instillation 

Dose/Concentration: Vehicle group: PBS; 

Ovalbumin (OVA) group: 1mg OVA; Nanoparticle 

groups: 50mg carbon black nanoparticles (14nm or 

56 nm); OVA + nanoparticle group: combined 

administration of nanoparticles and OVA 

Time to Analysis: Vehicle group - weekly for 6 wk

OVA group - biweekly for 6 wk 

Nanoparticle groups - weekly for 6 wk 

OVA+Nanoparticle group: same protocol as OVA 

and Nanoparticle studied 24 h after last 

administration 

Nanoparticles + OVA: Nanoparticles given with 

OVA enhanced airway inflammation, 

characterized by increased eosinophils, 

neutrophils, mononuclear cells and goblet cells. 

In addition, nanoparticles + OVA significantly 

increased local expression of IL-4, IL-5, eotaxin, 

IL-13, RANTES, MCP-1 and IL-6. The formation 

of 8-OHdG was enhanced by nanoparticles + 

OVA. 

14 nm Nanoparticles: All these effects were 

more prominent when 14 nm nanoparticles were 

used. The 14 nm nanoparticle + OVA group 

significantly raised levels of total IgE and 

antigen specific production of IgG1 and IgE. 

Reference: Inoue 

et al. (2006, 

190142) 

Species: Mouse 

Gender: Male 

Strain: ICR 

Age: 6 wk 

Weight: 29-33 g 

Whole DE (generated by 4-cylinder, 

3.059l, Isuzu diesel engine, Isuzu 

automobile, Tokyo, Japan) 

LPS 

Particle Size: 110 nm (peak particle 

size) 

Route: Whole-body Inhalation 

Dose/Concentration: 0.3 mgsoot/m3

1.0 mgsoot/m3

3.0 mg soot/m3

LPS: 125 mg/kg 

Time to Analysis: LPS prior to 

12 h exposure to exhaust 

BAL fluid, total cells, neutrophils, protein and 

gene levels (MCP-1 and KC) decreased 

compared to control with LPS, but were smaller 

with LPS + DE. Results are suggestive that 

short-term exposure to DE does not exacerbate 

LPS-related lung inflammation. 

Reference: Inoue 

et al. (2007, 

096702) 

Species: Mouse 

Gender: Male 

Strain: ICR 

Age: 6 wk 

Weight: 29-33 g 

Cell Type 

Splenocytes 

DEPs [4JB-1 type light-duty, fourcylinder, 2.74 liter Isuzu diesel engine 

(Isuzu Automobile Co., Tokyo Japan)] 

LPS 

Particle Size: PM2.5

Route: Cell Culture (Splenocytes resuspended to 

cell density of 1×106/mL and 1000 mL applied into 

each of 12-well plate) 

Dose/Concentration: DEP: 100 mg/mL; LPS: 1 

mg/mL; LPS(1mg/mL) + DEP (1, 10 or 100 mg/mL) 

Time to Analysis: 72 h 

Cell viability: No effect. 

Mononuclear cell response: Incubation with 

DEP alone inhibited basal cytokine production. 

LPS significantly increased protein levels of IFNγ, IL-2, and IL-10 compared to control. DEP 

suppressed the LPS-enhanced protein levels in 

a dose-dependent manner and moderately 

elevated the IL-13 level. 

Reference: Inoue 

et al. (2007, 

198885) 

Species: Mouse 

Gender: Male 

Strain: ICR 

Age: 6-7 wk 

Weight: 20-30 g 

Carbon nanoparticles (PrinteX 90, 

PrinteX 25; Dusseldorf, Germany) 

OVA 

Particle Size: CB14 = 14 nm, CB56 

= 56 nm 

Route: IT Instillation 

Dose/Concentration: 50 μg and/or 1 μg OVA in 

PBS 

Time to Analysis: 1×/wk for 6 wk; sacrifice 24 h 

after last exposure 

Lung Responsiveness: Respiratory system 

resistance, Newtonian resistance and tissue 

dampening were significantly higher in the 

nanoparticle + OVA groups. Elastance and 

tissue elastance were higher in these groups but 

not significantly so. Compliance was 

significantly lower in the nanoparticle + OVA 

groups compared to the control. 

Lung mRNA Level for Muc5ac: Levels were 

significantly higher in nanoparticle + OVA groups 

compared to the control. 

Reference: Inoue 

et. al. (2007, 

096692) 

Species: Mouse 

Gender: Male 

Strain: ICR 

Age: 6-7 wk 

Weight: 29-34 g 

DEP-OC collected from 4JB1 type, light 

duty, 4 cylinder, 2.74 liter Isuzu diesel 

engine, Isuzu Automobile Company, 

Tokyo, Japan) 

OVA 

Particle Size: 0.4 μm 

Route: IT Instillation 

Dose/Concentration: 50 μg and/or 1 μg OVA in 

PBS 

Time to Analysis: DEP or DEP-OC w/ or w/o OVA 

initially; OVA or vehicle every 2 wk for 6 wk; DEP 

components or vehicle 1×/wk for 6 wk; sacrifice 

24 h after last instillation 

Total respiratory system resistance, elastance, 

Newtonian resistance, tissue damping, tissue 

elastance displayed general positive trends and 

were significantly higher in OVA and OVA + 

DEP-OC groups. Compliance displayed a 

general negative trend and was significantly 

lower in the washed DEP + OVA group. 

December 2009 D-134 
Study Pollutant Exposure Effects 

Reference: Ito et 

al. (2006, 088391) 

Species: Rat 

Cell Line: L2 cells 

of alveolar 

epithelial cell type 

II origin 

DEP - generated from 2982-cc common 

rail direct injection diesel engine with 

oxidation catalyst and exhaust gas 

recirculation system. 

Particle Size: PM2.5

Route: Cell Culture 

Dose/Concentration: 1×106 

1,10 or 30 mg/mL 

Time to Analysis: 3 h 

ICAM-1 and LDL Receptor mRNA: Upregulation in a dose-dependent manner. 

Statistically significant at 30 mg/mL compared to 

control. 

HO-1 and PAF Receptor mRNA: Up-regulation 

in dose-dependent manner and statistically 

significant at all doses compared to control. 

Correlation Between HO-1 and ICAM-1, LDL, 

and PAF: Significant correlation between HO-1 

and each of these. 

Reference: Jang 

et al. (2005, 

155313) 

Species: Mouse 

Gender: Female 

Strains: BALB/c 

Age: 5-6 wk 

DEP -generated from 4JB1 type, light 

duty, four-cylinder diesel engine (Isuzu 

Automobile, Co, Tokyo, Japan) 

O3 - (generated with Sander Model 50 

ozonizers, Sander, Eltze Germany) 

OVA 

Particle Size: NR 

Route: Whole-body Inhalation 

Dose/Concentration: DEP: 2,000 μg/μL (sic) 

O3: 2 ppm (avg 1.98 ± 0.08 ppm) 

OVA sensitization: 10 mg 

Time to Analysis: OVA sensitization, 

DEP, O3 and OVA Challenge on d21- 23 

Exposed to O3 for 3 h and DEP for 1 h 

AH and BAL measured 1 day after last challenge 

Airway Responsiveness: OVA + O3 + DEP 

exposure group had significantly higher 

methacholine-induce Penh than sham group or 

OVA group. 

Total cells, proportion of eosinophils and 

neutrophils: The OVA + O3 + DEP group was 

significantly higher than OVA group and OVA+ 

O3 group. 

IL-4: OVA + O3, OVA + DEP and OVA + O3 + 

DEP IL-4 level increased compare to OVA 

group. 

IFN-γ: Levels significantly decreased in OVA + 

DEP and OVA + O3 + DEP compared to OVA + 

O3. 

Reference: 

Jaspers et al. 

(2005, 088115) 

Species: Human 

Cell Lines: A549 

cells, primary 

human bronchial 

and nasal epithelial 

cells 

DEas: aqueous-trapped solution of DE 

(emissions from Caterpillar diesel 

engine, model 3304) 

Influenza: A/Bangkok/1/79 (H3N2 

serotype) 

Particle Size: NR 

Route: Cell Culture

Dose/Concentration: Influenza: 3×105

 cells 

infected with 320 hemagglutination units (HAU) 

DEas: For A549 cells: 6.25, 12.5, 25 μg/cm2

. For 

bronchial and nasal cells: 22 or 44 μg/cm2

. 

Time to Analysis: 2 h incubation with DEas then 

virus added. 

HA RNA levels analyzed at 0, 15, 30, 60 or 120 min 

post infection. 

IFN and MxA responses: analyzed 24 h post 

infection. 

Fluorescence: some cells treated with GSH-ET 30 

min before DEas exposure. Measured 2 h postinfluenza infection. 

A549 Cells Increased Susceptibility: DEas 

enhances HA RNA levels in A549 cells in a 

dose-dependent manner. 25 μg/cm2 significantly 

enhanced levels in A549 cells compared to the 

influenza-infected controls. Viral protein levels 

were increased in A549 cells. Exposure to DEas 

increased the number of influenza-infected 

epithelial cells in A549 cells. 

Human Nasal and Bronchial Cells 

Susceptibility: Exposure to DEas increased HA 

RNA levels in the nasal and bronchial cells. 

Statistically significant at 22 μg/cm2 for nasal 

cells and approaching significance at 44 μg/cm2

for bronchial cells. Exposure of both types to 44 

μg/cm2 enhanced viral protein levels. 

Influenza Induced IFN Response in A549:

Exposure to DEas does not suppress but 

enhances IFN-ß mRNA levels. Treatment 

enhanced influenza-induced nuclear levels of 

both phospho-STAT-1 and ISFG3g. ISREpromoter activity was enhanced, but not 

significantly. Treatment enhanced myxovirus 

resistance protein (MxA) mRNA levels. This data 

suggest that DEas exposure enhances influenza 

virus replication without suppressing production 

of IFN-ß or IFN-ß-inducible genes. 

Influenza Induced IFN Response in Human 

Nasal and Bronchial Cells: Exposure to DEas 

increased IFN-ß and MxA levels. 

Oxidative Stress in A549: DEas exposure 

dose-dependently increases oxidative stress in 

A549 cells within 2-h post-exposure. Add the 

antioxidant GSH-ET and it reverses the effect. 

Pretreatment with GSH-ET A549 cells reversed 

the effects of DEas on the number of influenzainfected cells, and reduced HA RNA levels. 

Oxidative Stress in Human Bronchial Cells:

The results were the same as A549 cells 

pretreated with GSH-ET. Or Pretreatment with 

GSH-ET also reversed effects of DEas on HA 

RNA levels. 

December 2009 D-135 
Study Pollutant Exposure Effects 

Reference: Kaan 

and Hegele (2003, 

095753) 

Species: Guinea 

pig 

Gender: Female 

Strain: Cam 

Hartley 

Age: 22-29 days 

Weight: 250-300 g 

Cell Types: AM 

PM10 - EHC-93 obtained (Environmental 

Health Canada, Ottawa, ON, Canada) 

RSV - Human RSV (long strain/lot18D) 

(American Tissue Culture Collection, 

Bethesda, MD) 

Particle Size: PM10 (0.35 μm MMAD) 

Route: Cell Culture 

Dose/Concentration: PM10: 500 μl/well (100 μg/ml 

MEM) 

RSV exposure:: 1 ml/well (6×106

 pfu/ml MEM) 

Groups: PM10+RSV 

RSV+PM10

RSV only 

PM10 only 

negative control 

Time to Analysis: PM10 - 60 min; RSV - 90 min 

Parameters measured 24 h post treatment 

Interaction on Phagocytic Ability of AM: Not 

affected by sequential exposure to RSV and 

PM10. More than 95% of AM exposed to PM10

engulfed PM. AM exposed to PM10 showed 

significant increase in mean side scatter in 

comparison to negative control and RSVinfected AM. No significant difference between 

AM exposed only to PM10 and AM exposed to 

both agents. No significant side mean side 

scatter difference between AM exposed to PM 

only and to both agents. 

Interaction on RSV Immunopositivity: PM10

exposure inhibits. All RSV-treated groups 

showed significantly greater proportion of RSVimmunopositive cells compared with negative 

control. PM10+RSV showed significantly smaller 

proportion of RSV-immunopositive cells 

compared with RSV group. RSV+PM10 group 

similar to RSV group. Proportion of RSVimmunopositive AM was influenced by the 

sequence of exposure to RSV and PM10. 

Interaction on RSV Replication: PM exposure 

suppressed RSV replication. AM exposed to 

both agents produced 3 to 9 fold less RSV 

progeny compared with RSV alone group. 

Quantity of RSV progency was not significantly 

affected by the sequence of exposure RSV and 

PM10. Negative control and PM10 only did not 

propagate progeny. 

Interaction of RSV Yield: RSV alone group 

produced the highest RSV yield, those exposed 

to both agents, independent of sequence, 

showed a 5-fold decrease. 

Cytokine production: RSV infection stimulated 

all three cytokines measure (IL-6, IL-8 and TNFα) compared to negative control. IL-6: PM10

significantly reduced RSV-induced IL-6 

production. IL-6 was affected by the sequence 

of exposure to PM10 and RSV (PM10+RSV vs. 

RSV+ PM10). IL-8: PM10 significantly decreases 

RSV-induced IL-8 production and baseline. No 

affect on sequence of exposure. TNF-α: 

production was increased when exposed to 

RSV, PM10 or a combination of both agents. No 

differences among treatments. 

Reference: 

Kleinman et al. 

(2005, 087880) 

Species: Mouse 

Gender: Male 

Strains: BALB/c 

Age: 8-19 wk 

Weight: NR 

CAPS: fine (F) and ultrafine (UF) using 

VACES system; performed a 2 sites in 

Los Angeles, CA, one 50-m downwind 

and another 150-m downwind from a 

complex of three roadways, State Road 

CA60, Interstate 10, and Interstate 5 

F CAPS in 2001 and 2002, UF CAPS in 

2002 only 

OVA: Ovalbumin 

Particle Size: UF: dp ≤ 150 nm 

F: dp ≤ 2.5 μm 

Route: Whole-body Inhalation 

OVA sensitization: nasal instillation 

OVA challenge: inhalation 

Dose/Concentration: UF at 50 m: 433 μg/m3

-UF at 150 m: 283 μg/m3

F at 50 m or 150 m: average 400 μg /m3

 

OVA sensitization: 50 μg/5 μl 

OVA challenge: 30 mg/m3

 

Time to Analysis: CAPS: 4 h/day, 5 days/wk for 2 

wk 

Sensitization: On morning of each exposure 

1st Challenge: week after 10 days of treatment 

2nd Challenge: one week following 1st challenge 

Sacrificed: 24 h after 2nd challenge 

There were significantly higher concentrations of 

IL-5, IgE, IgG1 and eosinophils in mice exposed 

to either CAPS compared to air. Mice exposed 

to CAPS at 50-m downwind showed higher 

levels of IL-5, IgG1, and eosinophils than those 

exposed to CAPS 150-m downwind. 

December 2009 D-136 
Study Pollutant Exposure Effects 

Reference: 

Kleinman et al. 

(2007, 097082) 

Species: Mouse 

Gender: NR 

Strains: BALB/c 

Age: 6-8 wk 

CAPS - concentrated fine (F) and 

ultrafine (UF) using VACES system - 

performed a 2 sites in Los Angeles, CA, 

on 50-m downwind and another 150-m 

downwind from State Road CA60 and 

Interstate 5. Fall 2001-summer 2004 

OVA 

Particle Size: F: PM2.5; UF: PM0.15 

Route: Whole-body Chamber 

Dose/Concentration: 50 m - F: 394 ± 94 μg/m3 

50 m - UF: 297 ± 189 μg/m3 

150 m - F: 387 ± 68 μg/m3

 

150 m - UF: 213 ± 95 μg/m3

 

OVA - 50 mg in 5 mL saline 

Time to Analysis: 3, 4 h/day, 5 days/wk, 2wk 

OVA the morning of each exposure 

50m Site: higher levels and statistically 

significant concentration curves of IL-5 and IgG1 

in F-CAP mice at the 50 m site. 

150m Site: in no cases were responses greater 

than the 50m or control groups. 

F vs. UF: The study was not able to differentiate 

between the effects of F PM and UF PM 

exposures. 

Reference: KleinPatel et al. (2006, 

097092) 

Species: Cattle 

and Human 

Cell Types 

Bovine tracheal 

epithelial cells 

(BTE) and A549 

ROFA 

V2O5, VOSO4, SiO2 TiO2, Fe2(SO4)3, 

NiSO4, LPS 

Particle Size: 1.95 μm (MMAD) 

Route: Cell Culture 

Dose/Concentration: ROFA: 0, 2.5, 5, 10, 15, 20 

μg/cm2

LPS: 100 ng/mL 

V2O5: 0, 0.15, 0.3, 0.61, 1.25, 2.5, 5, 10, 20 μg/cm2

NiSO4, Fe2(SO4)3, TiO2, SiO2: 0, 1.23, 2.5, 5, 10, 20 

μg/cm2

VOSO4: 0, 0.145, 0.29, 0.58, 1.16, 2.32 μg/cm2

Time to Analysis: LPS: 0, 6, or 18 h 

ROFA: 0, 2,4,6 h 

V2O5: 0, 0.25, 0.5, 1, 2, 4, 6, 8h 

NiSO4, Fe2(SO4)3, TiO2, SiO2, VOSO4: 6 h 

ROFA in BTE: ROFA and ROFA leachate 

inhibition of LPS-induced TAP gene expression 

increases with exposure time and dose. Washed 

particles of ROFA at doses 2.5 to 10 mg/cm2

significantly increased inducible TAP 

expression. 

Soluble Metals in BTE: V2O5 inhibition of LPS 

and IL-1β induced TAP gene expression 

increases with exposure time and dose. NiSO4

exhibits non-significant dose dependent 

suppression of inducible TAP gene expression. 

Fe2(SO4)3, TiO2 and SiO2 were found to have no 

effect. 

A549: Results with ROFA and V2O5 in BTE were 

replicated using the A549 cell line and IL-1β to 

induce hBD2 gene expression. 

Cellular Viability: Was not significantly affected 

in ROFA doses below 20 μg/cm2

 and 

V2O5/VOSO4 doses below 2.5 μg/cm2

. 

Reference: Koike 

and Kobayashi 

(2005, 088303) 

Species: Rat 

Gender: Male 

Strains: Wistar 

Kyoto 

Age: 8-10 wk 

Weight: 280-350 g 

Cell Types: AM, 

PBM (peripheral 

blood monocytes), 

T-cells (antigen 

sensitized) 

Whole DEP: Diesel Exhaust Particles 

collected in the dilution tunnel of a diesel 

inhalation facility. (Ratio of organic 

extract to residual particles in the whole 

DEP was 3: 1.) 

Organic extract of DEP 

Residual particles of DEP 

OVA: Ovalbumin 

Particle Size: NR 

Route: Cell Culture (1×106 cells/ml) 

Dose/Concentration: Whole DEP: 10, 30, 100 

μg/mL 

Organic extract of DEP: 7.5, 22.5, 75 μg/mL 

Residual particles: 2.5, 7.5, 25 μg/mL 

Time to Analysis: 24 h post exposure 

Ia Antigen and Costimulatory Molecules: 

Most control AM did not express these 

molecules. Whole DEP did not cause any 

increase in expression level. 20% of control 

PBM expressed Ia and 10% B7; expression of 

these molecules was significantly increased by 

whole DEP. Organic extract significantly 

increased the expression of Ia and B7 

molecules on PBM similar to whole DEP. 

Residuals caused no effect. Organic extractinduced expression of Ia antigen in PBM was 

reduced by treatment with NAC. 

AP Activity: After exposure to organic extract, T 

cell proliferation was significantly increased by 

the addition of control PBM in a cell numberdependent manner. AP activity of PBM was 

increased over control by exposure to 3 μg/mL 

organic extract, although higher concentrations 

suppressed the activity of PBM. 

Cytokine Production: Organic extract 

treatment of PBM decrease IFN-γ production 

from T-cells stimulated by PBM. No significant 

effect on IL-4 observed. 

HO-1 Protein Level: Levels in PBM were 

significantly increased by exposure to whole 

DEP or organic extract. Levels induced by 

organic extract was diminished by NAC 

treatment. 

December 2009 D-137 
Study Pollutant Exposure Effects 

Reference: Last et 

al. (2004, 097334) 

Species: Mouse 

Gender: NR 

Strains: BALB/c 

Age: 6 wk 

Weight: 16-20 g 

PM - aerosol of soot and iron oxide 

OVA 

Particle Size: PM0.1 - PM2.5 

Route: Inhalation 

OVA - Intraperitoneal Injections; Aerosol Exposure 

Dose/Concentration: PM - 235-256 μg/m3

OVA - 10 μg/0.1 mL injection 

OVA aerosol - 10 mL of 10 mg/mL (1%) solution 

Time to Analysis: PM: 4 h/day, 3 days/wk; OVA: 2 

ip injections days 1 and 15. Aerosol on day 28 after 

first ip; 60 min 3x/wk 

2 Wk PM Exposure/4 Wk OVA Aerosol 

Treatment: The OVA alone group had 

significantly more airway collagen than the PM 

alone group. Histology showed significantly 

more collagen in the treatment than the air 

alone group. There was a significantly greater 

amount of goblet cells than the OVA alone 

group. 

4 Wk OVA Aerosol/ 2 Wk PM Treatment: The 

OVA treatment had significantly more goblet 

cells than the PM alone group. 

6 Wk Concurrent PM and OVA Treatment:

Significantly more cells were observed in the 

OVA alone group over the treatment. The 

treatment had significantly more lymphocytes 

and significantly less macrophages than groups 

exposed to PM before or after OVA. Histology 

showed significantly more collagen in the 

treatment than the air or PM alone groups. The 

treatment had significantly more goblet cells 

than the OVA alone group. 

Reference: Li et 

al. (2007, 093156) 

Species: Mouse 

Gender: Female 

Strain: BALB/c, 

C57BL/6 

Age: 9 wk 

Weight: NR

DEP (2369-cc diesel engine 

manufactured by Isuzu Motor, operated 

at 1050 rpm, 80% load, commercial light 

oil) 

Particle Size: NR 

Route: Inhalation 

Dose/Concentration: DEP: 103.1 ± 9.2 μg/m3, 

CO: 3.5 ± 0.1 ppm, NO2: 2.2 ± 0.3 ppm, SO2: 

 <0.01 ppm 

Time to Analysis: Protocol 1: Exposed 7h/day, 

5days/wk. Sacrificed at day 0, week 1, 4, 8. 

Protocol 2: DE alone or DE+NAC 7h/d, 1-5 days. 

Airway Hyperresponsiveness: Penh values 

increased in BALB/c mice compared to the 

control at day 0, but no significant changes 

occurred after this time. Penh values increased 

in C57BL/6 mice at 1wk compared to the control 

but returned to control levels at 8 wk. 

BALF: Compared to the other strain, the total 

number of cells and macrophages increased 

significantly at 1wk in C57BL/6 mice and at 8wk 

in BALB/c mice. Neutrophils, lymphocytes, 

MCP-1, IL-12, IL-10, IL-4, IL-13 increased 

significantly for both strains. No eosinophils 

were found. IL-1Β and IFN-γ increased 

significantly in BALB/c mice compared to 

C57BL/6 mice. 

HO-1 mRNA and Protein: HO-1 mRNA was 

more marked in BALB/c mice at 1wk and 

C57BL/6 mice at 4 and 8 wk. HO-1 protein

percentage changes from the control were 

greater in BALB/c mice at 1wk and C57BL/c 

mice at 8 wk. 

NAC: NAC inhibited the increased Penh values, 

total number of cells and macrophages in 

C57BL/6 mice at 1 wk and neutrophils and 

lymphocytes in both strains. 

Reference: Li et 

al. (2009, 190457) 

Species: Mouse 

Gender: Female 

Strain: BALB/c 

Age: 6-8 wk 

Weight: NR

CAPs (downtown Los Angeles, CA from 

major freeway, traffic mainly passenger 

cars and diesel trucks; Jan. 2007 or 

Sept. 2006) 

Ultrafine carbon black (UFCB; used as 

control) 

Particle Size: Fine- <2.5 μm (diameter), 

UF- <0.15 μm (diameter) 

Route: Intranasal Instillation 

Dose/Concentration: 0.5 μg PM in 50 μL 

suspension 

Time to Analysis: Day 1 exposed to PM or saline.

Day 2 exposed to PM+OVA or OVA or saline alone. 

Repeated on days 4, 7, 9. Different experiment: 

NAC ip injected 4 h pre-instillation on days 1, 2, 4, 

7, 9. All animals rested and OVA aerosol 

challenged 30 min on days 21, 22. Sacrificed day 

23. 

UFP alone had no effect on the lung. UFP+OVA 

significantly increased eosinophils, and OVAspecific IgG1 and IgE. The induction of 

eosinophils and IgG1 were inhibited by NAC. 

Generally, UFP+OVA mice had greater signs of 

inflammation than the other groups as 

determined by pulmonary histopathology and 

airway morphometry. UFP had a greater PAH 

content than fine particles. UFP significantly 

increased IL-5, IL-13, TNF-α, IL-6, KC, MCP-1, 

and MIP-1α. 

Reference: Li et 

al. (2009, 190457) 

Species: Mouse 

Cell Line: RAW 

264.7 

CAPs (downtown Los Angeles, CA from 

major freeway, traffic mainly passenger 

cars and diesel trucks; Jan. 2007 or 

Sept. 2006) 

Ultrafine carbon black (UFCB) 

Particle Size: Fine- <2.5 μm (diameter), 

UF- <0.15 μm (diameter) 

Route: Cell Culture 

Dose/Concentration: 1, 5, 8.3, 10 μg/mL 

Time to Analysis: NR

UFP induced greater HO-1 expression than fine 

particles. The higher PAH content of UFP 

correlated with HO-1 expression. 

December 2009 D-138 
Study Pollutant Exposure Effects 

Reference: Liu et 

al. (2008, 156709) 

Species: Mouse 

Gender: Female 

Strain: BALB/c 

Age: 11wk 

Weight: NR

DEP (5500-watt single-cylinder diesel 

engine generator (Yanmar, Model YDG 

5500E), 406 cc displacement air-cooled 

engine, Number 2 Diesel Certification 

Fuel, 40 weight motor oil) 

Particle Size: ~0.1 μm (MMAD) 

Route: Intranasal Exposure 

Dose/Concentration: Average particle 

concentration: 1.28 mg/m3

Time to Analysis: Four groups: saline+air control, 

saline+DEP, A. fumigatus+air, A.fumigatus+DEP. A. 

fumigatus exposure every 4 days for 6 doses. DEP 

exposure 5 h/dayfor 3 wk concurrent with A. 

fumigatus exposure. 

A.fumigatus+DEP increased IgE, the mean BAL 

eosinophil percentage, goblet cell hyperplasia, 

and eosinophilic and mononuclear cell 

inflammatory infiltrate around the airways and 

blood vessels compared to the A. fumigatus or 

DEP treatments. A.fumigatus+DEP also caused 

methylation at the IFN-γ promoter sites CpG-53, 

CpG-45, and CpG-205. 

Reference: Liu et 

al. (2007, 093093) 

Species: Mouse 

Gender: Female 

Strain: BALB/c 

Age: 11wk 

DEP: 5500-watt single-cylinder diesel 

engine. 

Particle Size: NR 

Route: Inhalation 

Dose/Concentration: Average particle 

concentration 1.28 mg/m3. 

Time to Analysis: 1. Aerosol vehicle (saline) + air

2. Aerosol vehicle (saline) + DEP 

3. A. fumigatus + air 

4. A. fumigatus + DEP 

A. fumigatus: 62.5 μg aerosolized protein extract in 

50 μL PBS; 6 total doses, every 4 d. 

DEP exposure 5 h/day 3wk concurrent with A. 

fumigatus. 

IgE Production: IgE production increased with 

the A.fumigatus treatment and increased further 

with the A.fumigatus and DEP treatment. 

Histopathology: A. fumigatus with DEP caused 

an increase in goblet cell hyperplasia and 

eosinophil and mononuclear cell infiltrate around 

the airways and blood vessels as compared to 

the control and DEP treatments. 

Gene Methylation: Greater methylation at the 

CpG-53 site of the IFN-γ promoter occurred 

under the A. fumigatus + DEP treatment 

compared to the A. fumigatus or DEP 

treatments. The DEP treatment did not induce 

methylation. Methylation correlated with 

increased IgE and hypomethylation with 

decreased IgE. Hypomethylation occurred in the 

IL-4 promoter under the A. fumigatus + DEP 

treatment. 

Reference: 

Lundborg et al. 

(2007, 096040) 

Species: Rat 

Gender: Male 

Strains: SD 

Age: NR 

Weight: 300-400 g 

Cell Line: AM 

Carbon-Black Particles (93% C) 

DEPs (97% C) - toluene-extracted 

10-fold Cr, Mn, N; 50-100 fold Al, Cd, 

Cu, Fe, Mg, Pb, Zn more in DEP 

aggregates 

Particle Size: Carbon aggregates: 0.17 

± 0.08 μm (mean diameter) 

Diesel Particles: 0.69 ± 0.46 μm (mean 

diameter) 

Primary particles: 0.044 ± 0.01 μm 

(mean diameter) 

Route: Cell Culture (0.5×106 AM/well) 

Dose/Concentration: 20 μg/mL 

surface area: 159 ± 4m2

/g 

Time to Analysis: 6 different experiments. AM preexposed to carbon or washed DEP. Loaded with 

particles. Incubated with S. pneumoniae, ATCC 

strain or clinical isolates. 

Effect of Time on Survival of S. Pneumoniae 

when Incubated with Carbon Loaded AM: 

Loading AM with carbon significantly increased 

the bacterial survival. Bacteria opsonization 

decreased bacterial survival. 

Effect of Carbon Load in AM on Survival of 

S. Pneumoniae: Bacterial survival increased in 

a dose-dependent manner as the carbon 

particle load of AM increased. 

Survival of S. Pneumoniae after Incubation 

with Carbon or Washed Diesel Loaded AM: 

Bacterial survival increased in carbon loaded 

AM compared to the control. No difference 

existed with the washed diesel particles. 

Survival of the ATCC Strain and Clinical 

Isolates of S. Pneumoniae when Incubated 

with Carbon Loaded AM or Control AM: 

Carbon significantly increased the CFU of 

opsonized and unopsonized bacteria for the 

ATCC strain and clinical isolates. 

Ability of carbon or washed diesel loaded 

AM, incubated with the ATCC strain of S. 

pneumoniae, to induce LPO of lung 

surfactant: A 97% increase in the surfactant 

LPO occurred after incubation with washed 

diesel loaded AM compared to control AM. The 

effect of washed diesel particles was 

significantly greater than that of carbon 

particles. 

LPO by carbon loaded AM incubated with the 

ATCC strain or clinical isolates in the 

presence of absence of surfactant: LPO 

induced by AM increased when incubated with 

carbon loaded AM compared to control AM. 

December 2009 D-139 
Study Pollutant Exposure Effects 

Reference: 

Matsumoto et al. 

(2006, 098017) 

Species: Mouse 

Gender: Female 

Strains: BALB/c 

Age: 6 wk 

Weight: 15-20 g 

DE (collected from a 2369 cm3

 diesel 

engine operated at 1050 rpm and 80% 

load with commercial light oil; engine 

exhaust passed through a particulate air 

filter and charcoal filer) Diluted DE 

introduced into the exposure chamber. 

Composition of the DE: 3.5 ± 0.1 ppm 

CO, 2.2 ± 0.3 ppm NO2, <0.01 ppm SO2

and 103.1 ± 9.2 μg/m3

 DEP. 

Particle Size: NR 

Route: Whole-body Inhalation 

Dose/Concentration: 100 μg/m3 DE 

Time to Analysis: Mice were initially sensitized w/ 

OVA (20ug absorbed to 2 mg alum diluted with 0.5 

mL saline) via ip injection on day 0, 6 and 7. Two 

wks later the mice were challenged with OVA 

(0.1mg in 0.1mL saline) intranasally on day 21. 

DE for 1d or 1,2, 3, 4 or 8 wk (at 7 h/day for 5 

days/wk). 

Airway Hyperresponsiveness: Exposure to 

DE significantly increased airway reactivity to 

methacholine after 1 wk in both 24 and 48 

mg/mL Mch and after 4 wk in the 48 mg/mL. DE 

exposure caused an increase in airway 

sensitivity after 1 wk of exposure, 4 wk and 8 wk 

of exposure did not result in a significant 

increase. 

BAL Cells: The total cell count was increased 

after 1 wk of DE exposure. This increase was 

mostly due to an increase in eosinophils. After 1 

wk the total cell count dropped drastically even 

after continuous exposure to DE. DE did not 

effect the number of CD3, CD4, CD8 or NK1 

cells at any point in time. 

Cytokine/Chemokine mRNA Levels: DE 

exposure on day 1 caused an increase in mRNA 

levels of IL-4, IL-5 and IL-13 when compared to 

the control mice but longer periods of DE 

exposure failed to cause an increase. Protein 

levels of IL-4 were significantly elevated at 

compared to control at day 1, but did not persist 

with time. mRNA levels of MDC were increased 

at 1 wk of exposure (compared to control) but 

also decreased at time periods after. mRNA 

levels of RANTES were increased at 2 and 3 wk 

after exposure and remained elevated at 4 wk 

but not significantly. The level of RANTES 

protein increased as the weeks went along, but 

increased significantly only at 8 wk. 

Histopathology: OVA sensitization caused an 

increase peribronchial and perivascular 

infiltration of inflammatory cells which peaked at 

1 wk after exposure and decreased afterward. 

DE exposure did not cause/show any additional 

signs of inflammation. 

Reference: 

Morishita et al. 

(2004, 087979) 

Species: Rat 

Gender: Male 

Strain: BrownNorway 

Age: 10-12 wk 

CAPs (generated from ambient air in an 

urban Detroit community). 

Particle Size: 0.1-2.5 μm 

Route: Whole-body Inhalation 

Dose/Concentration: July 676 μg/m3; September 

313 μg/m3

Time to Analysis: First rats were sensitized (days 

1-3) and challenged (days 14-16) with saline 

(control) or OVA by intranasal instillation (5% in 

saline, 150 μL/nasal passage). 

4 days after the last intranasal challenge, rats 

began exposure in the chambers. Exposures were 

10 h long. The July exposure was for 4 consecutive 

days. The September exposure was for 5 

consecutive days. 

Recovery of Trace Elements in Animal Lung 

Tissues: July Exposure- Anthropogenic trace 

elements were below limit of detection in 

pulmonary tissue of animals exposed to July 

CAPs. September Exposure- Several elements 

were recovered from pulmonary tissue during 

the Sept. exposure. La concentrations were 

increased in both control/CAPs exposure and in 

the OVA/CAPs exposure groups. V 

concentration was increased in OVA/CAPs 

exposed animals but not in rats exposed to just 

CAPs. S content was only significant in animals 

exposed to OVA/CAPs compared to the nonexposed control. 

Particle Characterization: July PM had an 

average mass concentration twice as high as 

the September mass concentration. S 

concentration was four-folds higher in July PM. 

In the September PM- the concentration of La 

was 12.5 fold higher than in July PM, V was 2.7 

fold higher than in July PM and Mn was 1.5 fold 

higher than in July PM. 

BALF Analysis: Eosinophil concentration was 

not significantly different when comparing rats 

exposed to CAPs only in either July or 

September (this was explained by the elapsed 

time between exposure and BALF collection). 

However OVA and CAP exposure in the 

September group led to elevated eosinophil 

levels. Similarly, the protein content was only 

significantly increased in the September 

OVA/CAP exposed rats, compared to the control 

group. 

December 2009 D-140 
Study Pollutant Exposure Effects 

Reference: 

Nygaard et al. 

(2005, 088655) 

Species: Mouse 

Gender: Female 

Strain: BALB/c 

Age: 6-7 wk 

Coarse and fine ambient air particles 

collected in Rome (spring), Oslo (1-

summer, fine only, 2- following spring, 

fine and coarse), Lodz (summer) and 

Amsterdam (spring). These represent 

areas with high population and 

dominance of traffic. 

DEP (Standard reference material 

1650a) 

Particle Size: Fine PM 0.1-2.5 μm; 

Coarse PM 2.5-10 μm 

Route: Subcutaneous Injection into mouse 

footpads. 

Dose/Concentration: 100 μg of particle 

Time to Analysis: Animals were in eight groups: 1. 

Control- Hank’s Balanced Salt Solution 

2. OVA- 50 ug 3. OVA (50 μg)+ Amsterdam Coarse 

PM (100 μg) 4. OVA (50 μg)+ Amsterdam Fine PM 

(100 μg) 5. OVA (50 μg)+ Lodz Coarse PM (100 

μg) 6. OVA (50 μg)+ Lodz Fine PM (100 μg) 7. 5. 

OVA (50 μg)+ Oslo Coarse PM (100 μg) 8. OVA (50 

μg)+ Oslo Fine PM (100 μg) 

Analysis 5 days after injection. 

Cell Numbers and Cell Phenotypes in the 

Lymph Node: The overall number of B 

lymphocytes, lymph node cells, PLN cells, and 

the expression of MHC class II, CD86 and CD23 

on B lymphocytes were increased by 

coexposure of OVA+ the particles compared to 

the OVA or particle groups alone. The OVA + 

particle groups displayed a significant decrease 

in T lymphocytes. Particles only significantly 

increased the number of lymph node cells and 

MHC Class II expression. There were no 

differences observed between coarse and fine 

PM fractions. 

Cytokine Production by Lymph Node (ex 

vivo culture of popliteal lymph node cells):

The OVA + particle (DEP and Oslo1 only) 

significantly increased IL-4 and IL-10 levels. No 

change was observed in IFN-γ. The particle 

groups only increased IL-4 and IL-10. All coarse 

and fine particle fractions co-exposed with OVA 

significantly increased IL-4 and IL-10 compared 

to OVA alone. There was no significant 

difference between coarse and fine particles. 

IFN-γ levels were not significantly affected by 

most of the groups, but the fine fractions of PM 

consistently produced higher levels of IFN-γ. 

Lymph Node Histology: OVA + particle groups 

resulted in significantly enlarged lymph nodes 

and the formation of germinal centers. 

Reference: 

Nygaard et al. 

(2005, 087980) 

Species: Mouse 

Gender: Female 

Strains: BALB/c 

Age: 6-8 wk 

Polysterene Particles (PSP) 

Particle Size: 0.1 μm (diameter) 

Route: Subcutaneous Injection into footpads. 

Dose/Concentration: 40 μg PSP (5.94×1010

particles) per injection suspended in HBSS. One 

injection per footpad 

Time to Analysis: 1. HBSS 

2. OVA (10 μg per injection) 

3. PSP (40 μg per injection) 

4. OVA (10 μg per injection) + PSP (40 μg per 

injection). 

Antibody experiments: reinjected with 10 μg OVA 

on day 21. Killed on day 26. 

Popliteal lymph node cell experiments-- animals 

injected. Killed 1 to 21 days post-injection. 

OVA-specific IgE, IgG1 and IgG2a 

Antibodies: Analysis at day 26 indicated IgE, 

IgG1 levels were significantly higher in mice 

exposed to OVA+PSP compared to mice 

injected with HBSS, OVA or PSP. No significant 

difference was observed for IgG2a levels. 

Number of Particle Containing Cells: There 

was no significant difference between PSP 

alone and OVA+PSP. Throughout days 0 -21 the 

number of particle-containing cells in the PSP or 

OVA+ PSP groups were significantly greater 

than the HBSS group. 

Total Cell Numbers, B and T Lymphocytes 

and MHC class II Expression: The total cell 

number and B lymphocytes significantly 

increased by coexposure to OVA+ PSP when 

compared to the other groups. Both OVA and 

OVA+PSP increased T lymphocytes on Days 1, 

3 and 5. MCH class II expression was 

significantly higher in the OVA+PSP group on 

days 5, 7 and 21 than other groups. 

Cell Types and Surface Markers: The number 

of CD40+ B Lymphocytes showed a slight but 

significant decrease with OVA+PSP and OVA 

compared to HBSS and PSP. CD86+, CD23+ 

and CD69+ B lymphocytes were significantly 

higher in OVA+PSP group than other groups. 

PSP alone did not affect CD86+ or CD23+ 

levels. 

Cytokine Production: IL-4 and IL-10 were 

significantly higher in the OVA+PSP group when 

compared to the other groups. OVA alone 

caused a slight increase compared to PSP. PSP 

did not alter IL-4 or IL-10 levels. 

December 2009 D-141 
Study Pollutant Exposure Effects 

Reference: 

Nygaard et al. 

(2004, 058558) 

Species: Mouse 

Gender: Female 

Strain: BALB/c 

Age: 6-7 wk 

Weight: NR 

CB (carbon black/DEP) 

Polystrene Particles (PSP) 

Particle Size: PSP diameter: 0.0588, 

0.202, 1.053, 4.64 or 11.14 μm 

Route: Single subcutaneous injection into footpad 

Dose/Concentration: 10 μg OVA + 40 μg (low 

dose) or 200 μg (high dose) of particles 

Time to Analysis: 5 days after OVA injection 

OVA Specific IgE and Ig2a: OVA with CB, DEP 

or PSP of diameters 0.0588 and 0.202 μm 

increased IgE compared to OVA alone, as well 

as the 1.053, 4.64 and 11.14 μm PSP. OVA with 

0.0588 μm PSP or CB significantly increased 

IgG2a compared to OVA alone. 

Primary Cellular Response: All OVA and PSP 

groups (except the low dose of 11.14 μm PSP) 

had more total lymph node cell numbers than 

the OVA alone group. The low and high does 

groups of 0.202 μm PSP had the greatest 

amount of cell proliferation and lymphoblasts. 

The OVA and 0.202 PSP treatment produced 

the greatest amounts of B lymphocytes, IL-4, IL10 and IFN-γ. IL-2 in the PLN cells was 

significantly lower in both dosage groups of OVA 

and 0.202 PSP than the OVA control. 

Particle Mass, Size, Number and Surface 

Area: Total particle surface area explained 64% 

of the variance in the IgE levels. 60-80% 

variance of the PLN cellular parameters (except 

CD23) were explained by total particle surface 

area, number and diameter. 

Reference: Reed 

et al. (2008, 

156903) 

Species: Mouse 

Gender: Male, 

Female (only 

BALB/c) 

Strain: C57BL/6, 

A/J, BALB/c 

Age: NR 

Weight: NR

GEE (two 1996 General Motors 4.3-L V6 engines; regular, unleaded, nonoxygenated, non-reformulated gasoline 

blended to US average consumption for 

summer 2001 and winter 2001-2002- 

Chevron-Phillips) 

Particle Size: 150 nm (MMAD) 

Route: Whole-body Inhalation 

Dose/Concentration: PM: Low- 6.6 ± 3.7 μg/m3, 

Medium- 30.3 ± 11.8 μg/m3

, High- 59.1 ± 28.3 

μg/m3

Time to Analysis: A/J- 6 h/day, 7days/wk, 3 days-6 

mo. C57BL/6- 1wk exposure. Instillation of P. 

aeruginosa. Killed 18 h postinstillation. BALB/cPregnant females exposed GD 1 and throughout 

gestation. Offspring exposures continued until 4wkold. Half of offspring sensitized to OVA. Tested for 

airway reactivity by methacholine challenge 48 h 

post-instillation and euthanized. 

The kidney weight of female A/J mice decreased 

at 6 mo. and was strongly related to PM by the 

removal of emission PM. PM-containing 

macrophages increased by 6 mo. 

Hypomethylation occurred in females at 1 wk. 

The clearance of P. aeruginosa was unaffected 

by exposure. Serum total IgE significantly and 

dose-dependently increased. OVA-specific IgE 

and IgG1 gave slight exposure-related evidence 

but were not significant. 

Reference: 

Roberts et al. 

(2007, 097623) 

Species: Rat 

Gender: Male 

Strain: SD 

Age: 10 wk 

Weight: 250-300 g 

R-Total = ROFA (Residual oily fish ash) 

R-Soluble = Soluble fraction of ROFA 

R-Chelex = R-Soluble+Chelex (insoluble 

resin) 

Particle Size: 2.2 μm (mean diameter) 

Route: IT Instillation 

Dose/Concentration: 10 mg/kg (2.5-3 mg) 

Time to Analysis: Pre-exposure to ROFA samples 

on Day 0. Inoculation with 5×104

 L. Monocytogenes 

or saline on day 3. Sacrifice on days 6, 8, 10. 

Uninfected Groups: Compared to the controls, 

the R-total and R-soluble groups had increased 

LDH, PMNs, lymphocytes and AMs. The R-total 

group had a slight, but significant increase in IL6 and the R-soluble group had a decrease in IL2. 

Infected Groups: The R-soluble group had 

increased levels of LDH (which also increased 

for the R-total group), albumin, BALF cells, NK 

cells, PMA-stimulated and zyomason-stimulated 

CL compared to all other groups at various time 

points. NOX was significantly elevated in the Rsoluble group at early time points, but in later 

time points R-soluble and R-total AMs produced 

less NOX than the controls. IL-10 and IL-6 

increased in the R-soluble group, while IL-12, 

IL-4 and IL-2 decreased. IL-12 also decreased 

in the R-total group. 

Reference: 

Saxena et al. 

(2003, 054395) 

Species: Mouse 

Gender: Female 

Strain: C57B1/6J 

Age: 18-30 wk 

Weight: NR 

DEPs (standard) 

Particle Size: NR 

Route: Intrapulmonary Instillation 

Dose/Concentration: 100 μg/mouse 

Time to Analysis: Pre-exposure to 2.5×104

bacillus Calmette-Guerin bacteria (BC G) with or 

without coadministration of DEP. Sacrifice 5 wk 

later. 

The BC G + DEP group had four times the BC G 

lung load than BC G alone. The load was 

significantly greater in other organs in the BC G 

+ DEP group. Interstitial lymphocytes, T, B and 

NK cells were increased in the BC G + DEP 

group over the DEP-alone group. DEP caused 

no release of NO by AMs, but inhibited the 

release of NO in response to IFN-γ. Except for 

CD8 cells, no increase in IFN-γ was seen in the 

BC G + DEP group. 

December 2009 D-142 
Study Pollutant Exposure Effects 

Reference: 

Schneider et al. 

(2005, 088368) 

Species: Mouse 

Strain: BALB/c 

Cell Line: RAW 

264.7 

SRM 1648 (greater than 63% inOC; 4-

7% OC; Si, S, Fe, Al, K greater than 1% 

by weight; Mg, Pb, Na, Zn, Cl, Ti, Cu, As, 

Cr, Ba, Br, Mn less than 1%) 

TiO2

Particle Size: TiO2 = 0.3 μm average, 

1.0 μm max 

SRM 1648 = 0.4 μm (mean diameter) 

Route: Cell Culture (625,000 cells/cm2 in 96 well 

plate) 

Dose/Concentration: 0, 7.8, 15.6, 31.2, and 62.5 

μg/cm2

Time to Analysis: 1, 3, 6, and 12 h 

No significant toxicity was exhibited by SRM 

1648. The rate of dye oxidation was significantly 

higher in SRM 1648-exposed cells. SRM 1648 

significantly increased reduced glutathione 

compared to the control at the 12-h time point. 

SRM 1648 increased GSH and concurrently 

caused significant PGE2 production compared 

to the no ester control at the 6-h and 12-h time 

points. 

Reference: 

Schober et al. 

(2006, 097321) 

Species: Human 

Gender: Male and 

Female 

Age: 21-39 yr 

treatment group; 

23-32 yr control 

group 

Tissue Type: 

Whole blood 

samples 

PM - organic extracts of airborne sample

AERex1d - urban aerosol 1 day sample 

(total air volume - 1270m3

) 

AERex5d - urban aerosol 5 day sample 

(total air volume - 6230m3

) 

rBet v 1 (birch pollen allergen 1a, 

Biomay, Vienna, Austria) 

Particle Size: NR 

Route: Cell Culture 

Dose/Concentration: 100 μL heparinized whole 

blood 

Time to Analysis: Blood stimulated with PBS/IL-3 

for 10 min. Incubated with rBet v 1 alone or with 

AERex for 20 min. Ice bath 5 min. Incubated with 

antibody reagent 20 min. 

Nine organic compound classes were identified 

in AERex1d and AERex5d, with AERex1d 

having 20 times more PAHs. Basophil activation 

increased in all treatment groups up to 90%, 

with AERex1d being the most pronounced. 5-50 

fold lower concentrations of AERex1d were 

needed to achieve the maximal effect on 

basophil activation. AERex-induced 

enhancement of CD63 upregulation of rBet v 1 

in sensitized basophils occurred in a dosedependent manner. The AERex-alone treatment 

did not affect CD63 expression. 

Reference: Shwe 

et al. (2005, 

111553) 

Species: Mouse 

Gender: Male 

Strains: BALB/c 

Age: 8 wk 

Weight: NR 

CB = carbon black particles (Degussa, 

Germany) 

CB14: 

C: 96.79% 

H: 0.19% 

N: 0.13% 

S: 0.11% 

Ash: 0.05% 

Others including O: 2.74% 

CB95: 

C: 97.98% 

H: 0.15% 

N: 0.28% 

S: 0.46% 

Others including O: 1.14% 

Particle Size: CB14 = 14 nm (primary 

particle size); CB95 = 95 nm (primary 

particle size) 

Route: IT instillation 

Dose/Concentration: 25, 125, or 625 μg in 1 mL 

saline solution 

Time to Analysis: 1/wk for 4 wk; 4 or 24 h after 

last instillation 

BALF Cells: In CB14, the total number of BAL 

cells increased significantly and dosedependently. In CB95, only the 625μg dose 

showed a significant increase. 

Cytokine and Chemokine: For CB14 and 

CB95, 125 or 625 μg showed a significant IL-1Β

increase in a dose-dependent manner. For 

CB14, only the 625 μg dose showed a 

significant IL-6 increase. No difference was 

observed in the CB95 group. For CB14, only 

larger doses showed a significant TNF-α increase. For CB95, no significant differences 

were observed. 

In BAL Fluid: CCL-2 production was 

significantly increased for the 625μg dose in 

both the CB14 and CB95 groups. CCL-3 

production was significantly increased for the 

larger doses in both the CB14 and CB95 

groups. 

Splenic Lymphocytes: No significant 

differences were detected among the CB14 

dosages, except for CD8+. No significant 

differences were observed among the various 

groups for CB95. 

Deposition in Lymph Nodes: For all dosages, 

greater deposition of CB14 than CB95 was 

observed. 

Chemokine mRNA Expression in Lungs and 

Lymph Nodes: At 125 μg, significant increases 

of CLL-3 mRNA expression was observed for 

CB14; for CB95, no differences were detected. 

December 2009 D-143 
Study Pollutant Exposure Effects 

Reference: Sigaud 

et al. (2007, 

096100) 

Species: Mouse 

Gender: Male 

Strains: BALB/c 

Age: 8-10 wk 

Weight: NR 

CAPs: Concentrated Ambient Particles 

(Collected from ambient Boston air on 

Teflon filters.) 

TiO2

IFN-γ

S. pneumoniae (ATCC 6303, American 

Type Culture Collection, Manassas, VA) 

Particle Size: CAPs: <2.5 μm 

Route: IFN-γ priming: aerosol 

Particle exposure and infection: Intranasal 

Instillation 

Dose/Concentration: CAPS or TiO2: 50 μg/50 μL 

PBS 

S. pneumoniae: 105CFU/25 μl saline 

Time to Analysis: Primed for 15 min

One time particle exposure 3 h post priming with 

lung RNA analyzed 3, 6, 24 h after exposure 

Sacrificed 24 h after exposure or one time infection 

Inflammation: Saline-primed and unprimed 

mice exposed to CAPs produced a significant 

increase in PMNs in the lung (100% more than 

mice exposed to TiO2.) Groups primed with IFNγ then exposed to CAPs produced a strong 

inflammatory response, a 2.5 increase in PMNs 

when compared to the increase caused by 

PBS+ CAPs exposure. 

Cytokine Levels: IFN-γ primed and CAPs 

exposed groups 

Inflammation+ S. Pneumo Infection: Salineprimed and unprimed mice exposed to CAPs 

produced a significant increase in PMNs in the 

lung (100% more than mice exposed to TiO2.) 

Groups primed with IFN-y then exposed to 

CAPs produced a strong inflammatory 

response, a 2.5 increase in PMNs when 

compared to the increase caused by 

PBS+CAPs exposure. 

Cytokine Levels: IFN-y primed and CAPs 

exposed groups showed a 1.5-fold increase 

over the control. 

PMNs: Treatment with CAPs enhanced 

inflammation, causing a 2-fold increase in PMN 

numbers as compared to the infected control. 

IFN-y+CAPs+S. pneumo produced a 3.5 fold 

increase compared to the infected control and a 

1.6-fold increase compared to 

PBS+CAPs+S.pneumo. Despite increased 

numbers of PMNs in the IFN-y+CAPs groups, 

the lungs were unable to clear the S. pneumo 

infection. 

Bacterial Load: Control groups showed 

efficient clearance of bacteria after infection. 

Unprimed, CAPs-treated, infected groups did 

not show a decrease in bacterial numbers. IFNy+CAPs showed a 2.5-fold increase in bacterial 

numbers. 

Histopathology: Indicated moderate 

pneumonia in PBS+CAPs and severe 

pneumonia in IFN-y+CAPs. The other groups 

did not indicate areas of pneumonia. 

Bacterial Uptake AM and PMN Cells: In all 

the treated groups, the bacterial content in AMs 

showed a decrease, with a more marked 

decrease in the IFN-y+CAPs group, but these 

decreases were not statistically significant. 

Groups exposed to CAPs showed a statistically 

significant decrease in bacterial uptake by 

PMNs. 

ROS Levels in AM and PMN Cells: 

Intracellular ROS significantly increased in AM 

cells in the IFN-y+CAPs group, approximately 

50% greater than controls. In PMNs, iROS 

increased 100% in the IFN-y+CAPs groups as 

compared to the controls. 

December 2009 D-144 
Study Pollutant Exposure Effects 

Reference: 

Steerenberg et al. 

(2004, 087474) 

Species: Rat 

Gender: Male 

Strain: Wistar 

Kyoto 

Age: 6-8 wk 

DEP:SRM1650a (NIST, Gaithersburg, 

MD 

EHC-93: ambient PM (Ottawa, Canada) 

O3 (positive control) 

L. mono: Listeria monocytogenes (strain 

L242/73 type 4B) 

Particle Size: DEP, EHC-93: NR 

Route: DEP/EHC-93: intranasal droplet; O3: 

Whole-body inhalation 

Dose/Concentration: DEP/EHC-93: 50 μg (1.0 

mg/ml) 

O3: 2mg/m3

L. mono: 0.2 or 0.3 ml (5x106 PFU/ml) *I have 

emailed author regarding correct dose 

Time to Analysis: DEP/EHC-93: 1/day for 7 days 

(-7 days to -1 days) 

O3 24 h/day for 7 days (-7 days to -1 daysR 

All rats infected on day 0. Sacrificed on days 3, 4, 

or 5. 

Body weight: Growth declined for O3 exposed 

group while DEP or EHC-93 groups grew 

progressively. Exposure to L. mono caused all 

groups to decline in weight. 

Bacterial Count in the Lung: The number of 

bacteria in the lung of those rats exposed to O3

was significantly greater than those exposed to 

saline. No differences in bacteria number were 

found for rats exposed to saline, EHC-93 or 

DEP at any time. 

Bacterial Count in the Spleen: The O3

exposed group exhibited statistically significant 

increases in bacteria numbers when compared 

to the saline-treated group. No differences in 

bacteria number were found for rats exposed to 

saline, EHC-93 or DEP at any time. Exposure to 

O3 decreases the defense of the respiratory 

tract against L. mono infection; however, DEP 

and EHC-93 did not appear to affect the host 

defense system in regards to clearing/fighting L. 

mono. 

Reference: 

Steerenberg et al. 

(2005, 088649) 

Species: Mouse 

Gender: Male 

Strain: 

BALB/cByJ.ico 

Age: 6-8 wk 

PM: collected from Rome, Oslo, Lodz, 

Amsterdam and De Zilk during the 

spring, summer and winter. 

Rome, Oslo, Lodz and Amsterdam 

represent areas with high population and 

dominance of traffic. De Zilk, selected as 

a negative control site, has low traffic 

emissions and natural allergens. 

EHC-93: used as a positive control 

OVA: Ovalbumin 

Particle Size: Coarse PM: 2.5 - 10.0 μm 

(MMAD); Fine PM: 0.1 - 2.5 μm 

(MMAD); Ultrafine: <0.1 μm (MMAD); 

EHC-93: NR 

Route: Intranasal Exposure 

OVA challenge: aerosol 

Dose/Concentration: PM: 450 μg PM (at 0, 3, or 9 

mg/ml) 

OVA sensitization: 50 μg (0.4 mg/ml) . 

OVA challenge: 20 μg (0.4 mg/ml) 

EHC-93 was administered at 

 0 - 900 μg to evaluate any dose-response 

relationship. 

Time to Analysis: Sensitization and PM exposure 

on days 0, 14

Challenged on days 35, 38, 41 for 20 min/day 

Sacrificed on day 42 

Effects of Coarse and Fine Particles: 

Immunoglobulins: 6/13 of the coarse and 9/13 

fine PM samples induced an increase in IgE and 

IgG1when compared to the control. IgG2a levels 

were increased in 3/13 of the coarse and 5/13 of 

the fine PM. Particles from De Zilk induced all 

three immunoglobulins, except the fine PM did 

not induce IgG2a. De Zilk was intended as a 

negative control (see Table 3). Analysis among 

the sites comparing the subclasses of 

antibodies indicated a rank as follows: Lodz 

>Rome ≥ Oslo. 

Histopathology: 9/13 of the coarse PM 

samples and 5/13 of the fine PM samples 

induced an inflammatory response. 

BALF Cells: Lodz (spring/ summer) coarse and 

fine PM induced a significant increase in 

eosinophils, neutrophils and monocytes. The 

coarse and fine PM from Rome (spring) induced 

an increase in neutrophils and the coarse PM induced an increase in eosinophils. Also both 

Lodz and Rome from the coarse PM from the 

spring induced an increase in macrophages. 

Other PM samples did not have an effect on 

BAL cell counts. 

Cytokine Production: None of the samples 

produced a significant effect on IL-4 levels. IFNγ levels were significantly decreased in mice 

exposed to the fine PM fraction (in 8/13 of the 

samples) when compared to control. Coarse 

particle exposure did not appear to affect IFN-γ

levels. TNF-α levels were significantly increased 

( in 2 of the 13 samples) when exposed to 

coarse PM; fine PM showed similar responses 

compared to the OVA only group. IL-5 was 

significantly increased in 4/13 of the coarse and 

fine PM samples. 

Analysis of PM Components: Samples from 

Lodz, Oslo and Rome (all spring) were 

evaluated and the water-soluble coarse PM 

fraction showed increased immunoglobulin and 

pathological responses and the water-insoluble 

fine PM fraction from Lodz (Spring) showed 

increased reactivity. Leukocytes and cytokines 

showed no major differences. 

December 2009 D-145 
Study Pollutant Exposure Effects 

Reference: 

Steerenberg et al. 

(2004, 087981) 

Species: Mouse 

Gender: Male 

Strain: 

BALB/cByJ.ico 

Age: 6-8 wk 

Treatment: 

1. C.D2-Vil6: 

Nramp1S and 

Nramp1R deficient 

2. B6.129P2: 

Nos2tmLau: iNOS 

deficient 

3. BALB/cIL4 

(tm2Nnt): deficient 

in IL-4 

4. BALB/c (wild 

type) pretreated 

with NAcetylcysteine 

(NAC) 

EHC-93 

OVA 

Particle Size: NR 

Route: Sensitization, Challenge: Intranasal 

NAC: IP injection 

Dose/Concentration: OVA: 200 μg (0.4 mg/ml) 

EHC-93: 150 μg (3 mg/ml) 

NAC: 320 mg/kg 

Time to Analysis: OVA-only or OVA+EHC-93 

sensitization on days 0 and 14. 

Some mice received NAC before intranasal 

exposure on days 0 and 14 

OVA challenge on days 35, 38 and 41 

Sacrificed on day 42 

Natural-Resistance-Associated Macrophage 

Protein 1 (Nramp1): When exposed to only 

OVA, Nramp1S evoked less of an antibody 

responses (IgE, IgG1 and IgG2a) compared to 

Nramp1RHowever when coexposed to OVA and 

EHC-93, the level of increased production of 

antibodies was similar in both groups. After 

coexposure, the wild-type showed increased 

histopathological lesions, whereas the 

macrophage-stimulation-deficient types showed 

only a slight increase (not significant). IL-4, IFNγ, TNF-α and IL-5 levels were similar in wildtype and the Nramp1 strains. 

Pretreatment with NAC: IgG2a concentration 

was increased further in the group pretreated 

with NAC. The wild-type mice and the NAC 

pretreated mice showed similar 

histopathological lesion patterns. IL-4 levels 

were similar in wild-type and the NAC pretreated 

mice. (IFN-γ, TNF-α and IL-5 levels not 

reported) 

Inducible Nitric Oxide Synthase (iNOS): The 

wild-type and the iNOS-deficient mice had 

similar levels of increased IgE antibody 

production. The IgG1 and IgG2a antibody 

response was twice as great in the iNOSdeficient mice compared to the wild type. The 

wild-type and the iNOS-deficient mice showed 

similar histopathological lesions. No differences 

in BAL cells or cytokines were observed 

between the wild-type and iNOS-deficient mice. 

IL-4: The IL-4-deficient mice did not produce an 

increase in IgE or IgG1 antibodies, as was seen 

in the wild-type mice. The IgG2a antibody 

response in the IL-4-deficient mice was similar 

to the wild type response resulting in adjuvant 

activity for the IgG2a antibodies. Overall the 

histological response of the wild-type mice was 

greater compared to the IL-4 deficient mice. 

There was no real difference between the two 

strains observed in the BAL cells, except IL-5 

was significantly lower in the IL-4-deficient mice.

December 2009 D-146 
Study Pollutant Exposure Effects 

Reference: 

Stevens et al. 

(2008, 155363) 

Species: Mouse 

Gender: Female 

Strain: BALB/c 

Age: 10-12 wk 

Weight: 17-20 g

DE: generated using a 30 kW 4-cylinder 

Deutz BF4M1008 diesel engine 

connected to a 22.3 kW Saylor Bell air 

compressor. The engine was operated 

on diesel fuel purchased from a service 

station in Research Triangle Park, NC. 

The engine was operated at a steadystate, approx. 20% of engine’s full load. 

High composition: 

O2: 4.3 ± 0.07 ppm 

NO: 9.2 ± 0.30 ppm 

NO2: 1.1 ± 0.05 ppm 

SO2: 0.2 ± 0.10 ppm 

Low composition: 

O2, NO, NO2, SO2 below detection limits 

Particle Size: NR 

Route: Whole-body Inhalation 

OVA immunization and challenge: intranasal 

Dose/Concentration: High = 2000 μg/m3

Low = 500 μg/m3

Time to Analysis: DE exposure for 4 h/day on 

days 0-4. 

OVA immunization 40 min after DE exposure on 

days 0-2 

Challenged on days 18 and 28. 

Sacrificed 4 h after last exposure of day 4 for gene 

set analysis or 18, 48, or 96 h after the last 

challenge 

IgE Antibody Production: In the absence OVA, 

IgE antibodies were not detected. 18, 48 and 96 

h following OVA, mice exposed to low and high 

doses of DE had an increase in antibodies over 

time. Mice exposed to high dose had an 

increase (non-significant) to the OVA exposed 

control at the 48 h time mark 

BAL Cells: Cell counts at 18 and 96 h after OVA 

treatment did not differ among treatment groups. 

At 48 h the number of eosinophils, neutrophils 

and lymphocytes were significantly increased in 

mice exposed to both high and low 

concentrations of DE. With DE exposure alone, 

only neutrophils were statistically increased in 

the high DE concentration. This indicates the 

combination exposure of DE and an antigen is 

essential to promote the development of allergic 

lung disease. 

BAL Cytokines: IL-6 production showed a 

dose-dependent and time-dependent increase, 

but was significantly increase in the high dose 

group at 96 h. The high dose group saw a non 

significant increase in IL-10 levels over time. 

The greatest increase in IL-10 for the low dose 

group occurred18 h after OVA stimulation. 

Pulmonary Inflammation and Lung Injury: No 

differences among the groups were observed 

for macrophage, lymphocyte, neutrophil and 

eosinophil counts. Protein and LDH levels were 

not found to be increased in the BALF of any 

group. 

Gene Analysis: Pair wise comparisons 

revealed significant gene set difference between 

the high DE and control groups. Comparison of 

the high DE/OVA versus air/OVA showed 

significant changes in 23 gene sets, including 

genes involved in oxidative stress responses. 

The high DE/saline versus the air/saline showed 

significantly altered pathways. Altered pathways 

include those for cell adhesion, cell cycle 

control, apoptosis, growth and differentiation, 

and cytokine signaling. The results show that 

relatively short exposures to DE cause mild 

increases in immunologic sensitization to 

allergen.

December 2009 D-147 
Study Pollutant Exposure Effects 

Reference: 

Takizawa et al. 

(2003, 157039) 

Species: Human 

Cell Lines: Normal 

Small Airway 

Epithelial Cells and 

Bronchial Epithelial 

Cells (BET-1A) 

Suspended DEP: collected using a 

2,300-cc Isuzu diesel engine using 

standard diesel fuel at 1,050 rpm under 

a load of 6 torque. 

DE exposure in vitro (air exposure): 

collected using a 2,300-ml Isuzu diesel 

engine at 1,050 rpm. 

Composition: 

Fine particles: 1 mg/m3

 

CO: 10.6 ppm 

NO2: 7.3 ppm 

SO2: 3.3 ppm 

Particle Size: NR 

Route: Cell Culture 

Dose/Concentration: Suspended DEP: varying 

doses from 0-50 μg/ml 

IL-13: varying doses from 0-25 ng/ml 

DE exposure in vitro (air exposure): 100 μg/m3

 

Time to Analysis: Cells were exposed to varying 

concentrations of suspended DEP for up to 24 h. 

NF-κB: analyzed at 6 h after suspended DEP 

exposure 

Air exposure at 0, 2, 4, 8 or 14 h 

Preliminary experiments indicated that DEP at 

0.1- 50 μg/mL had no significant cytotoxicity to 

BET-1A cells and human bronchial epithelial 

cells (as analyzed by LDH levels). 

Eotaxin Production: (Eotaxin is a cc 

chemokine that plays a role in eosinophil 

accumulation in a variety of allergic disorders) 

Epithelial and BET-1A cells treated with 

suspended DEP or IL- showed a dosedependent stimulatory effect on eotaxin release 

or production. Simultaneous exposure to 25 

ng/mL IL-13 and DEP depicted an additive effect 

for both cell types. 

Eotaxin mRNA: At 25 μg/mL, suspended DEP 

showed a time-dependent effect on eotaxin 

mRNA levels up to 12 h in both cell types. 

Extracted RNA from human bronchial epithelial 

cells exposed to varying doses of DEP showed 

a dose-dependent effect for both cell types (up 

to 25 μg/mL DEP) on eotaxin mRNA levels after 

12 h of exposure. IL-13 also induced a dosedependent increase on eotaxin mRNA levels in 

cells in both cell types. Combination of IL-13 

and DEP showed an additive effect on mRNA 

levels in BET-1A cells. DE exposure in vitro also 

showed a time-dependent stimulatory effect on 

eotaxin production in BET-1A cells. 

NF-κB / STAT6 Activation: (it has been 

suggested that NF-κB plays a role in the transcriptional regulation of eotaxin gene expression) 

Cells exposed to 1-25 μg/mL DEP for 6 h 

increased NF-κB. BET-1A cells treated with 

suspended DEP failed to activate STAT6. 

Effect of NAC and PDTC on Eotaxin mRNA 

Levels: (NAC and PDTC are antioxidant 

reagents with inhibitory effects on NF-κB 

activation) in BET-1A, both NAC and PDTC 

showed a dose-dependent inhibitory effect on 

DEP-induced eotaxin production. Both reagents 

also blocked DEP-induced eotaxin mRNA levels 

in BET-1A cells. NAC and PDTC did not 

suppress eotaxin production or eotaxin mRNA 

levels in IL-13 stimulated BET-1A cells. In 

addition pre-treatment with NAC attenuated NFκB activation induced by DEP but had no effect 

on STAT6 induction by IL-13. 

These findings suggest that DEP stimulated 

eotaxin gene expression via NF-κB dependent, 

but STAT6-independent, pathways. 

December 2009 D-148 
Study Pollutant Exposure Effects 

Reference: 

Tesfaigzi et al. 

(2005, 156116) 

Species: Rat 

Gender: Male 

Strain: BrownNorway 

Age: 6 wk 

PM: Wood smoke generated from a 

conventional wood stove that has a 

0.5m3

 firebox and a sliding gate air 

intake damper. The stove was operated 

over a 3-phase burn cycle that spanned 

6 h. Fire was started (initiated exposure) 

with unprinted / unbleached newspaper 

and a mix of black and white oak. 

Wood smoke components: organic 

material, small amounts of EC and 

metals and associated analytes. 

Particle Size: 0.36 μm (MMAD) 

Route: Whole-body Inhalation 

Dose/Concentration: PM: 1000 μg/m3

Time to Analysis: Exposed to wood smoke or 

filtered air 6 h/day for 70 consecutive days

OVA IP injection immunization on days 2, 9 

OVA aerosol exposure 2 h/day on days 67-70 

following daily exposure to wood smoke or filtered 

air 

Sacrificed day 70 

Body Weight and Respiratory Function: No 

difference in clinical signs or body weight was 

observed when comparing the two rat groups. 

The wood smoke exposed group had a 45% 

lower dynamic lung compliance when compared 

to those exposed to the filtered air group before 

the methacholine challenge. Challenging the 

rats with methacholine caused a decrease in 

dynamic lung compliance in both groups, but the 

decrease was greater in the air-exposed group. 

At the highest dose of methacholine, the 

dynamic lung compliance in controls was similar 

to the baseline value of the smoke-exposed 

group. No significant differences in total 

pulmonary resistance were observed. Wood 

smoke exposed rats had a 10% increase in 

functional residual capacity than the air-exposed 

group. 

BAL Cells and Cytokines: There was no 

difference in lymphocyte, eosinophil or 

neutrophils in the BALF of either group. There 

was an increase, though not statistically 

significant, in macrophages the wood smoke 

exposed group when compared to the filtered air 

group. In the BALF, IFN-γ and IL-1ß levels were 

significantly decreased, IL-4 and GRO-α levels 

were increased in rats exposed to wood smoke 

compared to filtered air. Serum IgE levels 

experienced a reduction trend in the wood 

smoke group, but it did not reach significance. 

Both groups showed mild signs of inflammation. 

The average eosinophils present in stained 

tissue was 21% higher in the wood smoke exposed group compared to the air exposed. 

Reference: Tomita 

et al. (2006, 

097827) 

Species: Mouse 

Gender: Female 

Strain: C57BL/6J; 

AHR-deficient; 

mEH-deficient; 

ARNT floxed (loxP 

sequences 

inserted in Arnt 

gene); 

Tcell-specific 

ARNT-deficient 

Age: 7 wk 

Weight: 20 g 

DEP: two independent preparations 

fractionated into 13 different fractions 

based on acidic and basic functionality 

(one from light-duty, 4-cylinder diesel 

engine using standard diesel fueled and 

other generated from A4JB-type, Isuzu 

automobile, Japan) 

Individual PAH tested (Osaka, Japan): 

BbF = benzo[b]fluoranthene 

BeP = Benzo[e]pyrene 

IDP = Indeno[1,2,3-cd]pyrene 

BpPe = Benzo[ghi]perylene 

BaP = Benzo[a]pyrene 

BkF = Benzo[k]fluoranthene 

Per = Perylene 

DBA = Dibenzo[a,h]anthracene 

Particle Size: NR 

Route: Intraperitoneal Injection 

Dose/Concentration: DEP, fractionated DEP or 

PAH compounds: 0.5 μg - 10 mg/kg bw in 50 μl of 

olive oil 

Time to Analysis: Single, sacrificed 3 days postexposure. 

Effect on Thymus: DEP treatment (10 mg/kg of 

body weight) caused severe atrophy of the 

thymus while the spleen and lymph nodes 

appeared normal. Three days following DEP 

treatment showed a marked reduction in thymus 

size. The total number of thymocytes was 

reduced by more than 70% mostly due to a 

massive reduction in DP cells (CD4+CD8+). 

DEP induced no significant alterations in the cell 

numbers of CD4/CD8 ratios in the spleen and 

lymph nodes. 

DEP Extracts: Only the WAC (carbonic acid 

fraction) and BE (weak basic fraction) did not 

produce a significant reduction in thymocyte 

numbers in vivo. Among the active fractions, 7 

produced a marked selective loss of immature 

DP thymocytes, similar to the crude extract of 

DEP. 

PAH Effects: Thymic involution was severely 

induced by the N and various other fractions. 7 

out of the 8 PAH compounds were significantly 

effective in decreasing the number of 

thymocytes upon in vivo exposure. Only BpPe 

did not have an effect. 

AHR/ARNT and mEH Deficient Mice (BaP and 

DEP only): In the absence of AHR, BaP 

treatment did not result in a loss of thymocytes. 

Like DEP, BaP produced severe thymic 

involution in mEH-deficient mice. DEP-mediated 

thymic involution was significantly enhanced in 

mEH-deficient mice. 

December 2009 D-149 
Study Pollutant Exposure Effects 

Reference: 

Verstraelen et al. 

(2005, 096872) 

Species: Human 

Tissue/Cell 

Types: Monocytederived dendritic 

cells (Mo-DC) 

Cord blood 

samples of seven 

women were 

collected from 

umbilical vessels 

of placentas of 

normal, full-term 

infants. 

DEP- SRM 2975 

Particle Size: NR 

Route: Cell Culture 

Dose/Concentration: DEP in varying 

concentrations: 0.2, 2, 20, 200, 2000 ng/mL 

LPS 100 ng/mL 

Time to Analysis: 24 h 

Biological Markers: Exposure to DEP alone did 

not alter expression levels of HLA-DR, CD86 or 

CD83. 

Treatment with LPS alone caused a nonsignificant increase in all three markers when 

compared to the control. 

Treatment with DEP+LPS caused a significant 

increase in the expression of CD83 and a nonsignificant increased expression of HLA-DR and 

CD86. DEP+ LPS induced a bell-shape doseresponse curve on the expression of all three 

markers, with a dose of 20 ng/mL DEP + 100 

ng/mL LPS causing the largest increase in 

upregulation. 

When only the results of the LPS-responsive 

donors (5 out of 7 blood cord samples) were 

included, the effects described above become 

more pronounced. 

Reference: 

WalczakDrzewiecka et al. 

(2003, 188803) 

Species: Mouse 

Cell Line: 

C1.MC/C57.1 

(C57) Mast Cells 

Metal and Transition Metal Ions: Sr2+, 

Ni2+, Cd2+, Al3+, Pb2+

Particle Size: NR 

Route: Cell culture, 

Dose/Concentration: 0.1- 5 μmol 

Time to Analysis: 10 min - 4 h 

Β-Hex Mediator Release in Mast Cells:

Incubation with SrCl2, NiSO4, CdCl2 or AlCl3

resulted in a 2-5% release of Β-hexoaminidase 

in mast cells. Incubation with a mixture of all 

these compounds induced a greater (11%) 

release in Β-hexoaminidase, indicating there 

might be a additive effect. 

Cell Viability: Incubation of cells at 

concentrations and incubation time employed 

did not result in decrease in cell viability. 

Antigen-Mediated Mediator Release in Mast 

Cells: Al3+ and Ni2+ enhanced antigen-mediated 

release. 10-7 M AlCl3 released 23% of Βhexoaminidase compared to antigen alone, 

which induced 11% release of Βhexoaminidase. Cd2+, Sr2+ and Pb2+ enhanced 

antigen-mediated release to a lesser extent. 

Ni2+, Al3+, Sr2+ and Cd2+ depicted a dosedependent relationship with antigen-mediated Βhexoaminidase release. 

Antigen-Induced Protein Phosphorylation: 

Addition of the antigen induced the anticipated 

phosphorylation of multiple proteins in C57 mast 

cells The presence of Ni2+ and Pb2+ mediated an 

increase in phosphorylation of several of the 

proteins and Al3+ mediated a decrease in 

phosphorylation of multiple proteins (specifically 

the 56 and 37 kD bands). 

Antigen-Mediated Cytokine Secretion (IL-4):

At certain concentrations all tested metal and 

transition metal ions were able to induce IL-4 

secretion or enhance antigen-induced IL-4 

secretion in mast cells, but no dose-dependent 

relationship was established. 

Reference: Wan 

and Yu (2006, 

157104) 

Species: Human 

Cell Lines: 

Human, B cell 

lymphocytes 

PMBC 

(>98.5% B cellsCD19+CD20+; <1 

% T cells (CD3+)) 

Human lymphocyte 

cell lines -- DG75 

NQO1 wild type 

DEP from 4 cyl Isuzu diesel methanol 

extracts 

Particle Size: NR 

Route: Cell Culture, PMBC = 1×106 cell 

DG 75 = 3×106

 cells 

IgE PMBC 1×106

/mL 

B-cells 0.5×106

/mL 

Dose /Concentration: 2.5, 5, 10, 20 μg DEPX/ 

plate (20 μg/mL) 

IgE DEPX 100 ng/mL 

sulfurophane at 0 - 30 μmol 

Time to Analysis: 6 h mRNA; 16 h protein assay. 

IgE 14 days. 

Induction of NQO1 by DEPX: In PBMCs and 

DG75DEPX dose-dependently induced NQO1 

mRNA expression NQO1 ARE was increased 

NAC inhibited NQO1 gene expression dose 

dependently. p38 MAPK and P13K inhibition 

partially blocked NQO1 mRNA and ARE 

induction by DEPX. 

Induction of phase II enzymes: DEPX induced 

IgE potentiation was reduced dose dependently 

by induced phase II enzymes. 

December 2009 D-150 
Study Pollutant Exposure Effects 

Reference: 

Whitekus et al. 

(2002, 157142) 

Species: Mouse 

Cell Line: RAW 

264.7

DEP (light-duty, four-cylinder engine4JB1 type, Isuzu Automobile, Japan; 

standard diesel fuel) (extracts) 

Particle Size: NR 

Route: Cell Culture 

Dose/Concentration: 50 μg/mL 

Time to Analysis: Exposed to antioxidants 5 h. 

HO-1 western blot, determination of cellular 

GSH:GSSG ratios, carbonyl protein content, lipid 

hydroperoxides performed.

DEP significantly reduced the GSH:GSSG ratio. 

This effect was prevented by adding thiol 

antioxidants NAC or BUC. DEP increased lipid 

peroxide levels, but the addition of all 

antioxidants decreased these levels. DEP 

increased carbonyl groups. NAC, BUC, and 

luteolin reduced HO-1 expression. 

Reference: 

Whitekus et al. 

(2002, 157142) 

Species: Mouse 

Gender: Female 

Strain: BALB/c 

Age: 6-8 wk 

Weight: NR

DEP (light-duty, four-cylinder engine4JB1 type, Isuzu Automobile, Japan; 

standard diesel fuel) (extracts) 

Particle Size: 0.5-4 μm 

Route: Inhalation 

Dose/Concentration: 200, 600, 2000 μg/m3

Time to Analysis: Exposed 1 h/day, 10 days. 

Animals receiving OVA had 20 min OVA exposure 

after DEP exposure.

DEP+OVA dose-dependently increased IgE and 

IgG1, being more effective than the OVA-alone 

treatment. This effect was significantly 

suppressed by thiol antioxidants NAC or BUC. 

DEP+OVA increased carbonyl protein and lipid 

peroxide over OVA. NAC or BUC suppressed 

lipid peroxide and protein oxidation. No general 

markers for inflammation were observed. 

Reference: Witten 

et al. (2005, 

087485) 

Species: Rat 

Gender: Female 

Strain: F344 

Age: 8 wk 

Weight: ~175 g

DEP (heavy-duty Cummins N14 

research engine operated at 75% 

throttle) 

Particle Size: 7.234-294.27 nm 

Route: Nose-only Inhalation 

Dose/Concentration: Low- 35.3 ± 4.9 μg/m3, High632.9 ± 47.61 μg/m3

 

Time to Analysis: Exposed 4 h/day, 5 days/wk, 

 3 wk. Pretreated with saline or capsaicin.

There were no differences for substance P. The 

low-exposure group had significantly less NK1. 

DEP reduced NEP activity. Plasma extraversion 

dose-dependently increased and was greatest 

in capsaicin animals. Respiratory permeability 

dose-dependently increased. IL-1β was 

significantly higher for the low-exposure group. 

IL-12 was significantly lower in the capsaicin 

high-exposure group. TNF-α increased in the 

high-exposure group and capsaicin lowexposure group. High exposure induced 

particle-laden AMs in the lungs, perivascular 

cuffing consisting of mononuclear cells, alveolar 

edema and increased mast cell number. 

Neutrophil and eosinophil influx was not seen.

Reference: Wong 

et al. (2003, 

097707) 

Species: Rat 

Gender: Female 

Strain: F344/NH 

Age: ~4 wk 

Weight: ~175 g

DEP (Cummins N14 research engine at 

75% throttle) (EC- 34.93-601.67 μg/m3

, 

OC- 1.90-11.25 μg/m3, Sulfates 0.94-

17.96 μg/m3

, Na- 4.07-4.78 ng/m3, Mg0.60-0.86 ng/m3

, Ca- 5.05-10.66 ng/m3, 

Fe- 3.17-6.44, Cr- 0.68-1.31 ng/m3, Mn0.11-0.22 ng/m3

, Pb- 0.97-1.24 ng/m3) 

Particle Size: 7.5-294.3 nm 

Route: Nose-only Inhalation 

Dose/Concentration: Low- 35.3 ± 4.9 μg/m3, High669.3 ± 47.6 μg/m3

 

Time to Analysis: Exposed 4 h/day, 5 days/wk, 

 3 wk. Pretreated with saline or capsaicin. 

DEP dose-dependently increased plasma 

extraversion, which was further increased by 

capsaicin. In the high-exposure group, particleladen AMs (which were reduced by capsaicin), 

inflammatory cell margination, perivascular 

cuffing with subsequent mononuclear cell 

migration and dispersal, increased mast cells, 

and decreased substance P were all seen. NK1R was downregulated in the low-exposure 

group and upregulated in the capsaicinpretreated high-exposure group. NEP 

decreased significantly for both groups. 

December 2009 D-151 
Study Pollutant Exposure Effects 

Reference: 

Yanagisawa et al. 

(2006, 096458) 

Species: Mouse 

Gender: Male 

Strain: ICR 

Age: 5 wk 

Weight: 25-28 g 

Washed DEP (carbonaceous core), 

DEP-OC(extracted organic chemicals) 

and Whole DEP 

Particles collected from: 4JB1-Type, 

four-cylinder, 2.74 L, Isuzu diesel 

engine, while operated on standard 

diesel fuel at 200 g under a load of 10 

torques. 

Particle Size: 0.4 μm (MMAD) 

Route: IT Instillation 

Dose/Concentration: 50 μg/0.1L 

1. Control- 0.1mL PBS 

2. DEP-OC- 50 μg 

3. Washed DEP- 50 ug 

4. Whole DEP- 50 ug DEP-OC + 50 ug Washed 

DEP5. OVA- 1 μg = 

6. DEP-OC- 1 μg + OVA 

7. Washed DEP- 50 μg + OVA 8. Whole DEP- 50 

μg DEP-OC + 50 μg Washed DEP + OVA 

Time to Analysis: All groups received OVA or PBS 

every 2 wk for 6 wk and the PM component or PBS 

once a week for 6 wk. 

BALF Cells: DEP-OC + OVA caused a 

significant increase in PMN infiltration in the 

BALF compared to the control. Exposure to 

Whole DEP+ OVA caused PMN count to rise 

further. OVA alone DEP-OC +OVA, Washed 

DEP + OVA and Whole DEP + OVA all caused a 

significant increase in macrophages compared 

to the control. 

Lung Histology: Exposure to OVA, Washed 

DEP, DEP-OC and Whole DEP caused a slight 

increase in PMNs, mononuclear cells and goblet 

cell proliferation. Treatment with all three DEP 

groups + OVA caused a significant increase in 

mononuclear cells, PMNs and goblet cell 

proliferation. Whole DEP + OVA had the 

greatest impact. 

Th1 and Th2 Cytokine Expression: Washed 

DEP+OVA caused a significant increase in IFNγ compared to control, whereas Whole 

DEP+OVA caused a significant decrease 

compared to control. No significant differences 

in IL-2 and IL-4 levels were seen among groups. 

DEP-OC+ OVA and Whole DEP+ OVA caused 

significant increases in IL-5 compared to control 

and compared to OVA Whole DEP+OVA caused 

significant increase in IL-13 compared to control 

Eotaxin and MIP-1α Expression: OVA 

increased eotaxin levels and DEP-OC+OVA 

caused a more significant increase in eotaxin. 

Whole DEP alone caused a significant increase 

in MIP-1α and Whole DEP+OVA caused an 

even greater increase in MIP-1α. 

IgG1 Levels: Exposure to DEP-OC+OVA 

caused an increase in IgG1 and exposure to 

Whole DEP+OVA caused greater elevation in 

IgG1 levels. 

Reference: Yang 

et al. (2003, 

087886) 

Species: Mouse 

Gender: Female 

Strain: B6C3F1 

Age: 6-8 wk 

DEP- SRM 1650 

Particle Size: 0.5 μm (MMAD) 

Route: IT Aspiration 

Dose/Concentration: 1, 5, or 15 mg /kg 

Time to Analysis: 3 times in 2 wk or 6 times in 

 4 wk. 

Toxicity of DEP Exposure: DEP did not have a 

significant effect on body, liver or spleen weight. 

The highest dose of DEP caused an increase in 

lung weight and lung weight relative to body 

weight. None of the hematological parameters 

were significantly different in the mice exposed 

for 2 wk; in the 4 wk group there was a 

significant decrease in platelet counts in mice 

exposed to 15 mg/kg. 

Exposure on Spleen IgM AFC: DEP exposure 

for 2 wk induced a dose-dependent decrease in 

spleen AFC in response to sRBC immunization. 

Mice exposed to 15 μg/kg depicted a 35% 

reduction in total spleen activity. In the group 

exposed to DEP for 4 wk, the decrease in AFC 

was not significantly different than the control. 

DEP Exposure on Spleen Cell 

Number/Lymphocyte Counts: Exposure for 2 

or 4 wk did not affect total number of nucleated 

splenocytes. DEP caused a 30% reduction in 

total T cells. The number of B cells were not 

significantly affected. 

DEP Exposure on Spleen T-Cell Function:

(evaluated in 2 wk exposure group only) DEP 

induced a dose-dependent decrease in spleen 

cell proliferation to ConA. DEP did not affect 

spleen cell proliferation in response to anti-CD3 

mAb. Production of IL-2 in response to ConA 

was reduced in a dose-dependent manner by 

DEP exposure. IFN-γ production was decreased 

by exposure to DEP. IL-4 production was not 

measured. 

December 2009 D-152 
Study Pollutant Exposure Effects 

Reference: Yin et 

al. (2005, 088133) 

Species: Rat 

Gender: Male 

Strain: BrownNorway 

(BN/CrlBR) 

Age: NR 

Weight: 200-250 g 

DEP = SRM 2975 (NIST) 

Listeria 

Particle Size: NR 

Route: Nose-only inhalation (DEP), IT instillation 

(Listeria) 

Dose/Concentration: 100,000 CFU (Listeria); 21.2 

± 2.3 mg/m3

 (DEP) 

Time to Analysis: DEP exposure for 4 h/day for 5 

days; infection with Listeria 7 days post-exposure; 

sacrifice 3 and 7 days postinfection 

Lung Deposit: Estimated mean lung deposit of 

DEP = 406 ± 29 μg/rat DEP prolonged growth of 

bacteria in lung 

Alveolar Macrophage (AM) Response: DEP 

significantly inhibited Listeria-induced IL-1β

secretion at day 7 and TNF-α and IL-12 at both 

day 3 and day 7 IL-10 production was enhanced 

at day 7. 

T-Lymphocyte Response: DEP significantly 

reduced the development of T cells in response 

to Listeria infection. These lymphocytes 

displayed increased production of IL-6 at day 7, 

but significantly diminished levels of IL-10, IL-2 

and IFN-γ. 

Reference: Yin et 

al. (2004, 097685) 

Species: Rat 

Gender: Male 

Strain: BrownNorway 

(BN/CrlBR) 

Age: NR 

Weight: 200-250 g 

DEP = SRM 2975 (NIST) 

Listeria 

Particle Size: NR 

Route: Inhalation (DEP), IT instillation (Listeria) 

Dose/Concentration: 20.62 ± 1.31 mg/m3 (DEP). 

100,000 CFU Listeria 

Time to Analysis: DEP exposure for 4 h/day for 5 

days; inoculation with bacteria 2 h postexposure; 

sacrifice 3, 7, 10 days postinfection 

Lung Deposit: Estimated mean lung deposit of 

DEP = 389 ± 25 μg/rat 

Pulmonary Responses and Bacterial 

Clearance: DEP significantly augmented 

Listeria-induced PMN infiltration, lung CFU and 

recoverable AM at all times post-infection. LDH 

activity was increased 3 days post-infection. 

Bacterial count in DEP exposed rats remained 

significantly higher through day 7. 

Cytokine Production by AM: DEP exposure 

significantly lowered Listeria-induced production 

of IL-1β, TNF-α and IL-12. Production of IL-10 

was strongly augmented. 

T-lymphocyte Responses: DEP moderately 

but not significantly lowered the total number of 

lymphocytes, CD4+ cells and lymphocyte IL-10 

production. Listeria-induced T-cell development 

was strongly inhibited, as were the development 

of CD8+ cells, IL-12 production and IFN-γ

secretion. DEP and Listeria exposure showed 

and increased production of IL-6 at day 3 and 

day 7 post-exposure. 

Reference: Yin et 

al. (2007, 198980) 

Species: Rat 

Gender: Male 

Strain: BrownNorway 

(BN/CrlBR) 

Age: NR 

Weight: 225-250 g 

Cell Line: AM 

DEP = SRM 2975 

eDEP = organic DEP extract 

wDEP = washed DEP 

CB = carbon black 

Particle Size: DEP: median diameter19.4 μm, surface area- 91 m2

/g; CB: 0.1-

0.6 μm 

Route: IT Instillation of Listeria; Cell Culture 

(2.5×105

 cells/well) 

Dose/Concentration: DEP: 10, 50, 100 μg/mL; 

CB: 50 μg/mL 

Time to Analysis: Sacrifice 7 days postinfection or 

no infection. Cell culture: 1, 4, 16, 24 h. 

AM Phagocytosis: None of the DEP or CB 

treatments were cytotoxic or affected the 

number of adherent cells. 10-100 μg/mL. DEP 

significantly decreased AM phagocytosis in a 

concentration- and time-dependent manner, with 

increased concentration and time decreasing 

activity. 

Bacterial Activity: The inhibition of AM 

bactericidal activity by DEP was time- and 

concentration-dependent. eDEP and wDEP 

inhibited the AM bactericidal activity but were 

less effective than DEP. The CB treatment was 

not significant. 

Cytokine Secretion by AM: DEP and eDEP 

concentration-dependently decreased TNF-α, 

IL-1Β and IL-12, but increased IL-10. wDEP and 

CB did not show a significant effect. 

Cytokine Secretion by Lymphocytes: DEP 

and cDEP concentration-dependently 

decreased IL-2 and IFN-γ. wDEP and CB had 

little effect, except high concentrations of wDEP 

decreased IFN-γ. 

December 2009 D-153 
Study Pollutant Exposure Effects 

Reference: Yin et 

al. (2004, 087983) 

Species: Rat 

Gender: Male 

Strain: BrownNorway 

(BN/CrlBR) 

Age: NR 

Weight: 225-250 g 

Cell Line: AM 

DEP = SRM2975 

eDEP = organic DEP extract 

wDEP = washed DEP 

CB = carbon black 

Particle Size: DEP- NR, CB- 0.1-0.6 μm 

Route: IT Instillation of Listeria; Cell Culture 

Dose/Concentration: 50 μg/mL (DEP or CB) 

Time to Analysis: Killed 7 days postinstillation. AM 

isolated then incubated. DEP treatments for up to 

24 h. 

DEP-Induced ROS Production: ROS was 

induced by DEP or eDEP and inhibited by eDEP 

with ANF or NAC. eDEP induction of ROS was 

time-dependent. wDEP or CB did not induce 

ROS. 

DEP-Induced HO-1 Expression: DEP- or 

eDEP-induced HO-1 expression was inhibited 

by ANF, NAC or SB203580. wDEP or CB did not 

induce ROS. DEP or eDEP exposure resulted in 

a 2.5- to 3-fold induction of HO-1 expression in 

uninfected AM. 

eDEP-Modulated Cytokine Production: eDEP 

exposure resulted in a time-dependent increase 

in LPS-stimulated IL-10 or TNF-α production, 

and both were inhibited by ANF or NAC. wDEP 

did not affect either. SOD pretreatment 

attenuated eDEP-upregulated HO-1 expression, 

inhibited IL-10, and reversed eDEP inhibition of 

IL-12. Znpp decreased IL-10. 

Reference: Yin et 

al. (2003, 096127) 

Species: Rat 

Gender: Male 

Strain: BrownNorway 

(BN/CrlBR) 

Age: NR 

Weight: 200-250 g 

DEP = SRM 1650a 

L. monocytogenes 

Particle Size: NR 

Route: Nose-only Inhalation (DEP); IT Instillation 

(Listeria) 

Dose/Concentration: 50 or 100 mg/m3 (DEP); 

100,000 bacteria per 500 μL sterile saline (Listeria) 

Time to Analysis: DEP exposure for 4 h. Bacterial 

inoculation. Sacrificed 3, 7 days post-exposure. 

Lymphocyte Population: DEP-alone exposure 

increased total lymphocytes, T cells and T-cell 

subsets. Elevated cell counts in the combined 

exposure were DEP dose-dependent, with the 

100 mg/m3

 treatment having significant 

increases in the cell number and CD8+/CD4+ 

ratio. 

IL-2: DEP exposure in noninfected rats at both 

doses increased IL-2 in the 24 h culture and 

decreased IL-2 in the 48 h culture. The increase 

in IL-2 at 3 days postinfection was not 

significant. DEP exposure increased IL-2Rα in 

response to ConA stimulation. DEP-treated 

infected rats had increases in ConA-inducible 

CD4+/IL-2Rα+ and CD8+/ IL-2Rα+. 

IL-6: IL-6 production was dose-dependent in 

DEP-treated uninfected rats and infected rats. 

The combined exposure produced less IL-6 than 

the DEP-alone or Listeria-alone treatments. 

IFN-γ: DEP decreased IFN-γ at 3 days postexposure, but increased at 7 days postexposure in a dose-dependent manner. 

Uninfected DEP-treated rats did not 

substantially respond to HKLM. HKLM-induced 

IFN-γ production is strongly inhibited at all 

tested DEP doses. 

Reference: 

Zelikoff et al. 

(2003, 039009) 

Species: Rat 

Gender: Male 

Strain: F344 

Age: 7-8 mo 

Weight: NR 

CAPS (concentrated ambient PM2.5 from 

New York City) 

S.pneumoniae 

Particle Size: 0.4 μm (MMAD) 

Route: Nose-only Inhalation (CAPS); IT Instillation 

(S.pneumoniae) 

Dose/Concentration: CAPS: Study 1- Mean- 345 

μg/m3

; 60-600 μg/m3. ,Study 2- Mean-107 μg/m3; 

65-150 μg/m3

(S.pneumoniae 2-4×107

) 

Time to Analysis: Study 1: Uninfected rats 

exposed to air or CAPS for 3 h. Sacrificed 3, 24, or 

72 h post-exposure or IT instilled 4, 24, 72, 120 h 

and sacrificed 4, 24, 72 h postinfection 

Study 2: Infection with bacteria. Exposed 48 h later 

to CAPS or filtered air for 5 h. Sacrifice 9, 18, 24, 

72, 120 h post-exposure. 

Study 1: CAPS did not effect cell numbers, 

viability, profiles, lavageable LDH activity, total 

protein, or total circulating WBC counts. 

Exposure to CAPs prior to infection significantly 

increased PMN and decreased lymphocytes. 

WBC levels returned to control levels by 4 h 

postinfection. CAPS had no effect on circulating 

monocyte values. CAPS significantly increased 

bacterial burdens at 24 h, but thereafter the 

burden decreased to below control levels. 

Study 2: In CAPS exposed rats, PMN 

decreased, Pam increased, and the cytokines 

TNF-α, IL-1β and IL-6 decreased. Lymphocytes 

and monocytes were unaffected. Bacterial 

burdens in CAP-exposed rats were about 10% 

greater than air controls at 9 h and >300% 

greater at 18 h. CAPS significantly increased 

the percent of affected lung area and severity of 

infection. 

December 2009 D-154 
Study Pollutant Exposure Effects 

Reference: 

Zelikoff et al. 

(2002, 037797) 

Species: Rat 

Gender: Male 

Strain: Fischer 

344 

Age: 7-9 mo. 

Weight: NR 

Ambient NYC PM 

Single transition metals of Fe, Mn, Ni 

Streptococcus pneumoniae 

Particle Size: NYC PM: PM2.5

Fe2+, Mn2+, Ni2+: 0.4 μm (MMAD) 

Route: Nose-only Inhalation, IT instillation (S. 

pneumoniae) 

Dose/Concentration: Single metals/NYC PM: 65-

90 μg/m3

; 15-20×106 (S.pneumoniae) 

Time to Analysis: Infection/no infection followed 

by 5 h exposure to NYC PM or single transition 

metal. Sacrifice 4, 5, 9, 18, 24, and 120 h after 

exposure. 

CAPs exposure to infected rats significantly 

increased pulmonary bacterial burdens of S. 

pneumo in a time-dependent manner. At 9 h, 

 18 h, 24 h, and 5 days after CAPs exposure, 

bacterial burdens were 10%, 300%, 70% and 

30% above controls. Uninfected rats exposed to 

the single transition metals showed significant 

alterations in PMNs and lymphocytes values at 

1 h post-exposure. 

Exposure to Fe of uninfected rats significantly 

increased superoxide anion production by 

pulmonary macrophages. Uninfected rats 

exposed to inhaled Fe significantly reduced Blymphocyte proliferation at 48 h, but did not 

affect T-lymphocyte production. Inhaled Ni, for 

the uninfected, significantly decreased Tlymphocyte production at 18 h, and did not 

affect B-lymphocyte production. Inhalation of Fe 

by infected rats facilitated an increase in 

bacterial numbers while Ni inhibited bacterial 

clearance. Inhaled Fe by infected also 

significantly decreased PMNs and lymphocyte 

numbers by 35% and increased pulmonary 

macrophage numbers by 29% when compared 

to the air exposed group. Results demonstrated 

that inhalation of Fe altered innate and adaptive 

immunity in uninfected hosts, and both Fe and 

Ni reduced pulmonary bacterial clearance in 

previously infected rats. 

Reference: Zhong 

et al. (2006, 

093264) 

Species: Mouse 

Gender: Male 

Strain: BALB/c 

Age: 6-8 wk 

Weight: NR 

Cell Line: 

J774A.1, IFN-yprimed AMs, 

unprimed AMs 

CAPs: Concentrated Air Particles 

(Boston, MA) 

Urban air particles (UAP) SRM1649 

(Washington, DC) 

TiO2 

Carbon Black (CB) (Sigma, St. Louis, 

MO) 

Streptococcus pneumoniae: strain 

ATCC6303

Particle Size: UAP = NR; 

TiO2/CB = NR; CAPs: ≤PM2.5

Route: Cell Culture 

Dose/Concentration: NR, 100 μg/mL 

Time to Analysis: CAPs for 1 h; bacteria for 1 h. 

Binding measured 15 h after bacteria exposure. 

Ingestion measured 2 h after bacteria exposure. 

Rate and number of killed bacteria measured 2 h 

after bacteria exposure. 

Binding, Internalization and Killing of 

Bacteria: CAPS significantly increased binding 

of bacteria by IFN-y-primed AMs, normal AMs 

and J774A.1. CAPS decreased internalization 

and absolute number of bacteria killed by 

macrophages of all types. The rate of killing of 

internalized bacteria was similar in the presence 

or absence of CAPs; however, CAPs did cause 

a decrease in the absolute number of bacteria 

killed by all three types of macrophages, due to 

the decrease in internalization. 

Effects of other particles: TiO2 and CB had no 

effect on J774 binding or internalization of S. 

pneumo. TiO2 and CB’s effects on primed and 

unprimed AMs were not reported. Testing with 

UAPs, however, showed effects similar to those 

observed with CAPs. 

Soluble components: The soluble fraction of 

CAPs, especially iron, is responsible for 

decreased internalization. 

Table D-5. Effects of the central nervous system. 

Reference Pollutant Exposure Results 

Reference: 

CalderónGarcidueñas et 

al. (2003, 

156316) 

Species: Dog 

Gender: Male, 

Female 

Strain: Mixed 

breed 

Age: 7d-10 yr 

Weight: 349 ± 

116g - 20 kg 

Urban Air (Mexico City-high PM 

region, Tlaxcala- low PM region) 

(PM, Pb, volatile organic 

compounds, formaldehyde, 

acetaldehyde, mutagenic PM, 

alkane hydrocarbons, Ni, V, Mn, Cr, 

peroxyacetyl NO4

2-, LPS, 

endotoxins) 

Particle Size: PM: 2.5, 10 μm 

Route: Ambient Air Exposure 

Dose/Concentration: Mexico City: PM10: 

78 μg/m3, PM2.5: 21.6 μg/m3, Pb in TSP: 

<0.4 μg/m3

 

Time to Analysis: Dogs raised in house or 

outdoor-indoor kennel. Lifetime exposure. 

Mexico City dogs had significantly greater apurinic and 

apyrimidic sites in the olfactory bulb and hippocampus. 

Histopathological changes in the respiratory and olfactory 

epithelium were greatest in Mexico City dogs. Mexico City 

dogs also had greater immunoreactivity than the controls for 

NF-κB, iNOS, cyclooxygenase-2, glial fibrillatory acidic protein, 

ApoE, amyloid precursor product and Β-amyloid. 

December 2009 D-155 
Reference Pollutant Exposure Results 

Reference:

Campbell et al. 

(2005, 087217) 

Species: Mouse 

Strain: BALB/c 

Age: 7 wk 

CAPs from Los Angeles, lacking 

reactive organic and H2O soluble 

gases, O3, NOX, SOX

Particle Size: F+UF: <2.5 μm; 

UF: <0.18 μm 

Route: Whole-body Inhalation 

Dose/Concentration: 20-fold 

concentration of near highway ambient air, 

avg UF concentration: 282.5 μg/m3, avg F 

concentration: 441.7 μg/m3

Time to Analysis: 4 h/day, 5 days/wk for 2 

wk 

Mice were challenged with OVA prior to exposure and 1 and 2 

wk following exposure, and then brains were assayed. F+UF 

and UF exposure increased NF-κB DNA binding in brain. TNFα increased with F+UF. IL-1α increased with UF and F+UF. 

This suggests a possible link between PM exposure and 

neurodegenerative disease processes. 

Reference: Che 

et al. (2007, 

096460) 

Species: Rat 

Strain: SD 

Gender: Male 

and Female 

Age: 9 wk 

Weight: 190- 

 220 g 

Gasoline exhaust (collected from 

1996 Guangzhou passenger car 

with Dongfeng Gasoline Series 155 

kw engine and no exhaust catalytic 

converter fuelled with 90-octane Pbfree gasoline from China 

Petroleum). 

Particle Size: NR 

Route: IT Instillation 

Dose/Concentration: 5.6, 16.7, or 50.0 

L/kg, final volume 0.3 mL/rat 

Time to Analysis: 1/wk for 4 wk; 24 h post 

-instillation. 

A dose-dependent increase was observed in brain DNA 

damage starting at 5.6 L/kg. Increase in lipid peroxidation and 

carbonyl protein was also observed at 50 L/kg. Decrease in 

brain SOD occurred at all exposures. GPx activity was 

unchanged with exposure. This suggests an association 

between gasoline exhaust and oxidative damage to the brain. 

Reference: 

Kleinman et al. 

(2008, 190074) 

Species: Mouse 

Gender: Male 

Strain: ApoE-/-

Age: 6 wk 

Weight: NR

CAPs (Los Angeles, CA) (OC, EC = 

~50%; sulfate, nitrate ~11%) 

Particle Size: NR 

Route: Whole-body Inhalation 

Dose/Concentration: High dose: Mass 

concentration- 114.2 μg/m3

, Low dose: 

Mass concentration: 30.4 μg/m3 

Time to Analysis: 5 h/day, 3days/wk, 6 

wk; 24 h postexposure.

Activated AP-1 dose-dependently increased. Activated NF-κB 

significantly increased with the high CAPs dose. GFAP (which 

represented activated astrocytes) and activated JNK 

significantly increased with the low CAPs dose. 

Reference: Liu 

et al. (2005, 

088650) 

Species: Rat 

Strain: Wistar 

Gender: Male 

Age: 8 wk 

CAPs from Taiyuan, China 

Particle Size: <2.5 μm 

Route: IT Instillation 

Dose/Concentration: 0, 1.5, 7.5, or 37.5 

mg/kg, final volume 0.2 mL/rat 

Time to Analysis: 24 h 

In the brain, SOD and CAT activity were significantly 

decreased at the 2 highest doses; GSH levels were 

significantly decreased at the highest dose. This suggests an 

association between PM exposure and oxidative damage 

mediated by prooxidant/antioxidant imbalance or high levels of 

free radicals. 

Reference: 

Sirivelu et al. 

(2006, 111151) 

Species: Rat 

Gender: Male 

Strain: Brown 

Norway 

Age: 12-13 wk

CAPs from Grand Rapids, MI 

Particle Size: <2.5 μm 

Route: Whole-body Inhalation 

Dose/Concentration: 500 μg/m3

Time to Analysis: 8h; assayed at 24-h PE 

PVN: CAPs alone or with OVA increased NE. 

MPA: CAPs increased Da when treated with OVA while no 

changes in NE, 5-HIAA and DOPAC were observed. 

Arcuate nucleus: OVA sensitization increased NE levels. 

OB: CAPs and OVA increased NE levels, but no changes in 

Da, DOPAC, or 5-HIAA were observed. 

Other areas: No differences in other areas of hypothalamus, 

substantia nigra, or cortex were observed. CAPs alone or with 

OVA increased serum corticosterone. These results suggest 

that CAPs can cause region-specific modulation of 

neurotransmitters in brain and that the stress axis may be 

activated causing aggravation of allergic airway disease. 

Reference: 

Veronesi et al. 

(2005, 087481) 

Species: Mouse 

Strain: ApoE-/- or 

C57Bl/6 

Age: Young 

adults 

CAPs from Tuxedo, NY 

Particle Size: <2.5 μm 

Route: Whole-body Inhalation 

Dose/Concentration: Average daily 

concentration 113 μg/m3

Time to Analysis: 6 h/day, 5days/wk for 4 

mo 

CAPs-exposed ApoE-/- mice had an 29% reduction in THstained neurons and a 8% increase in GFAP staining 

compared to air-exposed ApoE-/-. No differences were see in 

C57 mice. The results suggest that ApoE-/- mice, characterized 

by increased brain oxidative stress, are susceptible to PMinduced neurodegeneration. 

December 2009 D-156 
Reference Pollutant Exposure Results 

Reference: WinShwe et al. 

(2008, 190146) 

Species: Mouse 

Gender: Male 

Strain: BALB/c 

Age: 7 wk 

Weight: NR 

DEP (Nanoparticle-rich - NPDE; 81-

diesel engine, steady-state 

condition, 5 h/d, 2000rpm, 0 Nm) 

(CO, CO2, NO, NO2, SO2) 

Particle Size: 26.21 ± 1.50 nm 

(diameter) 

Route: Whole-body Inhalation 

Dose/Concentration: 148.86 ± 8.44 μg/m3

Time to Analysis: 5 h/day, 5 days/wk, 

4 wk. Some mice ip injected with 

lipoteichoic acid (LTA) 1×/wk, 4 wk. Morris 

water maze behavioral test: 3 days 

acquisition, 2 day probe trial. 

Mice in the LTA+NPDE group had significantly longer mean 

escape latencies, indicating impaired acquisition of spatial 

learning. NPDE directly increased NR1 and TNF-α. 

LTA+NPDE increased NR2A, NR2B, and IL-1β, however LTA 

was primarily responsible for the increases. 

Reference: 

Zanchi et al. 

(2008, 157173) 

Species: Rat 

Gender: Male 

Strain: Wistar 

Age: 45 days 

ROFA from Universidade de São 

Paulo, Brazil 

Particle Size: 1.2 ± 2.24 μm 

(MMAD) 

Route: Intranasal Instillation 

Dose/Concentration: 20 μg/10 μl saline 

Time to Analysis: 30 days 

Exposed rats had increased lipid peroxidation in striatum and 

cerebellum. This could be reversed with N-acetylcysteine 

treatment. ROFA treatment altered motor activity shown by 

decreased general exploration and peripheral walking, and 

was not prevented by NAC. Results suggests that chronic 

ROFA induces behavioral changes and brain oxidative stress. 

Table D-6. Reproductive and developmental effects. 

Reference Pollutant Exposure Effects 

Reference: Fedulov 

et al. (2008, 097482) 

Species: Mouse 

Gender: Female 

(pregnant), 

Offspring: NR 

Strain: BALB/c 

Age: NR 

Weight: NR 

DEP 

Carbon black (CB) 

TiO2

Particle Size: NR 

Route: Intranasal Instillation 

Dose/Concentration: DEP, TiO2: 50 μg in 50 μL, 

50 μg/mouse; CB: 250 μg in 50 μL 

Time to Analysis: Particle samples baked 3 h. 

Protocol 1a: Pregnant mice treated with DEP or 

TiO2. Analyzed 19 or 48 h later. Protocol 1b: 

Pregnant mice DEP, TiO2 or CB treated day 14 of 

pregnancy. 4 day-old offspring i.p. injected with 

OVA+alum. 12-14 days-old exposed aerosolized 

OVA. 

DEP increased BAL PMN counts in normal and pregnant mice. 

In pregnant mice, DEP and TiO2 increased IL-1β, TNF-α, IL-6 

and KC compared to nonpregnant controls. Offspring of DEP, 

CB or TiO2 exposed mice had increased AHR and airway 

inflammation. TiO2 exclusively altered the expression of 80 

genes in pregnant mice. 

Reference: Fujimoto 

et al. (2005, 096556) 

Species: Mouse 

Strain: Slc:ICR 

Gender: Females 

(pregnant mice and 

fetuses) 

Age: NR (pregnant 

females), 14 days of 

gestation (fetuses) 

DE: generated by 2369 

cc diesel engine at 1050 

rpm at 80% load with 

commercial light oil 

Particle Size: 0.4 μm 

(MMAD) 

Route: Inhalation 

Dose/Concentration: 0.3, 1.0 or 3.0 mg DEP/m3

Time to Analysis: 12 h/day, 7 days/wk from 2 day 

post coitum to 13 dpc. Sacrificed 14 dpc. mRNA 

expression examined in female fetuses. 

Significant increase in absorbed placentas were observed in 

the 0.3 and 3.0 concentration. A decrease in absorbed 

placentas was observed for the 1.0 concentration. Increased 

inflammatory cytokine mRNA in placentas from exposed 

offspring were observed. An increased number of absorbed 

placentas in DE-exposed offspring were seen. 

Reference: 

Hougaard et al. 

(2008, 156570) 

Species: Mouse 

Strain: C57Bl/6

Gender: Pregnant 

females, male and 

female offspring 

Age: 12 ,16 wk 

(female offspring), 

13, 17 wk (male 

offspring) 

DEP(SRM2975) 

Particle Size: 90 m2/g 

(SA) 

Route: Whole-body Inhalation 

Dose/Concentration: 20 mg DEP/m3 

Time to Analysis: Exposed 1 h/dayfrom gestation 

days 7-19. Mice separated for behavioral testing 

on PND 22 (day of delivery is PND 0). Behavioral 

testing at 12, 16 wk for female offspring and 13, 

17 wk for male offspring. 

Body weight of exposed unchanged at birth. Body weight 

decreased at weaning. 

Unchanged dams & pups at weaning. At 2 mo, exposed female 

pups required less time to locate platform in spatial Reversal 

task of Morris Water maze. 

December 2009 D-157 
Reference Pollutant Exposure Effects 

Reference:

Hougaard et al. 

(2008, 156570) 

Species: Mouse 

Gender: Female 

(pregnant), 

Offspring- male, 

female 

Strain: C57BL/6 

Age: NR 

Weight: NR

DEP (SRM 2975) 

Particle Size: 240 nm 

(MMAD); surface area 90 

mg2/g, density 2.1 g/cm3 

Route: Inhalation 

Dose/Concentration: 19.1 ± 1.13 mg DEP/m3

Time to Analysis: Pregnant dams exposed GD 7-

19, 1 h/day. GD 20 named PND 0 for pups. 

Weights recorded, 1 pup from each group 

sacrificed PND 2. Weights recorded PND 9. PND 

22 1 male and female removed from each group 

for behavioral testing. Dams and remaining 

offspring sacrificed PND 23 or 24. 

DEP females gained more weight during gestation. Generally, 

DEP pups weighed less. No significant DNA damage was 

measured, but DEP caused slightly higher IL-6, MCP-1, and 

MIP-2. Plasma thyroxin levels as well as learning and memory 

were similar amongst the groups. 

Reference: Huang 

et al. (2008, 156574) 

Species: Rat 

Gender: Male 

(adults), male and 

female (fetuses) 

Strain: Wistar 

Age: 8 wk (male 

adults), 20 days of 

gestation (fetuses) 

ME: Motorcycle Exhaust 

(generated from 1992 

Yamaha cabin motorcycle 

with two-stroke 50 cc 

engine). 

Particle Size: NR 

Route: Nose-only Inhalation 

Dose/Concentration: 1: 10 and 1: 50 dilutions 

Time to Analysis: 2 h/day (1 h in morning and 1 

h in afternoon), for 5 consecutive days/wk, for 4 

wk (1:50, 1:10 dilutions) and 2 wk (1:10 dilution). 

Male mated with untreated females. Pregnant 

females sacrificed on 20 days of gestation. Male 

and female fetuses observed. 

After exposure, decreased body weight and testicular 

spermatid number were observed. 1: 10 ME exposure for 4 wk 

(no recovery) decreased testicular weight and increased the 

inflammatory cytokine mRNA. Glutathione system and lipid 

peroxidation were not affected. 

Reference: 

Lichtenfels et al. 

(2007, 097041) 

Species: Mouse 

Gender: Male and 

Female 

Strain: Swiss 

Age: NR 

Ambient air in São Paulo, 

Brazil 

Particle Size: NR 

Route: Ambient Air Exposure 

Dose/Concentration: NA 

Time to Analysis: Males housed in open-top 

chambers for 24 h/day, everyday for 4 mo, 

beginning 10 days after birth. Males mated to nonexposed females immediately following exposure. 

Males sacrificed immediately following mating. 

Pregnant females remain in chamber and 

sacrificed on 19 days of pregnancy. 

Decreased testicular, epididymal sperm counts, decreased 

number of germ cells, and decreased elongated spermatids 

were observed. Decreased SSR, and a sex ratio shift (fewer 

males) also occurred after exposure. 

Reference: Mauad 

et al. (2008, 156743) 

Species: Mouse 

Gender: Male, 

Female 

Strain: BALB/c 

Age: 10 days 

Weight: Parental: 

21.4 ± 4.0 - 26.3 ± 

2.8 g; 15 day-old 

offspring: 7.8 ± 1.1 - 

9.0 ± 1.0 g; 90 daysold offspring: 20.3 ± 

2.3 - 27.4 ± 1.8 g

PM (busy traffic street 

São Paulo, Brazil; Aug. 

2005-April 2006) (NO2, 

SO2, CO) 

Particle Size: 2.5, 10 μm 

(diameter) 

Route: Ambient Air Exposure 

Dose/Concentration: PM2.5: filtered chamber- 2.9 

± 3.0 μg/m3, nonfiltered chamber- 16.9 ± 8.3 

μg/m3

; Outdoor concentration: PM10- 36.3 ± 15.8 

μg/m3

, CO- 1.7 ± 0.7 ppm, NO- 89.4 ± 31.9 μg/m3, 

SO2- 8.1 ± 4.8 μg/m3

Time to Analysis: Nonfiltered exposure 24 h/day 

for 4 mo. Mated at 120 days exposure. After birth, 

30 females and offspring transferred to filtered or 

nonfiltered chamber. Killed 15 or 90 days of age. 

Mild foci of macrophage accumulations containing black dots 

of carbon pigment occurred in the alveolar areas on 90 day-old 

mice. Surface-to-volume ratio decreased from 15 to 90 days of 

age and was higher in mice exposed to air pollution. PM 

exposure reduced inspiratory and expiratory volumes at higher 

levels of transpulmonary pressure. 

Reference: 

Mohallem et al. 

(2005, 088657) 

Species: Mouse 

Strain: BALB/c 

Gender: Female 

Age: 10 wk, 10 days 

Filtered or ambient air in 

downtown Sao Paulo 

situated at crossroads 

with high traffic density 

(predominant source of 

air pollution is 

automotive). 

Particle Size: NR 

Route: Whole-body Inhalation 

Dose/Concentration: PM10: 35.5 ± 12.8 μg/m3; 

CO: 2.2 ± 1.0 ppm; NO2: 107.8 ± 42.3 μg/m3; SO2: 

11.2 ± 5.3 μg/m3

Time to Analysis: Exposed for 24 h/7days/wk for 

4 mo. Newborns mated after reaching 

reproductive age of 12 wk. All pregnant females 

sacrificed between 19th and 20th day of 

pregnancy. 

No effects in adult exposed animals. Increased implantation 

failure of neonatal exposed-dams. 

Sex ratio, # of pregnancies, resorbtions, fetal deaths, and fetal 

placenta Weights unchanged after neonatal ambient air 

exposure. 

December 2009 D-158 
Reference Pollutant Exposure Effects 

Reference: Mori et 

al. (2007, 096564) 

Species: Mouse 

Strain: C57/BL 

Gender: Male 

Age: 6 wk 

DEP: generated by 4-

cylinder diesel engine 

Particle Size: NR 

Route: Dorsal Subcutaneous Instillation 

Dose/Concentration: 0.2 ml (of 1.1mg/ml or 0.37 

mg/ml) 

Time to Analysis: 2×/wk for 10 wk; 1 wk post last 

instillation. 

cDNA library screen after sub-cutaneous injection identified 

activated clones related to prostanoids and arachadonic acid 

(Platg2c2c, Acsl6) and sperm production (Stk35). However, the 

route of exposure was unconventional. 

Reference: Ono et 

al. (Ono et al., 2007, 

156007) 

Species: Mouse 

Strain: ICR 

Gender: Pregnant 

females, male 

offspring 

Age: NR (pregnant 

females), 12 wk 

(offspring) 

DE: generated from 4-cyl 

diesel Isuzu engine at 

1500 rpm using standard 

diesel fuel. 

Particle Size: NR 

Route: Inhalation 

Dose/Concentration: NR 

Time to Analysis: Exposed from 2 day post 

coitum to 16 dpc. Parameters for male offspring 

measured on days 8, 16, 21, 35, 84 and sacrificed 

at 84 days. 

PND 8 and 16 male reproductive accessory gland weight 

decreased. PND 21 decreased serum testosterone (T); PND 

84 increased serum T. FSHr, sTAR mRNA decreased PND 35 

and 84. Relative testis and epididymal weight unchanged. 

Sertoli cell degeneration observed. 

Reference: Ono et 

al. (Ono et al., 2007, 

156007) 

Species: Mouse 

Strain: ICR 

Gender: Male 

offspring, Pregnant 

females 

Age: 12 wk (male 

offspring) 

DE: generated from 4JB2type, light duty 3060 cc 

4-cyl Isuzu diesel engine 

under 1500 rpm 

Particle Size: NR 

Route: Whole-body Inhalation 

Dose/Concentration: 1.0 mg DEP/m3

Time to Analysis: Pregnant females exposed 

from 2 day postcoitum- 16 dpc. Without 

undergoing further exposure, male offspring 

sacrificed at 12 wk. 

Dose-dependent increase in seminiferous tubule degeneration 

and decreased DSP. After 1 mo recovery, DSP recovered at 

the lowest dose. 

Reference: 

Pinkerton et al. 

(2004, 087465) 

Species: Rat 

Gender: Female 

(pregnant), 

Offspring- NR 

Strain: SD 

Age: 10 days (pups), 

Pregnant females10-14 days of 

gestation 

Weight: NR

PM (Fe and soot from 

combustion of acetylene 

and ethylene in a laminar 

diffusion flame system) 

Particle Size: Median 

diameter: 72-74 nm; size 

range: 10-50 nm

Route: Inhalation 

Dose/Concentration: Mean mass concentration: 

243 ± 34 μg/m3

; Average Fe concentration: 96 

μg/m3

Time to Analysis: Exposed 10 days postnatal 

age, 6 h/day, 3 days (consecutive). 

Bromodeoxyuridine injected 2 h before necropsy. . 

A significant reduction of cell proliferation occurred only within 

the proximal alveolar region of exposed animals compared to 

controls. There were no significant differences between the 

groups for alveolar formation and separation within the 

proximal alveolar region 

Reference: Silva et 

al. (Silva et al., 

2008, 156981)

Species: Mouse 

Strain: Swiss 

Gender: Females 

(pregnant mice) 

Age: 1st, 2nd, 3rd 

wk of pregnancy 

(females), GD19 

(fetuses) 

Ambient air: Sao Paulo, 

Brazil 

Particle Size: NR 

Route: Ambient Air Exposure 

Dose/Concentration: NR 

Time to Analysis: 1st wk, 2nd wk, 3rd wk 

or combo of exposure during pregnancy. 

Decreased fetal weight with exposure in 1st wk of pregnancy. 

Decreased placental weight with exposure in any of the 3 wk of 

pregnancy. 

December 2009 D-159 
Reference Pollutant Exposure Effects 

Reference: Somers 

et al. (2002, 078100) 

Species: Mouse 

Strain: Swiss 

Webster 

Gender: Male and 

Female 

Age: 6-8 wk (adult 

male and females), 5 

days (pups) 

Ambient air: 2 sites in 

Canada (polluted 

industrial area 1km 

downwind from two 

integrated steel mills & 

rural location 30 km 

away) 

Particle Size: NR 

Route: Ambient Air Exposure 

Dose/Concentration: NR 

Time to Analysis: Exposed 24 h/day, 7 days/wk 

for 10 wk from September 10, 1999- November 

21, 1999. Exposed to clean air for 6 wk posttreatment. Paired with mice within exposure 

group. 5d old pups measured. 

ESTR germ line mutations following exposure. 

Heritable mutation rate increased 1.5 to 2 fold in urban vs. 

rural site. Increased frequency is paternal line dependent. 

Reference: Somers 

et al. (2004, 078098) 

Species: Mouse 

Gender: NR 

Strain: Sentinal Lab 

Age: NR 

Weight: NR

PM (rural or urbanindustrial) 

Particle Size: >0.1 μm 

Route: Ambient Air Exposure 

Dose/Concentration: Mean TSP: Rural- 16.2 ± 

8.3 - 31.7 ± 13.2 μg/m3, Urban-Industrial- 38.9 ± 

10.5 - 115.3 ± 25.3 μg/m3

Time to Analysis: Exposed 10 wk. Bred 9 wk 

postexposure. 

The offspring of urban-industrial mice inherited paternal ESTR 

mutations 1.9-2.1 times more than rural or HEPA-filtered 

offspring. Maternal ESTR mutations were not significant. 

Reference: 

Sugamata et al. 

(2006, 157025) 

Species: Mouse 

Strain: ICR 

Gender: Pregnant 

Females, male and 

female offspring 

Age: 11 wk 

(offspring), NR 

(pregnant females) 

DE 

Particle Size: NR 

Route: Inhalation 

Dose/Concentration: 0.3 mg DEP/m3

Time to Analysis: Pregnant females exposed 

from 2 day post coitum to 16 dpc. Offspring 

sacrificed 11 wk after birth. 

Exposed pups had increased caspase 3 positive cells and 

decreased purkinjie cell number (cerebellum), similar to human 

Autism brain phenotype. 

Reference: Tozuka 

et al. (2004, 090864) 

Species: Rat 

Strain: F344 

Gender: Pregnant 

females, male and 

female fetuses 

Age: Gestation day 

20 (fetuses), NR 

(pregnant females) 

DE: generated by diesel 

engine (309 cc Model 

NFAD-50) 

Particle Size: NR 

Route: Whole-body Inhalation 

Dose/Concentration: 1.73mg/m3

Time to Analysis: Exposed 6 h/day from GD 7-20 

with no exposure on Saturdays or Sundays (4 

non-exposure days total). Fetuses and maternal 

blood collected on GD20. PAHs: Exposed 6 h/day 

from GD 7-14 with no exposure on Saturdays or 

Sundays. Breast milk collected PND14. 

Gestational and lactational exposure to DE’s And PAHs. 7 milk 

PAHs increased in DE-exposed dams. DE exposure can lead 

to PAH pup exposure through breast milk. 

Reference: Tsukue 

et al. (2004, 096643) 

Species: Mouse 

Strain: Slc: ICR 

Gender: Pregnant 

females, female 

fetuses 

Age: Gestation day 

14 (fetuses) 

DE: generated by 2369 

cc Isuzu diesel engine 

operating at 1050 rpm 

with 80% load and using 

commercial light oil. 

Particle Size: NR 

Route: Whole-body Inhalation 

Dose/Concentration: 0.1 mg DEP/m3 (at 1:8 

dilution with clean air) 

Time to Analysis: Exposed for 8h/day from 2 day 

postcoitum to 13 dpc (with no exposure on days 4, 

5, 11, 12). Sacrificed 14 dpc. Only female fetuses 

studied. 

SF-1 & MIS mRNA did not change. Other steroidogenic genes 

were also unchanged. BMP-15 and oocyte differentiation 

mRNA decreased. 

December 2009 D-160 
Reference Pollutant Exposure Effects 

Reference: Tsukue 

et al. (2002, 030593) 

Species: Mouse 

Strain: C57/BL 

Gender: Females, 

male and female 

offspring 

Age: 6 wk, 70 days 

post natal (offspring) 

DE: generated by lightduty, 4-cyl Isuzu diesel 

engine at 1500 rpm. 

Particle Size: NR 

Route: Whole-body Inhalation 

Dose/Concentration: 0.3, 1.0 or 3.0 mg DEP/m3

Time to Analysis: Exposed 12 h/day, 7 days/wk 

for 4 mo. Some females sacrificed immediately 

following exposure. Remainder mated with 

unexposed males. Parameters measured in 

offspring at postnatal day 70. 

DE-exposed females had decreased uterine weight at 4 mo. 

Offspring had decreased body weight at 6 and 8 wk of age. 

Decreased rate of good nesting construction (3 mg/m3

). 

AGD decreased In males (30 and 70 days old). 

Organ weight decreased in females and female crown to rump 

length decreased. 

Reference: Ueng et 

al. (2004, 096199) 

Species: Mouse 

Gender: Female 

Strain: Wistar 

Age: 21days 

Cell Line: MCF-7 

ME: generated from a 

Yamaha Cabin 

motorcycle 2-stroke 50-

cc engine and variable 

venture carburetor 

Particle Size: NR 

Route: Intraperitoneal Instillation. Cell Culture. 

Dose/Concentration: IP: 1, 10, 50 μg/ml 

Cell Culture: 0.01, 0.1, 1, 10, 50, 100 μg/ml 

Time to Analysis: IP: 1/day for 3 days and 

sacrificed on 24 day. Cell Culture: 3, 24, 30, 48 h 

and 2 days. 

10 mg/kg +E2 induced anti-estrogenic uterine effects and 

antiestrogenic with in vitro (MCF-7 cells) E2 screen. 

Reference: Veras et 

al. (2008, 190493) 

Species: Mouse 

Gender: Male, 

Female 

Strain: BALB/c 

Age: 20 days, 

newborns 

Weight: NR

PM (downtown São 

Paulo, Brazil near 

crossroads with high 

traffic density, 67% PM2.5

comprises air pollution) 

Particle Size: 2.5 μm 

(diameter) 

Route: Open-Top Chamber 

Dose/Concentration: PM2.5- 27.5 μg/m3; NO2- 

101 μg/m3

; CO- 1.81 μg/m3; SO2- 7.66 ppm 

Time to Analysis: 20 days-old mice maintained in 

filtered or nonfiltered chamber until 60 days-old. 

Offspring maintained in respective chambers until 

21 days-old. Offspring mate at 60 days-old. 

Females euthanized 18th GD.

Fetal weight and maternal blood space volume and surfaces 

declined in the groups exposed to nonfiltered air. Fetal 

capillary surfaces were greater in nonfiltered air groups. There 

was a significant gestational effect on maternal:fetal surface 

ratios with values declining significantly in groups exposed 

during pregnancy to nonfiltered air. The total oxygen diffusive 

conductance of the intervascular barrier increased significantly 

during pregestational exposure to nonfiltered air. Mass-specific 

conductance increased during pregestational and gestational 

periods of exposure to nonfiltered air. 

Reference: Veras et 

al. (2009, 190496) 

Species: Mouse 

Gender: Male, 

Female 

Strain: BALB/c 

Age: 20 days 

Weight: NR

PM (São Paulo, Brazil; 

near crossroads with high 

traffic density) (Al, Ca, 

Cu, Fe, K, Na, Ni, P, Pb, 

S, Si, Ti, V, Zn, C) 

Particle Size: 2.5 μm 

(diameter) 

Route: Open-Top Chamber 

Dose/Concentration: Mean: Non-filtered- 27.5 

μg/m3

, Filtered- 6.5 μg/m3

Time to Analysis: 20 days-old mice maintained in 

filtered or non-filtered chamber. Allowed to mate at 

60 days. 2 generation model.

Ambient air pollution extended the estrus cycle, which reduced 

the number of cycles. Antral follicles decreased. Mating time 

increased and fertility and pregnancy indices decreased. The 

mean post-implantation loss rate increased, which was 

influenced by both pre- and post-gestational exposure. Fetal 

weight decreased and was also influenced by pre- and postgestational exposure, which exhibited a significant interaction. 

Reference: 

Watanabe (2005, 

087985) 

Species: Rat 

Gender: Female 

(pregnant), 

Offspring- male 

Strain: F344/DuCrj 

Age: 7 days of 

gestation - parturition 

(females), 96 days 

(offspring) 

Weight: 240-262 g 

(offspring)

DE (309cc engine, Model 

NFAD50, Yanmar Diesel 

Co., Osaka, Japan, 

1800rpm, 45% load) (PM, 

NO2) 

Particle Size: 90% <0.5 

μm 

Route: Inhalation 

Dose/Concentration: High dose total group: PM1.71 μg/m3

, NO2- 0.79 ppm; Low dose total group: 

PM- 0.17 μg/m3

, NO2- 0.10 ppm 

Time to Analysis: Pregnant rats exposed 

gestational day 7 to delivery 6 h/day. 5 groups: 

high dose total DE, high dose PM, NO2 filtered, 

low dose total DE, low dose PM, NO2 filtered, 

clean air control. Offspring sacrificed day 96 after 

birth. 

All groups had significantly less daily sperm production than 

the control. PM and NO2 in DE decreased spermatogenia but 

was not significant, however the high dose PM filtered group 

achieved significance. Pachytene cells, spermatids, and Sertoli 

cells were lower in all groups compared to the control. 

December 2009 D-161 
Reference Pollutant Exposure Effects 

Reference: Yauk et 

al. (2008, 157164) 

Species: Mouse 

Strain: C57BL/6 x 

CBA F1 hybrid 

Gender: Male 

Age: 7-9 wk 

HEPA-Filtered air (PM 

removed) and ambient air 

at 2 sites: 

-2 km from two integrated 

steel mills 

-1 km from major 

highway on Hamilton 

Harbor 

Components: 

Metals 3.6 ± 0.7 μg/m3

TSP 9.4+17 μg/m3

Particle Size: NR 

Route: Ambient Air Exposure 

Dose/Concentration: NR 

Time to Analysis: Parameters measured 3, 10 

wk, or 10 + 6 wk recovery following exposure. 

10+6 wk exposure induced increased ESTR mutations in 

sperm DNA of exposed v filtered. No testicular DNA adducts 

seen in exposed males. At 3 wk DNA increased adducts seen 

in lungs of exposed males, not in filtered males. Mutations 

were PM dependent, and gas-phase independent. 

Reference: Yokota 

et al. (2009, 190518) 

Species: Mouse 

Gender: Female 

(pregnant), Male 

(offspring) 

Strain: ICR 

Age: NR 

Weight: NR

DE (2369-cc diesel 

engine, Isuzu Motors, 

Ltd., Tokyo, Japan; 1050 

rpm, 80% load, 

commercial light oil) 

Particle Size: NR 

Route: Inhalation. Pre-natal Exposure 

Dose/Concentration: DE: 1.0 mg/m3; CO: 2.67 

ppm, NO2: 0.23 ppm, SO2: <0.01 ppm 

Time to Analysis: Pregnant mice exposed 8 h for 

5 days from GD 2-17. Mothers and pups kept in 

clean room. Pups weaned on PND 21 then 

transported to Tokyo University of Science. 2wk 

acclimation. Exposed 12 h light/dark cycle. Activity 

monitor with infrared ray sensor measured 

spontaneous motor activity (SMA), 10 min 

intervals 2 days. After behavioral test, mice 

decapitated.

Prenatal DE exposure decreased SMA in the male offspring. 

DE decreased locomotor activity during the light phase. 

Dopamine levels in the striatum and nucleus accumbens did 

not change, but HVA concentrations decreased in DE-exposed 

mice. 

Reference: Yoshida 

et al. (2006, 156170) 

Species: Mouse 

Strain: ICR, 

C57Bl/6J or DDY

Gender: Pregnant 

Females, Male 

fetuses 

Age: 14 days of 

gestation (fetuses), 

2-13 days of 

gestation (pregnant 

females) 

DE(generated from a 4-

cyl., 2300 cc diesel Isuzu 

engine at 1050 rpm and 

80% load). 

Particle Size: NR 

Route: Whole-body Inhalation 

Dose/Concentration: 0.1 mg DEP/m3

Time to Analysis: Exposure on 2-13 days of 

gestation. Parameters measured on 14 days of 

gestation. 

Responses to exposure showed strain-related variations with 

ICR as the most sensitive followed by C57 and ddY as the 

least sensitive. MIS mRNA expression, a factor in male 

gonadal differentiation, was significantly decreased in the ICR 

and C57 strains. Ad4BP/SF-1 expression was significantly 

decreased in the ICR strain only. 

Reference: Yoshida 

et al. (2006, 097015) 

Species: Mouse 

Strain: ICR 

Gender: Pregnant 

females and male 

offspring 

Age: 2-16 days 

postcoitum (pregnant 

females), 28 days 

(male offspring) 

DE: generated by 4Jb1-

type, light duty 4-cylinder 

Isuzu diesel engine using 

standard diesel fuel at 

1500 rpm. 

Particle Size: NR 

Route: Whole-body Inhalation 

Dose/Concentration: 0.3, 1.0 or 3.0 mg DEP/m3

Time to Analysis: Pregnant females exposed 12 

h/days, 7 days/wk from 2-16 days postcoitum. 

Offspring sacrificed on postnatal day 28. 

NOAEL 0.3 mg DEP/m3

. 

DE exposure induced increased reproductive gland weight 

(two higher doses) in male mice. mRNA decreases in 

aromatase and 3 μ-hD (3.0 mg DEP/m3

). 

No change in sex ratio. Two higher doses induced significant 

increased reproductive organ weights. 

Male pup weight Increased at PND 28. Increased serum T was 

observed in pups exposed to 1.0mg DEP/m3

. 

Serum T positively correlated with DSP, testis weight, steroid 

enzyme mRNA. 

Reference: Yoshida 

et al. 2004 (2004, 

097760) 

Species: Mouse 

Gender: Female 

(pregnant), 

Offspring- male 

Strain: ICR 

Age: 4, 6 wk 

Weight: NR

DE 

Particle Size: NR 

Route: Inhalation 

Dose/Concentration: 6wk-old males, embryos: 

0.3, 1.0, 3.0 mg DEP/m3, Pregnant mice: 0.1, 3.0 

mg DEP/m3

Time to Analysis: 6 wk-old males: Exposed 12 

h/day, 6 mo. 1 mo clean air exposure. Pregnant 

mice: Exposed 2-13 p.c. 8 h/day. Male embryos: 

Exposed 2-16 p.c. Examined at 4 wk-old. 

6wk-old Males: In the seminiferous tubules, DE dosedependently caused degenerative and necrotic changes, 

desquamation of the seminiferous epithelium, and loss of 

spermatozoa. Spermatogenesis was still inhibited after a 1m 

clean air exposure. 

Pregnant Mice: Ad4BP/5F-1 and MIS mRNA significantly and 

dose-dependently decreased in male fetuses exposed to DE. 

4wk-old Male Newborns: Tissue weight of the testis and 

accessory reproductive glands were significantly greater in DEexposed mice. Blood testosterone concentration was 8X 

higher than the control at 1.0 mg DEP/m3

. No significant 

differences occurred for testosterone synthetase mRNA. 

December 2009 D-162 
Table D-7. Mutagenic/genotoxic effects in bacterial cultures. 

Reference Pollutant Exposure Effects 

Reference:

Binkova et al. 

(2007, 156273) 

Species:

Salmonella (±S9 

(rat liver)) 

Cell Line: Calf 

thymus DNA 

PM (Prague, Košice, Sofia, 

Czech Republic; summer, 

winter) (organic extracts) 

Particle Size: Diameter: <10 

μm 

Route: Cell Culture 

Dose/Concentration: 100 μg EOM/mL 

Time to Analysis: PM collected 24 h daily 3 

mo, extracted. 24 h incubation BaP, c-PAH, 

EOM, with or without S9. 32P-Postlabeling 

4 h. Autoradiography 1-24 h.

DNA adducts in EOM treatments were greater with S9 than 

without. Positive correlations were found between the amount of 

DNA adducts and the PAH content (notably BaP) in the EOM 

treatment. 

Reference: Brits 

et al. (2004, 

087397) 

Species: S. 

typhimuriam 

Strain: TA98 ± 

S9 (Ames); 

TA104 recN2-4 

and TA104pr1 

(Vitotox) 

Cell Line:

Human whole 

blood (Comet, 

MN assays)

PM (Flanders, Belgium; 

urban, rural, industrial sites) 

(organic extracts) 

Particle Size: 10 μm 

(diameter) 

Route: Cell Culture 

Dose/Concentration: 2.5, 5, 10, 20m3 air 

equivalents/mL 

Time to Analysis: Air samples extracted. 

Ames assay 48 h. Vivotox test. Comet assay 

24 h. MN assay. 

Ames: S9 induced mutagenicity of all extracts from all areas in a 

dose-dependent manner. Without S9, only extracts from the urban 

and industrial areas were mutagenic at the highest dose. 

Vitotox: Extracts were toxic at the highest dose. 

Comet: Significant DNA damage in the extracts was seen and 

enhanced by S9. 

MN: A dose-response relationship was seen in the urban extracts 

for increased micronucleated binuclear cells.

Reference:

Brown et al. 

(2005, 095919) 

Species: S. 

typhimuriam 

Strain:TA98 

Cell Line: Rat 

hepatoma H4IIE

PM (New Zealand, summer, 

winter) (extracts) 

Particle Size: 10 μm 

(diameter) 

Route: Cell Culture 

Dose/Concentration: 9.7-20.8 μg/m3

(summer), 21.8-61 μg/m3

 (winter) 

Time to Analysis: Air samples collected 15 

days, extracted. Ames test: Bacteria growth 

12 h, incubated 24 h. Hepatoma bioassay: 

24 h incubation 2x. EROD assay.

Generally, the mutagenic rate was positively correlated to PM10, as 

well as PAH and BaP. PM10 levels were higher and more 

mutagenic in winter than summer.

Reference: 

Bunger et al. 

(2006, 156303) 

Species: 

Salmonella 

typhimuriam 

Strain: TA98, TA 

100

DEP (diesel fuel (DF), lowsulfur diesel fuel (LSDF), 

rapeseed oil methyl ester 

(RME), and soybean oil 

methyl ester (SME)) (SOFsoluble organic fractions) 

Particle Size: Total particulate 

matter (no OCC) (gh-1): Mean 

DF- 4.0 ± 0.2; 2.8 ± 0.5; 1.8 ± 

0.0; 3.4 ± 0.2; 1.2 ± 0.1 

Route: Cell Culture 

Dose/Concentration: Log 2 dilutions of 

extracts: 1.0, 0.5, 0.25, 0.125 

Time to Analysis: SOF extracted 12 h. 

Plates incubated 48 h. 

No OCC: Without oxidation catalytic converter (OCC), DF extract 

produced the highest number of revertant colonies at all load 

modes in both TA98 and TA100 ± S9. RME, SME, and LSDF 

extracts caused lower or no mutagenic effects, seen especially at 

partial load modes and idle motion. 

OCC: With OCC, all extracts reduced the number of revertant 

colonies in TA98 and TA100 ±S9 at partial load modes B, C, and 

D. At load mode A (rated power), there was an increase of the 

number of revertant colonies in all assays -S9, significant for 

extracts from RME (TA98, TA100) and SME (TA98). S9 lowered 

frequency of mutations. At load mode E (idling), number of 

revertant colonies of DF extracts increased ±S9.

Reference: 

Bunger et al. 

(2007, 156305) 

Species: 

Salmonella 

typhimuriam 

Strain: TA98, TA 

100

Diesel engine emissions 

(DEE)—rapeseed oil (RSO) 

and rapeseed methyl ester 

(RME, biodiesel), natural gas 

derived synthetic fuel (GTL), 

and diesel fuel (DF) 

(SOF- soluble organic 

fractions) 

Particle Size: NR 

Route: Cell Culture 

Dose/Concentration: Log 2 dilutions of 

extracts: 1.0, 0.5, 0.25, 0.125 

Time to Analysis: SOF extracted 12 h. 

Plates incubated 48 h. 

Compared to DF, RSO significantly increased mutagenic effects of 

particle extracts (i.e., revertants) by 9.7-59 in TA98 and by 5.4-

22.3 in TA100. (mRSO, RSO with lowered viscosity and fuel 

preheating in tank, produced highest number of revertant colonies 

in both strains ±S9.) RSO fuels condensates had 13.5 times 

stronger mutagenicity than DF. RME extracts had moderate but 

significantly higher mutagenic response in TA98 +S9 and TA100 

-S9. Effects of GTL did not differ significantly from DF.

December 2009 D-163 
Reference Pollutant Exposure Effects 

Reference: de 

Kok et al. (2005, 

088656) 

Species: S. 

typhimurium 

Strain: TA98 

(with and without 

rat liver S9) 

Cell Line:

Salmon testis 

DNA

TSP (Total suspended 

particulate , Maastricht, The 

Netherlands; PM10 and PM2.5

from 6 urban locations with 

different traffic intensities.) 

(organic extracts) 

Particle Size: NR 

Route: Cell Culture 

Dose/Concentration: Mutagenicity assay: 

2.5, 9, or 18m3 sampled air in 100 μL DMSO; 

DNA adduct assay: 5 μL DMSO containing 

PM10 or TSP from equivalent 50m3

 sampled 

air. PM2.5 concentration equivalent to 35m3

sampled air. 

Time to Analysis: Mutagenicity assay: Cells 

incubated 1 h with extracts. DNA adduct 

assay: DNA incubated 4 h with extracts. 

Overall, the direct mutagenicity and DNA reactivity of PM2.5

extracts were higher compared to PM10 and TSP. S9 generally 

reduced mutagenic activity in TA98 but increased reactivity to 

Salmon testis DNA. Total PAH and total carcinogenic PAH levels 

correlated with the mutagenicity of TSP and the S9-mediated 

mutagenicity of PM2.5. Neither transition metal composition nor 

radical generating capacity of PM correlated with mutagenic 

potential. Total PAH and carcinogenic PAH levels from PM10 and 

PM2.5 correlated with direct and S9-mediated DNA adducts; for 

TSP these levels correlated with direct DNA reactivity only.

Reference: 

DeMarini et al 

(2004, 066329) 

Species: 

Salmonella 

Strain: TA98, 

TA98NR, TA98/1, 

8-DNP6, 

YG1021, 

YG1024, TA100

A-DEP and forklift DEP (SRM 

2975) 

DEP (EOM) 

Particle Size: 0.4 μm (mean 

diameter) 

Route: Cell Culture 

Dose/Concentration: 0, 0.25, 0.5, 1.0, 2.0 

EOM μg/plate 

Time to Analysis: DEPs sonicated 20min. 

Centrifuged 10 min. Organic material 

extracted and concentrated. Ames assay. 

Incubated 3 days.

A-DEPs were more mutagenic in both TA98 and TA100 than SRM 

2975. There was 22× more PAH-related and 8-45× more 

nitroarene-related activity. 

Reference: El 

Assouli et al. 

(2007, 186914) 

Species: S. 

typhimuriam 

Strain: TA98 

(±S9)

PM (Jeddah, Saudi Arabia; 11 

sites, urban, winter) (organic 

extracts) 

Particle Size: 10 μm 

(diameter) 

Route: Cell Culture 

Dose/Concentration: 2.5, 50, 100 μg/plate; 

EOM range: 6-40 μg/m3 

Time to Analysis: 24 h air samples, 

extracted. Refluxed 18-24 h. GC-MS. Comet 

assay. 48 h incubation. Ames assay.

PAHs varied from 0.83 to 0.18 ng/m3. Only 2 locations of heavy 

petrol driven cars showed strong genotoxic responses. A 

correlation existed between DNA damage and the amount of 

pollutants and PAHs. Toxicity and mutagenicity occurred only in 

the presence of S9. Only 3 of the 11 sites exhibited moderate 

mutagenic activities. 

Reference: Endo 

et al. (2003, 

097260) 

Species: S. 

typhimuriam 

Strain: YG1024 

(±S9)

PM (Tokyo, Japan; winter) 

(organic extracts) 

Particle Size: Diameter: 

>12.1 - 0.06> μm; Bimodal 

mass concentration: 1-2 μm 

Route: Cell Culture 

Dose/Concentration: 2.5, 5, 10 μL; 0.30 - 

22.76 μg/m3

Time to Analysis: Air samples collected, 

extracted. 90 min pre-incubation. 48 h 

incubation.

Mutagenicity tests showed dose-response relationships that were 

higher without S9 and increased with decreasing size. 

Reference: 

Erdinger et al. 

(2005, 156423) 

Species: S. 

typhimurium 

Strain: TA98. 

TA100, TA98NR 

PM (Baden-Württemberg, 

Germany; urban, 8 locations, 

glass fiber filters) (organic 

extracts) 

Particle Size: NR 

Route: Cell Culture 

Dose/Concentration: 0.25, 2.5, 5, 12.5, 25 

m3/plate 

Time to Analysis: Standard Ames test 

protocol followed.

Extracts were mutagenic in all strains evaluated. No significant 

difference in response with or without metabolic activation. Activity 

in TA98NR suggests that the mutagenicity correlates with 

concentrations of air pollutants such as NOX. 

Reference: Iba et 

al. (2006, 

156582) 

Species: S. 

typhimuriam 

Strain: TA98, 

TA100 (±S9 (rat 

liver)) 

PM (wood smoke (WS) (New 

Jersey) and cigarette smoke 

(CS) (Tobacco Research and 

Health Institute, University of 

Kentucky) (organic extracts) 

Particle Size: 10 μl aliquots of 

organic extracts 

Route: Cell Culture 

Dose/Concentration: 62.5, 12.5 μg TPM 

equivalent/plate 

Time to Analysis: Incubation, shaking 25 

min. Agar added. 48 h incubation. Rat lung 

explants incubated 18 h. 12 h incubation with 

treatments. 

WS and CS were equally mutagenetic to TA98, but CS was 3-fold 

more mutagenetic to TA100 than WS. CS induced CYP1A1 in the 

explants, but WS did not.

Reference: Liu et 

al. (2005, 

097019) 

Species: S. 

typhimurium 

Strain: YG1024, 

YG1029 

Cell Line: 

Chinese hamster 

lung V79 cells 

DEP extract (DP), gasoline 

engine exhaust particulate 

extract (GP), diesel exhaust 

SVOC extract (DSVOC), 

gasoline engine SVOC extract 

(GSVOC), NIST SRM 1650a

Particle Size: Gasoline PM: 

0.554 mg extract (mg PM)-1; 

Diesel PM: 0.363 mg extract 

(mg PM)-1 

Route: Cell Culture 

Dose/Concentration: 1.48, 4.44, 13.3, 40, 

120, 360, 1080 μg/plate

Time to Analysis: 30 min preincubation. 48 

h (YG1029). 66 h (YG1024). Overnight 

preincubation 20 h. 

Mutations: All samples induced mutations in both strains. The 

increase was highly significant and dose-dependent. Response 

with S9 was generally greater than without S9. PM extract was 

more mutagenic than SVOC extract. 

DP, GP, and GSVOC: Dose-response was seen for DNA damage 

and micronuclei induction. GP, GSVOC and SRM 1650a were 

stronger inducers of micronuclei than DP.

December 2009 D-164 
Reference Pollutant Exposure Effects 

Reference:

Matsumoto et al. 

(2007, 187020) 

Species: S. 

typhimuriam 

Strain: TA98, 

TA100 (±S9) 

APM (airborne particulate 

matter) 

APE (airborne particulate 

extracts) 

(Hokkaido, Japan; residential) 

Particle Size: NR 

Route: Cell Culture 

Dose/Concentration: Crude APE: 979mg/m3

air (CALUX BaP Equivalent (BaPEq)), 21 

mg/m3 air (CALUX TCDD Equivalent 

(TCDDEq)); Cleaned APE: 7.87 mg/m3

 air 

(CALUX BaPEq), 0.614 mg/m3

 air (CALUX 

TCDDEq) 

Time to Analysis: Air samples collected, 

extracted. Preincubation with S. typhimuriam. 

3, 24 h exposure in CALUX assay. RNA 

extracted from mice 6 days after last 

application. 

Most of the CALUX BaPEq for crude APE was derived from PAHlike compounds, as suggested by the CALUX BaPEq of cleaned 

APE accounting for 0.80% of CALUX BaPEq for crude APE. 

CALUX TCDDEq showed TCDD and similar compounds to have a 

low contribution. The TA100 strain was more mutagenic to APE, 

with and without S9. S9 increased mutagenicity in both strains.

Reference:

Pastorkova et al. 

(2004, 087431) 

Species: S. 

typhimuriam 

Strain: TA98, 

YG1041 (±S9)

PM (EOM) (Plzeň, Prague, 

Ústí, Zďár - Czech Republic) 

Particle Size: 10 μm 

(diameter) 

Route: Cell Culture 

Dose/Concentration: TA98 (4 doses): 20-

200 μg/plate, YG1041 (4 doses): 4-20 

μg/plate 

Time to Analysis: Collected 24 h every 18th 

day, Oct-Mar, 1999-2003. Extracted. Ames 

assay. 70 h incubation. 

Significant dose-response effects in mutagenic potency of EOM 

occurred. Prague, one of the most polluted cities, had the highest 

mutagenicity values. Increasing time-trends were observed in the 

TA98 ± S9 mutagenicity and PAH concentrations.

Reference:

Rivedal et al. 

(2003, 097684) 

Species: S. 

typhimurim 

Strain: TA100, 

TA98, TA100NR, 

TA98NR, 

TA98/1,8-DNP6

DEP (SRM 1650)(organic 

extracts) (fractionated into 

PAH, nitro-PAH, dinitro-PAH, 

aliphatics, polar fraction) 

Particle Size: NR 

Route: Cell Culture 

Dose/Concentration: Ames: 300, 600 

DEP/plate; Gap junction: 100, 200 μg/mL 

DEP 

Time to Analysis: Extracted 16 h. 

Fractionated. Ames assay. Gap junction 

intracellular communication: exposed 1-6 h. 

Western blot. 

TA100 was the most mutagenic without S9 activation. GJIC was 

dose- and time-dependently inhibited. The polar fraction was the 

most potent inhibitor. Nitro-PAH and dinitro-PAH were the most 

responsive fractions in the Ames assay.

Reference: 

Seagrave et al. 

(2003, 054979) 

Species: 

Salmonella 

Strain: TA98, 

TA100 

Compressed natural gas 

(CNG) emissions (heavy-duty 

vehicles): High emitter (HE), 

Normal emitter (NE), New 

technology (NT)

Particle Size: NR 

Route: Cell Culture 

Dose/Concentration: PM (mg/mi)- NE- 7.0, 

NT- 5.0, HE- 406; Recovered PM (mg/mi)- 

NE-1.26, NT- 0.71, HE- 57.1; Recovered 

SVOC- NE- 58, NT- 26.4, HE- 227.5 

Time to Analysis: Samples collected in 

filters 7x/day over several days. Recovered 

PM, recovered SVOC extracts combined. 

Ames assay. 

All three CNG emissions were mutagenic in both strains. 

Mutagenicity was reduced by S9 in TA100 but not in TA98. Activity 

ranking in both strains was HE>NE>NT. 

Reference: 

Sharma et al. 

(2007, 156975) 

Species: S 

typhimurium 

Strain: TA98, 

YG1041, YG5161 

Cell Line: 

Human A549 lung 

epithelial cells 

PM (airborne, 4 sites: an oven 

hall and receiving hall in a 

waste incineration plant; 

heavy-traffic street; 

background; Mar-June 2005) 

Particle Size: 2.5 μm

(diameter) 

Route: Cell Culture 

Dose/Concentration: 0.25 mg/ml 

Time to Analysis: Samples taken over 7-16 

days. A549 cells incubated 24 h. Comet and 

microsuspension assays performed. 

DNA damage: Samples from all four sites induced DNA damage 

in the comet assay with the street samples more damaging than 

the oven hall sample. 

Mutations: Microsuspension assay was used to assess 

mutagenic activity. No mutagenic activity was observed for any of 

the non-polar fractions from any sample sites. The moderately 

polar fractions were all mutagenic, except for the oven hall 

sample, only when S9 was added. Comparatively, the polar and 

crude fractions were mutagenic without metabolic activation, 

suggesting a direct mutagenic effect. 

Reference: Song 

et al. (2007, 

155306) 

Species: S. 

typhimurium 

Strain: TA98, 

TA100 

Cell Line: Rat

fibrocytes L-929 

cells

PM (soluble organic fraction 

(SOF) extracts from diesel 

engines using fuels blended 

with ethanol by volume: E0 - 

base diesel fuel; E5 - 5%; E10 

- 10%; E15 - 15%; E20 - 20%)

Particle Size: Density 

(g/cm3

): E0- 0.8379; E5- 

0.8349; E10- 0.8324; E15- 

0.8301; E20- 0.8279 

Route: Cell Culture 

Dose/Concentration: Ames Assay: 0.025, 

0.05, 0.1 mg/plate; Comet Assay: 0.125, 

0.25, 0.5, 1.0 mg/mL 

Time to Analysis: Samples extracted 24 h. 

Ames and comet assays performed

All PM extracts induced higher mutational response in TA98 (3- to 

5-fold increase over spontaneous) than in TA100 (2-to 3-fold 

increase). The highest brake specific revertants (BSR) ±S9 in both 

strains occurred with E20 and lowest BSR was in E5 (except in 

TA98 -S9). E0 and E20 caused more significant DNA damage 

(similar in effect) than the other extracts. Damage was dosedependent but variable with increasing ethanol volume. 

December 2009 D-165 
Reference Pollutant Exposure Effects 

Reference: 

Zhang et al. 

(2007, 157186) 

Species: S. 

typhimurium 

Strain: TA98, 

TA100 

Cell Line: A549 

Gasoline engine exhaust 

(GEE) 

Methanol engine exhaust 

(MEE) 

Particle Size: NR 

Route: Cell Culture 

Dose/Concentration: MTT Assay- 0.05-0.8 

GEE or MEE L/ml; MN Assay- 0.025, 0.05, 

0.1, 0.2 GEE or MEE L/ml; Comet Assay0.025, 0.05, 0.1, 0.2, 0.4 GEE or MEE L/ml; 

Ames Assay- GEE: 0.625, 1.25, 2.5, 5.0, 10, 

20 L/plate; MEE: 0.3125, 0.625, 1.25, 2.5, 

5.0, 10, 20 L/plate 

Time to Analysis: Organic extracts from 

GEE and MEE. MTT assay- 24 h incubation, 

followed by 2 or 24 h incubation, followed by 

4 h incubation. MN assay- 24 h incubation. 

Comet assay. Ames assay- 72 h incubation. 

Mutagenicity: GEE was mutagenic in TA98 but not TA100, -S9 at 

10 and 20 L/plate and +S9 at ≥1.25 L/plate. Mutagenicity was 

higher with S9 than without at 0.625-10 L/plate and a doseresponse was reported. MEE had no effect in either strain. 

MN: GEE significantly and dose-dependently induced MN. MEE 

had no significant effect at any dose. 

DNA damage: GEE significantly induced DNA damage at all 

doses compared to controls. MEE had no effect at any dose. 

Reference: Zhao 

et al. (2004, 

100972) 

Species: Rat 

Gender: Male 

Strain: SD 

Age: NR 

Weight: ~200 g 

Cell Line: S. 

typhimurium 

YG1024 (±S9)

DEP (SRM 2975) 

DEPE (SRM 1975) 

Carbon black (CB) (Elftex-12 

furnace black, Cabot, Boston, 

MA) 

Particle Size: NR 

Route: IT Instilled. Cell Culture. 

Dose/Concentration: DEP or CB: 35mg/kg; 

S9: 25, 50, 100, 200 μg/plate; Cytosolic 

protein: 20, 40, 80, 160 μg/plate; Microsomal 

protein: 5, 10, 20, 40 μg/plate 

Time to Analysis: Rats instilled. Sacrificed 

1, 3, 7 days post-exposure. S9, cytosolic, 

microsomal fractions prepared from lung 

homogenates. Ames assay: 72 h incubation. 

DEP and CB-exposed lung S9 time-dependently decreased 2-

aminoanthracene (2-AA) mutagenicity. Metyrapone and αnapthoflavone inhibited the S9-activation of 2-AA in DEP and CB 

exposed rats. Lung S9 increased the mutagenicity of DEPE but 

not of DEP or CB. Liver S9 reduced DEPE dose-dependently. 

CYP2B1 and CYP1A1 activated DEPE, with CYP2B1 being more 

effective. 

Reference: Zhao 

et al. (2006, 

100996) 

Species: S. 

typhimuriam 

Strain: YGL024 

(±S9) 

DEP (SRM 2975) 

DEPE (SRM 1975) 

Aminoguanidine (AG) 

Particle Size: NR 

Route: Cell Culture 

Dose/Concentration: NR 

Time to Analysis: Lung S9 obtained from 

rats used in in vivo experiment. Ames test. 

Modified microsuspension assay. All assays 

in duplicate plates. Repeated 3x.

AG significantly lowered 2-aminoanthracene mutagenic activity of 

DEP or DEPE-exposed lung samples, with DEP being lowered the 

most. 

Table D-8. Mutagenicity and genotoxicity data summary: In vitro and in vivo. 

Reference Particle Exposure Effects 

Reference: Abou 

Chakra et al. (2007, 

098819)

Species: Human 

Gender: Male, 

Female 

Age: 6-13 yr and 

Adults 

Participant 

Characteristics: 

Non-smokers 

Cell Line: HeLa S3 

cells 

PM (3 French metropolitan cities: Urban 

PM2.5 and PM10 from “Residential 

Sector,” “Proximity Sector," “Industrial 

Sector”) 

(organic extracts)

Particle Size: 2.5, 10 μm (diameter) 

Route: Cell Culture 

Dose/Concentration: 200 μL organic 

extract; 20 μL aphidicoline 

Time to Analysis: 24 h 

Seasonal variation was observed with genotoxic 

effects being greater in winter. PM2.5 was more 

active than PM10 extracts. Samples from the 

“Proximity Sector” (downtown area with heavy traffic) 

exhibited the strongest genotoxic responses. 

Reference: Arrieta et 

al. (2003, 096210) 

Species: Rat 

Cell Line: Hepatoma 

(H4IIE) 

Species: Mouse 

Cell Line: Hepatoma 

H1l1.1c2 

PM (El Paso, Texas; Juarez, 

Chihuahua, Mexico; Sunland Park, New 

Mexico) (organic extracts) 

Particle Size: 10 μm (diameter) 

Route: Cell Culture 

Dose/Concentration: EROD test: 0.03, 

0.17, 0.34, 0.50, 0.68, 4.96, 9.93 extract 

equivalents (m3

 air); Luciferase: 0.17, 0.51, 

1.26, 5.01 extract equivalents (m3

 air) 

Time to Analysis: 24 h 

EROD activity declined at higher extract amounts, 

but luciferase activity was not inhibited. Cytotoxicity 

occurred only at extract equivalents to 0.47 m3

 air. 

PAH concentration increased with PM mass.

December 2009 D-166 
Reference Particle Exposure Effects 

Reference: Bao et al. 

(2007, 097258) 

Cell Line: Humanhamster hybrid (AL)

DEP (organic extracts) (SRM 2975) 

Particle Size: NR 

Route: Cell Culture 

Dose/Concentration: 10, 20, 50, 100 μg/mL

Time to Analysis: Phagocytosis inhibitors: 

Exposed 24 h with or without cytochalasin B 

or ammonium chloride. Cytotoxicity: 24, 48 h 

incubation. Mutations: Exposed 24 h. 5-7 

days culture. Incubated additional 7-8 days. 

The nucleus of DEP-treated cells was condensed 

and shrunken compared to controls. DEPs 

accumulated in cells, disrupting the mitochondrial 

cristae, and were lodged in large cytoplasmic 

vacuoles. DEP produced minimal toxicity. CD59 

locus mutations dose-dependently increased but 

decreased when simultaneously treated with 

cytochalasin B or ammonium chloride.

Reference: CarvalhoOliveria et al. (2005, 

077898) 

Species: T. pallida; A. 

cepa

PM (Sao Paulo, Brazil; spring, bus strike 

and non-strike days) (organic extracts) 

Particle Size: 2.5 μm (diameter) 

Route: Cell Culture 

Dose/Concentration: Strike day: 47.32 

μg/m3; Non-strike day: 43.01 μg/m3 

Time to Analysis: 8 h. 24 h recovery. A. 

cepa roots induced 5 days. Exposed 30 h. 

Fixed 24 h.

Element concentrations, sulfur and BTEX decreased 

on the strike day. Micronuclei decreased in T. pallida 

during the strike. Toxicity measured in A. cepa was 

not significant, but higher on strike days. 

Reference: Dybdahl 

et al. (2004, 089013) 

Species: Human 

Cell Line: A549

DEP (SRM 1650) 

Particle Size: NR 

Route: Cell Culture 

Dose/Concentration: 10, 50, 100, 500 μg 

DEP/mL 

Time to Analysis: 2, 5, 24 h incubation.

DEP induced dose-dependent increases of IL-1α, IL6, IL-8, TNF-α. The cytokines increased 4-18-fold at 

the highest dose. Cell viability did not decrease. 

Comet tail length increased at 100 and 500 μg/mL 

for 2, 5, 24 h. 

Reference: Gabelova 

et al. (2007, 156458) 

Species: Human 

Cell Line: Hepatoma 

Hep G2

PM (PRG-SM, PRG-LB, Košice, Sofia; 

winter, summer) (organic extracts) 

Particle Size: 10 μm (diameter) 

Route: Cell Culture 

Dose/Concentration: 5-150 μg/mL 

Time to Analysis: 2, 24, 48 h 

Cell viability significantly decreased in the 24, 48 h 

exposure groups compared to the 2 h exposure 

group. DNA migration significantly dose-dependently 

increased at most concentrations. In general, 

oxidative DNA damage did not significantly increase.

Reference: Gabelova 

et al. (2007, 156457) 

Species: Human 

Cell Line: Hepatoma 

Hep G2 cell line

PM10 (Prague (Czech Republic), Koˇsice 

(Slovak Republic) and Sofia (Bulgaria); 

urban, winter, summer) 

(organic extracts) 

Particle Size: 10 μm (diameter) 

Route: Cell Culture 

Dose/Concentration: 5 -150 μg/ml 

Time to Analysis: 24 h DNA adduct 

formation. 2 h Comet assay. Oxidative DNA 

damage measured by Fpg-sensitive sites.

Total DNA adducts ranged from ~60 to 200 adducts 

per 108 nucleotides. Extracts also produced 

approximately the same levels of strand breaks. 

Results suggested that the genotoxic potential of 

ambient air was at least 6-fold greater in the winter 

compared to summer. No substantial difference was 

reported for oxidative DNA damage induced by 

summer vs. winter samples. 

Reference: Gong et 

al. (2007, 091155) 

Species: Human 

Cell Line:

Microvascular 

endothelial (HMEC)

DEP (aggregates, exhaust 4JB1-type 

LD,274 1,4-cylinder Isuzu diesel engine, 

10 torque load, cyclone impactor, 

dilution tunnel constant volume sampler)

Particle Size: <1 μm (diameter) 

Route: Cell Culture 

Dose/Concentration: 5, 15, 25 μg/mL 

Time to Analysis: Cells treated with DEP, 

ox-PAPC (oxidized 1-palmitoyl-2-

arachidonyl-sn-glycero-3-

phosphorylchlorine), DEP+ox-PAPC 

HO-1 expression was dose-dependent and greatest 

with the DEP+ox-PAPC treatment. DEP significantly 

dose-dependently upregulated or downregulated a 

number of genes and was shown to have a 

synergistic effect with co-treatment of ox-PAPC. The 

most varying genes were significantly enriched for 

EpRE, inflammatory response, UPR, immune 

response, cell adhesion, lipid metabolism, apoptosis 

and protein folding genes. 

Reference:

Greenwell et al. 

(2003, 097478) 

Species: Rat 

Cell Line: Epithelial 

fluid; icosahedral 

bacteriophage 

φX174-RF DNA 

PM (South Wales, UK) (urban, 

industrial) 

Particle Size: Coarse diameter: 10-2.5 

μm, Fine diameter: 2.5-0.1 μm 

Route: Cell Culture 

Dose/Concentration: Urban mean: 18.7 ± 

4.7 mg/day; Industrial mean: 22.6 ± 2.5 

mg/day 

Time to Analysis: 24 h air samples 4-11 

days. Substrates vortexed 1 h, suspended 

4 h, centrifuged 1 h. Oxidation assay.

Industrial PM was more bioreactive than urban PM. 

Coarse fractions had greater oxidative potential and 

bioreactivity than fine fractions. 

Reference: Gu et al. 

(2005, 195923) 

Species: Hamster 

Strain: Chinese 

Cell Line: Lung 

fibroblast (V79)

DPM (1980 model General Motors 5.7-L 

V-8 engine) 

Particle Size: NR 

Route: Cell Culture 

Dose/Concentration: 25, 50, 100, 150 

μg/mL; 10 μg DPM in 10 μg in DPPC/mL; 10 

μg DPM in 10 μg DMSO/mL 

Time to Analysis: Chromosomal aberration: 

24 h incubation. Treated 24 h. Incubated 

again 24 h. MN assay: 24 h treatment. Gene 

mutation: 24 h treatment. Cells replated. 7 

days expression times. Staining at 8, 10 

days.

DPM significantly and dose-dependently increased 

aberrant cells at 25-100 μg/mL. DPM increased MN 

formation dose-dependently. Mutant frequencies 

were not significant and showed no dose-dependent 

trends. DPM was toxic to cells at the highest 

concentration. 

December 2009 D-167 
Reference Particle Exposure Effects 

Reference: Gualtieri 

et al. (2005, 097841) 

Species: Human 

Cell Line: A549

TD (Tire debris, generated by rotating 

new vehicle wheel against a steel brush, 

significant component of PM10) (organic 

extracts) 

Particle Size: 10-80 μm (diameter) 

Route: Cell Culture 

Dose/Concentration: 50, 60, 75 μg/mL 

Time to Analysis: Particles extracted 6 h. 

Cells subcultured every 3-4 days. After 24 h, 

TD treatments 24, 48, 72 h.

A time- and dose-dependent inhibitory effect on the 

reduction of MTT was seen. Mortality increased 

dose-dependently and was significantly greater than 

the controls. DNA strand breaks increased 

significantly in a dose-dependent manner. A 

significant cell cycle block in the G1 phase with a 

consequent decrease in the cell number in the S and 

G2/M phases was seen. Exposed cells had a 

modified morphology. 

Reference: 

Gutierrez-Castillo et 

al. (2006, 089030) 

Species: Human 

Cell Line: A549 

PM2.5 and PM10 (4 monitoring stations in 

Mexico City: (1) downtown high auto 

traffic, (2) two industrial areas with high 

levels of auto traffic and low vegetation, 

(3) medium-traffic residential area) 

(winter, spring , 4 sampling days in each 

period) 

(aqueous and organic extracts) 

Particle Size: 2.5 or 10 μm (diameter) 

Route: Cell Culture 

Dose/Concentration: 0.05, 0.07, 0.1m3/ml 

equivalents PM2.5; 0.82, 1.25, 1.63m3

/ml 

equivalents PM10

Time to Analysis: 48 h 

Higher amounts of water-soluble metals were found 

in samples collected during winter. Water-soluble 

extracts increased DNA damage 1.7-fold over the 

background. Similar results were observed with 

organic extracts. In general, PM2.5 extracts had 

greater genotoxic potential than PM10 extracts, and 

water soluble fractions form both particle sizes were 

more genotoxic than the corresponding organic 

extracts. 

Reference: Izawa H 

et al. (2007, 190387) 

Cell Line: NA 

DEPE (4JB-1 Isuzu 4-cylinder directinjection 2740cc diesel engine; 1500 

rpm, 10 kg/m load) 

Particle Size: NR 

Route: Cell Culture 

Dose/Concentration: DEP: Ah-1 

experiment- 111, 55.5, 27.8, 13.9, 6.9, 3.5, 

1.7 μg/mL; Foods, polyphenols experiment27.8 μg/mL 

Time to Analysis: DEPE incubated 2 h for 

dioxin toxicity measurement. Absorbance at 

405 nm measured. Food, polyphenol 

inhibitory effects: food extract or polyphenol 

solution added to cytosol solution, shaken 5 

min. DEPE added, shaken 5 min. 2 h 

incubation. Absorbance at 405 nm 

measured. 

The dioxin toxicity equivalent was 6,479 ± 58 ng 

DEQ/g of DEP. The absorbance showed a sigmoid 

curve and dose-dependently increased from 6.9 to 

27.8 μg DEP/mL. The Ginkgo biloba extract 

significantly inhibited AhR activation significantly 

more than the other foods, and was followed by 

green tea, onions, and garlic. Quercetin and 

myricetin dose-dependently inhibited AhR activation. 

Ginkgolides A and B had weak inhibitory effects and 

resveratol was the weakest. 

Reference: Jacobsen 

et al. (2008, 156597) 

Species: Mouse 

Cell Line: FE1-

MutaTM lung 

epithelial cells 

DEP (SRM 1650b) 

Carbon black (CB) (Printex 90) 

Particle Size: DEP: 18-30 nm; CB: 14 

nm; Agglomerates in suspensions: DEP 

Peaks- 249 nm, CB Peaks- 476 nm 

Route: Cell Culture 

Dose/Concentration: 37.5, 75 μg/mL 

Time to Analysis: 8 repeated 72 h 

incubations. 

Mutagenicity: The 75 μg/mL dose was significantly 

increased compared to the 37.5 μg/mL dose. Linear 

regression showed a significant increasing trend by 

increasing exposure. There was no change in the 

total cell numbers. 

ROS: ROS production increased in DEP-treated 

cells after 3 h of exposure. CB-treated cells showed 

a dose-dependent increase. 

Reference: Karlsson 

et al. (2004, 198976) 

Species: Human 

Cell Line:

Fibroblasts; calf 

thymus DNA with 

human liver 

microsomes or rat 

liver S9 

PM (urban dust particles, SRM 1649) 

(extracted with DCM, acetone, DMSO, 

water) (Fe 3% w/w, Ti 0.32% w/w, V 

0.04% w/w, Mn 0.03% w/w, Cu 0.025% 

w/w) 

Particle Size: <10 μm (mean diameter) 

Route: Cell Culture 

Dose/Concentration: 0.1, 1.0, 10, 100 

μg/cm2

Time to Analysis: Fibroblasts exposed 

24 h. Comet assay. Calf thymus incubated 2 

h with microsomes or S9. 32P-labelled.

DNA damage increased dose-dependently, and a 

significant amount of DNA-damaged cells had 

particle interactions. DNA damage induced by the 

insoluble particle core significantly increased after 

each extraction. Native particles were more 

genotoxic than those extracted with DMSO, DCM 

and water, but not with acetone or hexane. DMSO 

extracts had the most adduct-forming PACs, and 

water extracts had the most oxidizing substances. 

Reference: Karlsson 

et al. (2005, 086392) 

Species: Human 

Cell Line: A549 

PM (subway station, urban street) 

Subway particles: O2, Fe (Fe from 

Fe3O4) Street particles: Fe from Fe2O3

Particle Size: 10 μm (diameter) 

Route: Cell Culture 

Dose/Concentration: Comet: 5, 10, 20, 40 

μg/cm2; 8-oxodG: 10 μg/cm2

Time to Analysis: 4 h. 

Both PM types induced concentration-dependent 

DNA damage, but subway particles were more 

potent. Subway particles caused more 8-oxodG 

formation and oxidation of dG, the latter of which 

was inhibited by deferoxaminemesylate. Oxidation 

from subway particles was due to nonsoluble, redox 

active substances, and soluble substances from 

street particles.

Reference: Karlsson 

et al. (2006, 156625) 

Species: Human 

Cell Line: A549; 

monocytes from 

heparinized whole 

blood 

PM (wood- old, modern boiler; pelletspellets burner, electrical ignition; tireroad simulator studded, friction tires; 

Street- busy street, Stockholm; Subwayplatform near street) 

Particle Size: 2.5, 10 μm (diameter) 

Route: Cell Culture 

Dose/Concentration: 40 μg/cm2 

Time to Analysis: Cells grown 24 h. Comet 

assay. Monocytes incubated 10 days. 

Macrophages incubated 18 h.

All particles tested caused DNA damage, but there 

was no significant difference between the size 

fractions. Subway particles were the most genotoxic. 

The urban street particles were the most potent 

inducers of the cytokines. On the Teflon filters, PM10

was somewhat more potent than PM2.5.

December 2009 D-168 
Reference Particle Exposure Effects 

Reference: Kubátová 

et al. (2004, 087986) 

Species: Monkey 

Cell Line: African 

green kidney COS-1 

(CV-1 cells with 

origin-defective SV40 

mutants) (±S9)

PM (DE from diesel bus, wood smoke 

(WS) from chimney, hardwood smoke) 

(organic extracts) 

Particle Size: NR 

Route: Cell Culture 

Dose/Concentration: 25, 50, 100, 200 

μg/mL; 50mg of each material used for all 

experiments 

Time to Analysis: 24 h cytotoxicity. 2 h SOS 

chromotest. 

WS had significantly increased cytotoxicity in 

fractions of 25-250°C, and DE in nonpolar fractions 

of 250 and 300°C and polar fractions of 50°C. The 

cytotoxicity of DE PM nonpolar fractions 

corresponded to increased concentrations of PAHs. 

WS was not genotoxic and DE was genotoxic in 

midpolarity fractions (50-250°C). Genotoxic 

response was not increased after S9 activation. 

Reference: Landvik 

et al. (2007, 096722) 

Species: Mouse 

Cell Line: Hepatoma 

Hepa1c1c7 cells 

DEP extracts (DEPE in the paper) 

Particle Size: NR 

Route: Cell Culture 

Dose/Concentration: 10, 20, 30, 50, 70 

μg/mL 

Time to Analysis: 24 h 

50 and 70 μg/mL DEPE did not induce DNA 

fragmentation but did cleave caspase 3 to a minor 

extent. 

Reference: Mehta et 

al. (2008, 190440) 

Species: Human 

Cell Line: A549

PM (SRM 1949a) 

Particle Size: ≤ 0.18 μm (diameter) 

Route: Cell Culture 

Dose/Concentration: 0, 50, 100, 200, 400 

μg/mL 

Time to Analysis: Cell culture and cell 

viability assay: PM treatment 24 h. 10 days 

incubation. Host cell reactivation assay: 

pGL3-luciferase plasmid UV irradiated 20 

min. PM treatment 24 h. 16 h transfection. 

24 h PM incubation. DNA repair synthesis 

assay: PM treatment 24 h. Proteinase K 

treatment 30 min. supf mutagenesis assay: 

PM treatment 24 h. PM culture 60 h. DNA 

extracted. Overnight incubation of 

transformed bacteria. 

PM reduced colony-forming ability and repair 

synthesis capacity was proportional to the PM 

concentration. PM dose-dependently decreased 

HCR capacity and decreased more than TSP. PM 

induced cyclobutane dimmers and pyrimidine<6-

4>pyrimidones mutations in UV-irradiated supf. 

Reference: Meng 

and Zhang (2007, 

198963) 

Species: Rat 

Gender: Male 

Strain: Wistar Kyoto 

Age: NR 

Weight: Mean: 230g; 

Range: 200-250g 

Cell Line: AMs from 

treated rats

PM (Baotou, Wuwei, China) (normal 

weather, dust storms, Mar 1-31) 

(organic extracts, water soluble 

fractions) 

Particle Size: 2.5 μm 

Route: Cell Culture 

Dose/Concentration: AM: 0, 33.3, 100, 300 

μg/mL; Water-soluble: 0, 75, 150, 300 

μg/mL; Organic extracts: 0, 25, 50, 100 

μg/mL; Mass concentration normal day: 

68.49 ± 28.83 μg/m3

; Mass concentration 

dust storm day: 221.83 ± 69.89 μg/m3

 

Time to Analysis: 24 h; cultures 4 h. 

OC, NH4

+

, NO3

-

 were higher in normal weather 

PM2.5. SO4

2-, Ca2+ were higher in dust storm PM2.5. 

Fe, Al, Ca, Mg were 5x higher in dust storm PM2.5. 

Cell viability reduced in a concentration-dependent 

manner, with normal weather being slightly more 

cytotoxic. DNA damage was dose-dependently 

induced, with normal weather and organic extracts 

showing the greatest damage. 

Reference: Motta et 

al. (2004, 198953) 

Species: Hamster 

Strain: Chinese 

Cell Line: Epithelial 

liver, ovary 

PM (Catania, Sicily; spring) (organic 

extracts) 

Particle Size: NR 

Route: Cell Culture 

Dose/Concentration: 0.60, 1.21, 2.42, 4.85, 

9.70, 19.40 μg/mL; 0.78, 1.56, 2.12, 6.25, 

12.50, 25.00 μg/mL 

Time to Analysis: 24 h 

The treatment was only slightly cytotoxic at the 

highest dose. DNA damage and aberrant cells 

generally increased with dose. No effect was seen in 

the Chinese hamster ovary cells without metabolic 

activation. 

Reference: Oh and 

Chung (2006, 

088296) 

Cell Line: A549 

(Comet), CHO-K1 

(CBMN), H4IIE 

(EROD-microbiassay) 

Crude extract (CE) DEP and fractions of 

CE of DEP (organic extracts: F1 - 

organic bases, F2 - organic acids, F3 - 

aliphatic, F4 - aromatic, F5 - slightly 

polar, F6 --moderately polar, F7 - high 

polar) 

Particle Size: Diameter: <2.5 μm, 

87.71%, 2.5-10 μm, 3.87%, >10 μm, 

8.42% 

Route: Cell Culture 

Dose/Concentration: 100 μg/mL

Time to Analysis: DEP generated, 

extracted. Comet assay- 24 h incubation, 

CE, DEP exposed 24 h. MN assay- cultured 

24 h, 4 h treatment, growth medium 

incubation 20 h. EROD-microbioassay- 48 h.

DNA damage: CE significantly increased the 

amount of DNA damage in A549 cells with and 

without SKF-525A, a CYP450 inhibitor, and in CHOK1 cells. It significantly increased MN formation ±S9 

compared to controls. 

Organic Extracts: Organic base (F1) and neutral 

(F3-F7) fractions of CE of DEP significantly induced 

DNA damage without SKF-525A compared to 

controls. Adding SKF-525A completely inhibited 

damage caused by F3, F4, F6 and F7 but kept the 

effect of F1 similar to that without SKF and only 

partially inhibited that of F5. F2 did not induce DNA 

damage with or without SKF. All fractions except F6 

induced MN formation ±S9.

December 2009 D-169 
Reference Particle Exposure Effects 

Reference: Poma et 

al. (2006, 096903) 

Species: Mouse 

Cell Line: RAW 264.7 

PM (L’Aquila, Italy; urban); air samples 

collected weekly basis Jan-Mar 2004. 

Carbon black (CB) 

Particle Size: 2.1-0.43 μm (diameter) 

Route: Cell Culture 

Dose/Concentration: 1, 3, 10 μg/cm2

Time to Analysis: Cells cultured 48 h. 

Treatment 48 h. MN assay: 44 h incubation, 

28 h incubation. 

PM and CB dose-dependently reduced cell 

proliferation and induced micronuclei. PM and CB 

also reduced cellular metabolism of the 

macrophages and induced significant amounts of 

apoptosis. PM produced more micronuclei than 

equally-weighted CB. 

Reference: Roubicek 

et al. (2007, 156929) 

Species: Human 

Cell Line: A549 

PM (Mexico City from an industrial area 

with high-traffic and a medium-traffic 

residential area) 

(aqueous or organic extracts) 

Particle Size: 10 μm (diameter) 

Route: Cell Culture 

Dose/Concentration: 1.25, 1.63, 2.5 m3/ml 

equivalents of PM10

Time to Analysis: Cells treated 24 h 

followed by 48 h incubation with 

cytochalasin B. Micronuclei frequency 

determined. 

Water and organic extracts induced a significant 

dose-dependent increase in the micronuclei 

frequency. After doses of PM from different regions 

were normalized for mass differences, the genotoxic 

potency was higher for samples from the industrial 

area. 

Reference: Salonen 

et al. (2004, 187053) 

Species: Mouse 

Cell Line: RAW 264.7 

PM (Vallila, Finland; busy traffic site; 

spring, winter) 

Particle Size: <10 μm (diameter) 

Route: Cell Culture 

Dose/Concentration: 15, 50, 150, 500, 

1000 μg/mL of RPMI 

Time to Analysis: 24 h

PAHs decreased from winter to spring. TNF-α dosedependently increased and was higher in spring 

samples. IL-6 generally increased in spring but not in 

winter. NO dose-dependently increased and was 

higher in winter. Cell viability generally decreased 

but there were no consistent potency differences 

between the samples. Generally, proinflammatory 

activity, cytotoxicity and IL-6 were associated with 

the insoluble PM fractions. Polymyxin B inhibited IL6 and TNF-α. ·OH and 8-hydroxy-2’-deoxyguanosine 

dose-dependently increased and were higher in the 

spring and winter, respectively. 

Reference: Seaton et 

al. (2005, 198904) 

Species: Human 

Cell Line: A549

PM (3 busy London underground (LU) 

stations and cabs) (LU dust in PM2.5

samples: iron oxide 64-71%, chromium 

0.1-0.2%, manganese 0.5-1%, copper 

<0.1-0.9%; respirable dust samples: 1-

2%) 

Particle Size: Diameter: <2.5 μm, 10 

μm, Median diameter: 0.4 μm 

Route: Cell Culture 

Dose/Concentration: Assays: 1, 10, 50, 

100 μg/mL 

Time to Analysis: 8, 24 h. 

PM10 caused less LDH release, IL-8 stimulation and 

free radical activity than LU dust particles that 

contained PM2.5. Chelation had little effect on PM10

soluble components.

Reference: 

Sevastyanova et al. 

(2007, 156969) 

Species: Human 

Cell Line: HepG2 cell 

line, embryonic lung 

diploid fibroblasts 

(HEL), or acute 

monocytic leukemia 

cells (THP-1)

PM10 (Prague, Czech Republic; Koˇsice; 

Slovak Republic; Sofia, Bulgaria) 

(urban, summer, winter) 

(organic extracts) 

Particle Size: 10 μm (diameter) 

Route: Cell Culture 

Dose/Concentration: 10-100 μg/ml 

Time to Analysis: 24 h 

DNA adducts were observed in all cell types 

evaluated. Highest adduct levels were observed in 

HepG2 cells, followed by HEL and THP-1 cells. A 

correlation between DNA adduct levels and 

carcinogenic PAH content was observed in HepG2 

cells at 50 μg/ml.

Reference: Shi et al. 

(2003, 088248) 

Species: Human 

Cell Line: A549 

PM (Düsseldorf, Germany, July-Dec.) 

Weekly samplings July-Dec 1999. 

Particle Size: Fine diameter: <2.5 μm; 

Coarse diameter: 10-2.5 μm 

Route: Cell Culture 

Dose/Concentration: Fine: 0.57-2.49 mg; 

Coarse: 0.66-1.89 mg; Concentration: 0.57 

mg/mL 

Time to Analysis: NR

Coarse and fine particles generated ·OH, but coarse 

particles had significantly higher ·OH formation as 

well as 8-hydroxy-2’-deoxyguanosine formation. 8-

hydroxy-2’-deoxyguanosine and ·OH had a 

significant correlation. 

Reference: Skarek et 

al. (2007, 096814) 

Species: Rat 

Cell Line: Modified 

hepatoma H4IIE.luc; 

SOS: E. coli PQ37 

(±S9) 

PM (urban: Ústí and Laben, Karviná; 

background: Červenohorské sedlo, 

Košetice - Czech Republic; July) 

(organic extracts, TSP); GP (gas 

phase). 24 h samples July 2002 

Particle Size: <2.5 μm (diameter) 

Route: Cell Culture 

Dose/Concentration: SOS: 8, 4, 2, 1 m3/ml; 

Dioxin: TSP+GP- 8, 1.33, 0.22, 0.04 m3/ml, 

PM2.5+GP: 4, 0.66, 0.11, 0.02 m3

 ml-1 

Time to Analysis:. SOS chromotest: 22 h 

incubation. Dioxin toxicity test: 24 h 

exposure.

The urban areas had a much greater level of 

carcinogenic PAHs and overall number of PAHs than 

the background areas. Significant genotoxic activity 

was only detected at TSP+GP without S9 from urban 

areas. PM2.5+GP had lower dioxin activity at the 

urban areas, but similar levels of toxicity were seen 

for both treatments in the background areas. 

Reference: Song et 

al. (2007, 155306) 

Species: S. 

typhimurium 

Strain: TA98, TA100 

Cell Line: Rat

fibrocytes L-929 cells 

PM (soluble organic fraction (SOF) 

extracts from diesel engines using fuels 

blended with ethanol by volume: E0 - 

base diesel fuel; E5 - 5%; E10 - 10%; 

E15 - 15%; E20 - 20%)

Particle Size: Density (g/cm3): E0- 

0.8379; E5- 0.8349; E10- 0.8324; E15- 

0.8301; E20- 0.8279 

Route: Cell Culture 

Dose/Concentration: Ames Assay: 0.025, 

0.05, 0.1 mg/plate; Comet Assay: 0.125, 

0.25, 0.5, 1.0 mg/mL 

Time to Analysis: 24 h 

All PM extracts induced higher mutational response 

in TA98 (3- to 5-fold increase over spontaneous) 

than in TA100 (2-to 3-fold increase). The highest 

brake specific revertants (BSR) ±S9 in both strains 

occurred with E20 and lowest BSR was in E5 

(except in TA98 -S9). E0 and E20 caused more 

significant DNA damage (similar in effect) than the 

other extracts. Damage was dose-dependent but 

variable with increasing ethanol volume. 

December 2009 D-170 
Reference Particle Exposure Effects 

Reference: Ueng et 

al. (2005, 097054) 

Species: Human 

Cell Line: Lung 

epithelium CL5 

(cancerous), BEAS2B, WI-38 normal 

lung fibroblast

MEP (Yamaha cabin motorcycle 2-strok 

50-cc engine) 

Particle Size: NR 

Route: Cell Culture 

Dose/Concentration: 1, 10, 100, 200 μg/mL

Time to Analysis: microarray analysis. RTPCR: 2 h. ELISA: 12 h incubation. 

Centrifuged 24 h post-treatment. Bioactivity: 

12 h incubation. Centrifuged 24 h posttreatment. Medium replaced 48 h postincubation. Fibroblasts determined 96 h 

post-incubation. Time response studies: 3-

48 h treatment. Concentration response 

studies: 6 h treatment. 

Drug Metabolism Array Study: MEP increased 

CYP1A1, CYP3A7 and UGT2B. 

Cytokine Array Study: MEP increased fibroblast 

growth factor (FGF)-6, FGF-9, IL-1α, IL-22 and 

vascular endothelial growth factor (VEGF)-D mRNA. 

Oncogene, Tumor Suppressor, Estrogen 

Signaling Pathway: MEP increased fra-1, c-src, 

SHC, p21, COX7RP, and decreased p53 and Rb 

expression. 

RT-PCR: MEP increased CYP1A1, CYP1B1, IL-6, 

IL-11, IL-1α, FGF-6, FGF-9, VEGF-D, fra-1 and p21. 

Concentration and Time Responses:

Concentration and time-dependent increases 

occurred for FGF-9, IL-1α, IL-6, IL-11, but decreased 

time-dependently after 6 h exposure. 

BEAS-2B Cells: MEP had concentration-dependent 

increases on CYP1A1 and CYP1B1 but did not 

affect anything else. 

Peroxide, MEP+NAC, WI-38 Cells: MEP increased 

peroxide production. The MEP+NAC treatment 

reduced MEP-elevated levels of IL-1α, IL-6, FGF-9, 

VEGF-D to control levels. Fibroblasts increased in 

WI-38 cells. 

Reference: 

Umbuzeiro et al. 

(2008, 190491) 

Species: Salmonella 

typhimurium 

Strain: TA98, 

YG1041 (+/- S9) 

PM (urban; São Paulo, Brazil- Cerqueira 

César street station, Ibirapuera park 

station) (winter- June 17, 18; average 

temperature: 16oC) (EOM) 

Particle Size: NR 

Route: Cell Culture 

Dose/Concentration: Cerqueira César: 

UPM- 156 μg/m3

, EOM- 57.7 mg/total UPM; 

Ibirapuera Park: UPM- 32 μg/m3

, EOM- 41.7 

mg/total UPM; Salmonella assay- 0.5, 1, 5, 

10, 50, 100 UPM equiv/plate (μg) 

Time to Analysis: Organic extraction 20 h. 

PAH fractionation. 

The TSP and EOM were similar for both sites. The 

PAH fraction had very low mutagenicity for the 

Cerqueira César sample in the YG1041 strain and 

no mutagenicity for the Ibirapuera sample. Nitro-PAH 

and oxy-PAH had similar mutagenetic activities from 

both samples. S9 decreased mutagenicity in nitroPAH but was increased in oxy-PAH. DNA adduct 

levels were dose-dependent and not different 

between the two sites. 

Reference:

Upadhyay et al. 

(2003, 097370) 

Species: Human 

Cell Line A549

PM (Dusseldorf, Germany) (Particles 

contain carbon (19.70%), 

hydrogen(1.4%),nitrogen (<.05%), 

oxygen(14.12%), sulfur (2.09%), ash 

(63.24%)) (Ionizable metals 

concentrations (ppm): Co(103), 

Cu(48),Cr(104),Fe(14,521), Mn(21.3), 

Ni(1,519),Ti(131), V(2,767) 

Particle Size: NR 

Route: Cell Culture 

Dose/Concentration: 1, 5, 25, 100 μg/cm2; 

10, 25, 50, 100 μg/cm2

Time to Analysis: 1, 4, 8, 12, 24 h.

PM induced dose- and time-dependent reductions in 

ds-DNA due to the formation of DNA-SB. The 

soluble component caused higher DNA damage. 

Apoptosis and DNA fragmentation increased dosedependently. ΔΨm decreased dose-dependently in 

control cells, but not in cells with Bcl-xl 

overexpression. PM caused activation of caspase 9. 

Pretreatment with iron chelators or a free radical 

scavenger reduced PM-induced DNA-SB formation, 

DNA fragmentation, caspase 9 activation, and 

weakened ΔΨm reductions. 

Reference:

Valavanidis et al. 

(2005, 096432) 

Cell Line: NR

PM (TSP: high volume pumps, Athens; 

DEP: 2.0L engine GM Astra; GEP: 1.6L 

passenger vehicle Ford; Wood smoke 

soot: domestic fireplace exhaust 

chimney; PM10: high volume sampling 

system, Athens; PM2.5: high volume 

cascade impactor (Anderson) system 

Particle Size: >10.2 - <0.41 μm 

(diameter) 

Route: Incubation 

Dose/Concentration: 20, 40 mg/5mL 

Time to Analysis: PM incubated with H2O2

and 2’-deoxyguanosine (dG). Stored 3-7 

days at -20°C. 

PM generated ·OH by a Fenton reaction, which is 

increased by the addition of EDTA but inhibited by 

deferoxamine. PM dose-dependently induced dG 

hydroxylation and 8-hydroxy-2’-deoxyguanosine 

formation. Transition metals Ni, V, Co, Cr that are 

capable of redox cycling electron producing ROS 

were found in the PM samples. 

Reference: Xu and 

Zhang (2004, 

097231) 

Species: Human 

Cell Line: A549 

PM (Taiyuan, Beijing; Nov-Feb) 

(Taiyuan: coal-fume pollution; Beijing: 

coal-fume and vehicle exhaust) 

Particle Size: 2.5 μm (diameter) 

Route: Cell Culture 

Dose/Concentration: 5, 50, 200 μg/mL 

Time to Analysis: 12-24 h

Taiyuan had a significantly higher daily PM2.5

average than Beijing. It was shown that the smaller 

the particulate diameter, the higher the concentration 

of BaP and Pb. A dose- and time-response 

relationship was seen in DNA fragmentation. 

December 2009 D-171 
Annex D References 

Aam BB; Fonnum F (2007). ROS scavenging effects of organic extract of diesel exhaust particles on human neutrophil 

granulocytes and rat alveolar macrophages. Toxicology, 230: 207-18. 155123

Abou Chakra OR; Joyeux M; Nerriere E; Strub MP; Zmirou-Navier D (2007). Genotoxicity of organic extracts of urban 

airborne particulate matter: an assessment within a personal exposure study. Chemosphere, 66: 1375-81. 098819

Adamson IYR; Vincent R; Bakowska J (2003). Differential production of metalloproteinases after instilling various urban 

air particle samples to rat lung. Exp Lung Res, 29: 375-388. 087943

Agopyan N; Head J; Yu S; Simon SA (2004). TRPV1 receptors mediate particulate matter-induced apoptosis. Am J Physiol 

Lung Cell Mol Physiol, 286: 563-572. 156198

Agopyan N; Li L; Yu S; Simon SA (2003). Negatively charged 2- and 10-"mu"m particles activate vanilloid receptors, 

increase cAMP, and induce cytokine release. Toxicol Appl Pharmacol, 186: 63-76. 056065

Ahn E-K; Yoon H-K; Jee Bo K; Ko H-J; Lee K-H; Kim Hyung J; Lim Y (2008). COX-2 expression and inflammatory 

effects by diesel exhaust particles in vitro and in vivo. Toxicol Lett, 176: 178-187. 156199

Ahsan MK; Nakamura H; Tanito M; Yamada K; Utsumi H; Yodoi J (2005). Thioredoxin-1 suppresses lung injury and 

apoptosis induced by diesel exhaust particles (DEP) by scavenging reactive oxygen species and by inhibiting DEPinduced downregulation of Akt. Free Radic Biol Med, 39: 1549-1559. 156200

Alfaro-Moreno E; Martinez L; Garcia-Cuellar C; Bonner JC; Murray JC; Rosas I; Rosales SP; Osornio-Vargas AR (2002). 

Biologic effects induced in vitro by PM10 from three different zones of Mexico City. Environ Health Perspect, 110: 

715-720. 156204

Amakawa K; Terashima T; Matsuzaki T; Matsumaru A; Sagai M; Yamaguchi K (2003). Suppressive effects of diesel 

exhaust particles on cytokine release from human and murine alveolar macrophages. Exp Lung Res, 29: 149-164. 

156211

Amara N; Bachoual R; Desmard M; Golda S; Guichard C; Lanone S; Aubier M; Ogier-Denis E; Boczkowski J (2007). 

Diesel exhaust particles induce matrix metalloprotease-1 in human lung epithelial cells via a NADP(H) 

oxidase/NOX4 redox-dependent mechanism. Am J Physiol Lung Cell Mol Physiol, 293: L170-181. 156212

Andre E; Stoeger T; Takenaka S; Bahnweg M; Ritter B; Karg E; Lentner B; Reinhard C; Schulz H; Wjst M (2006). 

Inhalation of ultrafine carbon particles triggers biphasic pro-inflammatory response in the mouse lung. Eur Respir J, 

28: 275-285. 091376

Anselme F; Loriot S; Henry J-P; Dionnet F; Napoleoni J-G; Thnillez C; Morin J-P (2007). Inhalation of diluted diesel 

engine emission impacts heart rate variability and arrhythmia occurrence in a rat model of chronic ischemic heart 

failure. Arch Toxicol, 81: 299-307. 097084

Anseth JW; Goffin AJ; Fuller GG; Ghio AJ; Kao PN; Upadhyay D (2005). Lung surfactant gelation induced by epithelial 

cells exposed to air pollution or oxidative stress. Am J Respir Cell Mol Biol, 33: 161-168. 088646

Antonini JM; Taylor MD; Leonard SS; Lawryk NJ; Shi X; Clarke RW; Roberts JR (2004). Metal composition and 

solubility determine lung toxicity induced by residual oil fly ash collected from different sites within a power plant. 

Mol Cell Biochem, 255: 257-265. 097199

Apicella C; Custidiano A; Miranda S; Novoa L; Dokmetjian J; Gentile T (2006). Differential macrophage modulation of 

asymmetric IgG antibody synthesis by soluble or particulate stimuli. Immunol Lett, 103: 177-185. 096586

Arantes-Costa F; Lopes F; Toledo A; Magliarelli-Filho P; Moriya H; Carvalho-Oliveira R; Mauad T; Saldiva P; Martins M 

(2008). Effects of residual oil fly ash (ROFA) in mice with chronic allergic pulmonary inflammation. Toxicol 

Pathol, 36: 680-686. 187137

 

Note: Hyperlinks to the reference citations throughout this document will take you to the NCEA HERO database (Health and 

Environmental Research Online) at http://epa.gov/hero. HERO is a database of scientific literature used by U.S. EPA in the process of 

developing science assessments such as the Integrated Science Assessments (ISA) and the Integrated Risk Information System (IRIS). 

December 2009 D-172 
Archer AJ; Cramton JL; Pfau JC; Colasurdo G; Holian A (2004). Airway responsiveness after acute exposure to urban 

particulate matter 1648 in a DO1110 murine model. Am J Physiol, 286: L337-L343. 088097

Arimoto T; Takano H; Inoue K; Yanagisawa R; Yoshino S; Yamaki K; Yoshikawa T (2007). Pulmonary exposure to diesel 

exhaust particle components enhances circulatory chemokines during lung inflammation. Int J Immunopathol 

Pharmacol, 20: 197-201. 097973

Arrieta DE; Ontiveros CC; Li W-W; Garcia JH; Denison MS; McDonald JD; Burchiel SW; Washburn BS (2003). Aryl 

hydrocarbon receptor-mediated activity of particulate organic matter from the Paso del Norte airshed along the USMexico border. Environ Health Perspect, 111: 1299-1305. 096210

Auger F; Gendron MC; Chamot C; Marano F; Dazy AC (2006). Responses of well-differentiated nasal epithelial cells 

exposed to particles: role of the epithelium in airway inflammation. Toxicol Appl Pharmacol, 215: 285-294. 156235

Bachoual R; Boczkowski J; Goven D; Amara N; Tabet L; On D; Lecon-Malas V; Aubier M; Lanone S (2007). Biological 

effects of particles from the Paris subway system. Chem Res Toxicol, 20: 1426-1433. 155667

Bagate K; Meiring JJ; Cassee FR; Borm PJA (2004). The effect of particulate matter on resistance and conductance vessels 

in the rat. Inhal Toxicol, 16: 431-436. 055638

Bagate K; Meiring JJ; Gerlofs-Nijland ME; Cassee FR; Borm PJA (2006). Signal transduction pathways involved in 

particulate matter induced relaxation in rat aorta--spontaneous hypertensive versus Wistar Kyoto rats. Toxicol In 

Vitro, 20: 52-62. 097608

Bagate K; Meiring JJ; Gerlofs-Nijland ME; Cassee FR; Wiegand H; Osornio-Vargas A; Borm PJA (2006). Ambient 

particulate matter affects cardiac recovery in a Langendorff ischemia model. Inhal Toxicol, 18: 633-643. 096157

Bao L; Chen S; Wu L; Hei Tom K; Wu Y; Yu Z; Xu A (2007). Mutagenicity of diesel exhaust particles mediated by cellparticle interaction in mammalian cells. Toxicol Sci, 229: 91-100. 097258

Barrett EG; Henson RD; Seilkop SK; McDonald JD; Reed MD (2006). Effects of hardwood smoke exposure on allergic 

airway inflammation in mice. Inhal Toxicol, 18: 33-43. 155677

Bartoli CR; Wellenius GA; Coull BA; Akiyama I; Diaz EA; Lawrence J; Okabe K; Verrier RL; Godleski JJ (2009). 

Concentrated ambient particles alter myocardial blood flow during acute ischemia in conscious canines. Environ 

Health Perspect, 117: 333-337. 179904

Bartoli CR; Wellenius GA; Diaz EA; Lawrence J; Coull BA; Akiyama I; Lee LM; Okabe K; Verrier RL; Godleski JJ 

(2009). Mechanisms of Inhaled Fine Particulate Air Pollution-Induced Arterial Blood Pressure Changes. Environ 

Health Perspect, 117: 361-366. 156256

Batalha JR; Saldiva P H; Clarke RW; Coull BA; Stearns RC; Lawrence J; Murthy GG; Koutrakis P; Godleski JJ (2002). 

Concentrated ambient air particles induce vasoconstriction of small pulmonary arteries in rats. Environ Health 

Perspect, 110: 1191-1197. 088109

Baulig A; Blanchet S; Rumelhard M; Lacroix G; Marano F; Baeza-Squiban A (2007). Fine urban atmospheric particulate 

matter modulates inflammatory gene and protein expression in human bronchial epithelial cells. Front Biosci, 12: 

771-82. 151733

Bayram H; Ito K; Issa R; Ito M; Sukkar M; Chung KF (2006). Regulation of human lung epithelial cell numbers by diesel 

exhaust particles. Eur Respir J, 27: 705-713. 088439

Becher R; Bucht A; Ovrevik J; Hongslo JK; Dahlman HJ; Samuelsen JT; Schwarze PE (2007). Involvement of NADPH 

oxidase and iNOS in rodent pulmonary cytokine responses to urban air and mineral particles. Inhal Toxicol, 19: 

645-655. 097125

Beck-Speier I; Dayal N; Karg E; Maier KL; Schumann G; Schulz H; Semmler M; Takenaka S; Stettmaier K; Bors W; Ghio 

A; Samet JM; Heyder J (2005). Oxidative stress and lipid mediators induced in alveolar macrophages by ultrafine 

particles. Free Radic Biol Med, 38: 1080-1092. 156262

Becker S; Dailey LA; Soukup JM; Grambow SC; Devlin RB; Huang YC (2005). Seasonal variations in air pollution 

particle-induced inflammatory mediator release and oxidative stress. Environ Health Perspect, 113: 1032-1038. 

088592

Becker S; Mundandhara S; Devlin RB; Madden M (2005). Regulation of cytokine production in human alveolar 

macrophages and airway epithelial cells in response to ambient air pollution particles: further mechanistic studies. 

Toxicol Appl Pharmacol, 207: 269-275. 088590

December 2009 D-173 
Bhattacharyya SN; Dubick MA; Yantis LD; Enriquez JI; Buchanan KC; Batra SK; Smiley RA (2004). In vivo effect of 

wood smoke on the expression of two mucin genes in rat airways. Inflammation, 28: 67-76. 088095

Binkova B; Chvatalova I; Lnenickova Z; Milcova A; Tulupova E; Farmer PB; Sram RJ (2007). PAH-DNA adducts in 

environmentally exposed population in relation to metabolic and DNA repair gene polymorphisms. Mutat Res Fund 

Mol Mech Mutagen, 620: 49-61. 156273

Bitterle E; Karg E; Schroeppel A; Kreyling WG; Tippe A; Ferron GA; Schmid O; Heyder J; Maier KL; Hofer T (2006). 

Dose-controlled exposure of A549 epithelial cells at the air-liquid interface to airborne ultrafine carbonaceous 

particles. Chemosphere, 65: 1784-1790. 156276

Blanchet S; Ramgolam K; Baulig A; Marano F; Baeza-Squiban A (2004). Fine particulate matter induces amphiregulin 

secretion by bronchial epithelial cells. Am J Respir Cell Mol Biol, 30: 421-427. 087982

Bonvallot V; Baeza-Squiban A; Baulig A; Brulant S; Boland S; Muzeau F; Barouki R; Marano F (2001). Organic 

compounds from diesel exhaust particles elicit a proinflammatory response in human airway epithelial cells and 

induce cytochrome p450 1A1 expression. Am J Respir Cell Mol Biol, 25: 515-521. 156283

Brits E; Schoeters G; Verschaeve L (2004). Genotoxicity of PM10 and extracted organics collected in an industrial, urban, 

and rural area in Flanders, Belgium. Environ Res, 96: 109-118. 087397

Brown DM; Hutchison L; Donaldson K; Stone V (2007). The effects of PM10 particles and oxidative stress on 

macrophages and lung epithelial cells: modulating effects of calcium-signaling antagonists. Am J Physiol Lung Cell 

Mol Physiol, 292: 1444-1451. 156300

Brown LE; Trought KR; Bailey CI; Clemons JH (2005). 2,3,7,8-TCDD equivalence and mutagenic activity associated with 

PM10 from three urban locations in New Zealand. Sci Total Environ, 349: 161-174. 095919

Bunger J; Krahl J; Munack A; Ruschel Y; Schroder O; Emmert B; Westphal G; Muller M; Hallier E; Bruning T (2007). 

Strong mutagenic effects of diesel engine emissions using vegetable oil as fuel. Arch Toxicol, 81: 599-603. 156305

Bunger J; Krahl J; Weigel A; Schroder O; Bruning T; Muller M; Hallier E; Westphal G (2006). Influence of fuel properties, 

nitrogen oxides, and exhaust treatment by an oxidation catalytic converter on the mutagenicity of diesel engine 

emissions. Arch Toxicol, 80: 540-546. 156303

Burchiel SW; Lauer FT; Dunaway SL; Zawadzki J; McDonald JD; Reed MD (2005). Hardwood smoke alters murine 

splenic T cell responses to mitogens following a 6-month whole body inhalation exposure. Toxicol Appl Pharmacol, 

202: 229-236. 088090

Burchiel SW; Lauer FT; McDonald JD; Reed MD (2004). Systemic immunotoxicity in AJ mice following 6-month whole 

body inhalation exposure to diesel exhaust. Toxicol Appl Pharmacol, 196: 337-345. 055557

Calcabrini A; Meschini S; Marra M; Falzano L; Colone M; De Berardis B; Paoletti L; Arancia G; Fiorentini C (2004). Fine 

environmental particulate engenders alterations in human lung epithelial A549 cells. Environ Res, 95: 82-91. 

096865

Calderón-Garcidueñas L; Maronpot RR; Torres-Jardon R; Henriquez-Roldan C; Schoonhoven R; Acuna-Ayala H; 

Villarreal-Calderon A; Nakamura J; Fernando R; Reed W; Azzarelli B; Swenberg JA (2003). DNA damage in nasal 

and brain tissues of canines exposed to air pollutants is associated with evidence of chronic brain inflammation and 

neurodegeneration. Toxicol Pathol, 31: 524-538. 156316

Campbell A; Oldham M; Becaria A; Bondy SC; Meacher D; Sioutas C; Misra C; Mendez LB; Kleinman M (2005). 

Particulate matter in polluted air may increase biomarkers of inflammation in mouse brain. Neurotoxicology, 26: 

133-140. 087217

Campen MJ; Babu NS; Helms GA; Pett S; Wernly J; Mehran R; McDonald JD (2005). Nonparticulate components of 

diesel exhaust promote constriction in coronary arteries from ApoE -/- mice. Toxicol Sci, 88: 95-102. 083977

Campen MJ; McDonald JD; Gigliotti AP; Seilkop SK; Reed MD; Benson JM (2003). Cardiovascular effects of inhaled 

diesel exhaust in spontaneously hypertensive rats. Cardiovasc Toxicol, 3: 353-361. 055626

Campen MJ; McDonald JD; Reed MD; Seagrave J (2006). Fresh gasoline emissions, not paved road dust, alter cardiac 

repolarization in ApoE-/- mice. Cardiovasc Toxicol, 6: 199-210. 096879

Cao D; Tal TL; Graves LM; Gilmour I; Linak W; Reed W; Bromberg PA; Samet JM (2007). Diesel exhaust particulateinduced activation of Stat3 requires activities of EGFR and Src in airway epithelial cells. Am J Physiol Lung Cell 

Mol Physiol, 292: L422-L429. 156322

December 2009 D-174 
Cao Q; Zhang S; Dong C; Song W (2007). Pulmonary responses to fine particles: Differences between the spontaneously 

hypertensive rats and wistar kyoto rats. Toxicol Lett, 171: 126-137. 097491

Carter JM; Corson N; Driscoll KE; Elder A; Finkelstein JN; Harkema JN; Gelein R; Wade-Mercer P; Nguyen K; 

Oberdorster G (2006). A comparative dose-related response of several key pro- and antiinflammatory mediators in 

the lungs of rats, mice, and hamsters after subchronic inhalation of carbon black. J Occup Environ Med, 48: 1265-

1278. 095936

Carvalho-Oliveira R; Pozo RMK; Lobo DJA; Lichtenfels AJFC; Martins-Junior HA; Bustilho JOWV; Saiki M; Sato IM; 

Saldiva PHN (2005). Diesel emissions significantly influence composition and mutagenicity of ambient particles: a 

case study in Sao Paulo, Brazil. Environ Res, 98: 1-7. 077898

Cassee FR; Boere AJF; Fokkens PHB; Leseman DLAC; Sioutas C; Kooter IM; Dormans JAMA (2005). Inhalation of 

concentrated particulate matter produces pulmonary inflammation and systemic biological effects in compromised 

rats. J Toxicol Environ Health A Curr Iss, 68: 773-796. 087962

Chan RC-F; Wang M; Li N; Yanagawa Y; Onoe K; Lee JJ; Nel AE (2006). Pro-oxidative diesel exhaust particle chemicals 

inhibit LPS-induced dendritic cell responses involved in T-helper differentiation. J Allergy Clin Immunol, 118: 455-

465. 097468

Chang C-C; Chen S-H; Ho S-H; Yang C-Y; Wang H-D; Tsai M-L (2007). Proteomic analysis of proteins from 

bronchoalveolar lavage fluid reveals the action mechanism of ultrafine carbon black-induced lung injury in mice. 

Proteomics, 7: 4388-4397. 097475

Chang C-C; Chiu H-F; Wu Y-S; Li Y-C; Tsai M-L; Shen C-K; Yang C-Y (2005). The induction of vascular endothelial 

growth factor by ultrafine carbon black contributes to the increase of alveolar-capillary permeability. Environ 

Health Perspect, 113: 454-460. 097776

Chang C-C; Hwang J-S; Chan C-C; Cheng T-J (2007). Interaction effects of ultrafine carbon black with iron and nickel on 

heart rate variability in spontaneously hypertensive rats. Environ Health Perspect, 115: 1012-1017. 155720

Chang C-C; Hwang J-S; Chan C-C; Wang P-Y; Hu T-H; Cheng T-J (2004). Effects of concentrated ambient particles on 

heart rate, blood pressure, and cardiac contractility in spontaneously hypertensive rats. Inhal Toxicol, 16: 421-429. 

055637

Chauhan V; Breznan D; Goegan P; Nadeau D; Karthikeyan S; Brook JR; Vincent R (2004). Effects of ambient air particles 

on nitric oxide production in macrophage cell lines. Cell Biol Toxicol, 20: 221-239. 096682

Chauhan V; Breznan D; Thomson E; Karthikeyan S; Vincent R (2005). Effects of ambient air particles on the endothelin 

system in human pulmonary epithelial cells (A549). Cell Biol Toxicol, 21: 191-205. 155722

Che W; Zhang Z; Zhang H; Wu M; Liang Y; Liu F; Shu Y; Li N (2007). Compositions and oxidative damage of 

condensate, particulate and semivolatile organic compounds from gasoline exhausts. Environ Toxicol Pharmacol, 

24: 11-18. 096460

Chen LC; Hwang JS (2005). Effects of subchronic exposures to concentrated ambient particles (CAPs) in mice IV 

Characterization of acute and chronic effects of ambient air fine particulate matter exposures on heart-rate 

variability. Inhal Toxicol, 17: 209-216. 087218

Chen LC; Nadziejko C (2005). Effects of subchronic exposures to concentrated ambient particles (CAPs) in mice V CAPs 

exacerbate aortic plaque development in hyperlipidemic mice. Inhal Toxicol, 17: 217-224. 087219

Cheng MD; Malone B; Storey JM (2003). Monitoring cellular responses of engine-emitted particles by using a direct aircell interface deposition technique. Chemosphere, 53: 237-243. 156337

Chin BY; Trush MA; Choi AM; Risby TH (2003). Transcriptional regulation of the HO-1 gene in cultured macrophages 

exposed to model airborne particulate matter. Am J Physiol Lung Cell Mol Physiol, 284: L473-480. 156340

Cho H-Y; Jedlicka AE; Clarke R; Kleeberger SR (2005). Role of Toll-like receptor-4 in genetic susceptibility to lung injury 

induced by residual oil fly ash. Physiol Genomics, 22: 108-117. 156344

Churg A; Brauer M; del Carmen Avila-Casado M; Fortoul TI; Wright JL (2003). Chronic exposure to high levels of 

particulate air pollution and small airway remodeling. Environ Health Perspect, 111: 714-718. 087899

Churg A; Xie C; Wang X; Vincent R; Wang RD (2005). Air pollution particles activate NF-kappaB on contact with airway 

epithelial cell surfaces. Toxicol Appl Pharmacol, 208: 37-45. 088281

December 2009 D-175 
Ciencewicki J; Gowdy K; Krantz QT; Linak WP; Brighton L; Gilmour MI; Jaspers I (2007). Diesel exhaust enhanced 

susceptibility to influenza infection is associated with decreased surfactant protein expression. Inhal Toxicol, 19: 

1121-1133. 096557

Corey LM; Baker C; Luchtel Daniel L (2006). Heart-rate variability in the apolipoprotein E knockout transgenic mouse 

following exposure to Seattle particulate matter. J Toxicol Environ Health A Curr Iss, 69: 953-965. 156366

Costa DL; Lehmann JR; Winsett D; Richards J; Ledbetter AD; Dreher KL (2006). Comparative pulmonary toxicological 

assessment of oil combustion particles following inhalation or instillation exposure. Toxicol Sci, 91: 237-246. 

088438

Courtois A; Andujar P; Ladeiro Y; Baudrimont I; Delannoy E; Leblais V; Begueret H; Galland MAB; Brochard P; Marano 

F (2008). Impairment of NO-Dependent Relaxation in Intralobar Pulmonary Arteries: Comparison of Urban 

Particulate Matter and Manufactured Nanoparticles. Environ Health Perspect, 116: 1294-1299. 156369

Cozzi E; Hazarika S; Stallings HW; Cascio WE; Devlin RB; Lust RM; Wingard CJ; Van Scott MR (2006). Ultrafine 

particulate matter exposure augments ischemia-reperfusion injury in mice. Am J Physiol, 291: H894-H903. 091380

Dagher Z; Garcon G; Billet S; Verdin A; Ledoux F; Courcot D; Aboukais A; Shirali P (2007). Role of nuclear factor-kappa 

B activation in the adverse effects induced by air pollution particulate matter (PM25) in human epithelial lung cells 

(L132) in culture. J Appl Toxicol, 27: 284-290. 097566

Dai J; Xie C; Vincent R; Churg A (2003). Air pollution particles produce airway wall remodeling in rat tracheal explants. 

Am J Respir Cell Mol Biol, 29: 352-358. 087944

Day KC; Reed MD; McDonald JD; Keilkop SK; Barrett EG (2008). Effects of gasoline engine emissions on preexisting 

allergic airway responses in mice. Inhal Toxicol, 20: 1145-1155. 190204

DeMarini DM; Brooks LR; Warren SH; Kobayashi T; Gilmour MI; Singh P (2004). Bioassay-directed fractionation and 

Salmonella mutagenicity of automobile and forklift diesel exhaust particles. Environ Health Perspect, 112: 814-819. 

066329

de Haar C; Hassing I; Bol M; Bleumink R; Pieters R (2005). Ultrafine carbon black particles cause early airway 

inflammation and have adjuvant activity in a mouse allergic airway disease model. Toxicol Sci, 87: 409-418. 

097872

de Haar C; Hassing I; Bol M; Bleumink R; Pieters R (2006). Ultrafine but not fine particulate matter causes airway 

inflammation and allergic airway sensitization to co-administered antigen in mice. Clin Exp Allergy, 36: 1469-

1479. 144746

de Haar C; Kool M; Hassing I; Bol M; Lambrecht BN; Pieters R (2008). Lung dendritic cells are stimulated by ultrafine 

particles and play a key role in particle adjuvant activity. J Allergy Clin Immunol, 121: 1246-1254. 187128

De Kok TM; Hogervorst JG; Briede JJ; Van Herwijnen MH; Maas LM; Moonen EJ; Driece HA; Kleinjans JC (2005). 

Genotoxicity and physicochemical characteristics of traffic-related ambient particulate matter. Environ Mol 

Mutagen, 46: 71-80. 088656

Dick CAJ; Brown DM; Donaldson K; Stone V (2003). The role of free radicals in the toxic and inflammatory effects of 

four different ultrafine particle types. Inhal Toxicol, 15: 39-52. 036605

Doherty SP; Prophete C; Maciejczyk P; Salnikow K; Gould T; Larson T; Koenig J; Jaques P; Sioutas C; Zelikoff JT; 

Lippmann M; Cohen MD (2007). Detection of changes in alveolar macrophage iron status induced by select PM25-

associated components using iron-response protein binding activity. Inhal Toxicol, 19: 553-562. 096532

Dong CC; Yin XJ; Ma JYC; Millecchia L; Barger MW; Roberts JR; Zhang X-D; Antonini JM; Ma JKH (2005). Exposure 

of Brown Norway Rats to diesel exhaust particles Prior to ovalbumin (OVA) sensitization elicits IgE adjuvant 

activity but attenuates OVA-induced airway inflammation. Toxicol Sci, 88: 150-160. 088079

Dong CC; Yin XJ; Ma JYC; Millecchia L; Wu Z-X; Barger MW; Roberts JR; Antonini JM; Dey RD; Ma JKH (2005). 

Effect of diesel exhaust particles on allergic reactions and airway responsiveness in ovalbumin-sensitized Brown 

Norway Rats. Toxicol Sci, 88: 202-212. 088083

Doornaert B; Leblond V; Galiacy S; Gras G; Planus E; Laurent V; Isabey D; Lafuma C (2003). Negative impact of DEP 

exposure on human airway epithelial cell adhesion, stiffness, and repair. Am J Physiol Lung Cell Mol Physiol, 284: 

L119-L132. 156410

December 2009 D-176 
Dostert C; Petrilli V; Van Bruggen R; Steele C; Mossman BT; Tschopp J (2008). Innate Immune Activation Through Nalp3 

Inflammasome Sensing of Asbestos and Silica. Science, 320: 674-677. 155753

Doyle M; Sexton KG; Jeffries H; Bridge K; Jaspers I (2004). Effects of 1,3-butadiene, isoprene, and their photochemical 

degradation products on human lung cells. Environ Health Perspect, 112: 1488-1495. 088404

Drela N; Zesko I; Jakubowska M; Biernacka M (2006). CD28 in thymocyte development and peripheral T cell activation in 

mice exposed to suspended particulate matter. Toxicol Appl Pharmacol, 215: 179-188. 096352

Duvall RM; Norris GA; Dailey LA; Burke JM; McGee JK; Gilmour MI; Gordon T; Devlin RB (2008). Source 

apportionment of particulate matter in the US and associations with lung inflammatory markers. Inhal Toxicol, 20: 

671-683. 097969

Dvonch JT; Brook RD; Keeler GJ; Rajagopalan S; D'Alecy LG; Marsik FJ; Morishita M; Yip FY; Brook JR; Timm EJ; 

Wagner JG; Harkema JR (2004). Effects of concentrated fine ambient particles on rat plasma levels of asymmetric 

dimethylarginine. Inhal Toxicol, 16: 473-480. 055741

Dybdahl M; Risom L; Bornholdt J; Autrup H; Loft S; Wallin H (2004). Inflammatory and genotoxic effects of diesel 

particles in vitro and in vivo. DNA Repair (Amst), 562: 119-131. 089013

Dybing E; Lovdal T; Hetland RB; Lovik M; Schwarze PE (2004). Respiratory allergy adjuvant and inflammatory effects of 

urban ambient particles. Toxicol Sci, 198: 307-314. 097545

ElAssouli S; AlQahtani M; Milaat W (2007). Genotoxicity of air borne particulates assessed by comet and the samonella 

mutagenicity test in Jeddah, Saudi Arabia. Int J Environ Res Publ Health, 4: 216-223. 186914

Elder A; Gelein R; Finkelstein J; Phipps R; Frampton M; Utell M; Kittelson DB; Watts WF; Hopke P; Jeong CH; Kim E; 

Liu W; Zhao W; Zhuo L; Vincent R; Kumarathasan P; Oberdorster G (2004). On-road exposure to highway 

aerosols. 2. Exposures of aged, compromised rats. Inhal Toxicol, 16 Suppl 1: 41-53. 087354

Elder A; Gelein R; Finkelstein JN; Driscoll KE; Harkema J; Oberdorster G (2005). Effects of subchronically inhaled 

carbon black in three species I Retention kinetics, lung inflammation, and histopathology. Toxicol Sci, 88: 614-629. 

088194

Elder ACP; Gelein R; Azadniv M; Frampton M; Finkelstein J; Oberdorster G (2004). Systemic effects of inhaled ultrafine 

particles in two compromised, aged rat strains. Inhal Toxicol, 16: 461-471. 055642

Endo O; Sugita K; Goto S; Amagai T; Matsushita H (2003). Mutagenicity of size-fractioned airborne particles collected 

with Andersen low pressure impactor. Eisei Kagaku, 49: 22-27. 097260

Erdinger L; Durr M; Hopker KA (2005). Correlations between mutagenic activity of organic extracts of airborne particulate 

matter, NOx and sulphur dioxide in southern Germany: results of a two-year study. Environ Sci Pollut Res Int, 12: 

10-20. 156423

Evans S-A; Al-Mosawi A; Adams RA; Berube KA (2006). Inflammation, edema, and peripheral blood changes in lungcompromised rats after instillation with combustion-derived and manufactured nanoparticles. Exp Lung Res, 32: 

363-378. 097066

Farraj AK; Haykal-Coates N; Ledbetter AD; Evansky PA; Gavett SH (2006). Inhibition of pan neurotrophin receptor p75 

attenuates diesel particulate-induced enhancement of allergic airway responses in C57/B16J mice. Inhal Toxicol, 

18: 483-491. 088469

Farraj AK; Haykal-Coates N; Ledbetter AD; Evansky PA; Gavett SH (2006). Neurotrophin mediation of allergic airways 

responses to inhaled diesel particles in mice. Toxicol Sci, 94: 183-192. 141730

Fedulov AV; Leme A; Yang Z; Dahl M; Lim R; Mariani TJ; Kobzik L (2008). Pulmonary exposure to particles during 

pregnancy causes increased neonatal asthma susceptibility. Am J Respir Cell Mol Biol, 38: 57-67. 097482

Finkelman FD; Yang M; Orekhova T; Clyne E; Bernstein J; Whitekus M; Diaz-Sanchez D; Morris SC (2004). Diesel 

exhaust particles suppress in vivo IFN-gamma production by inhibiting cytokine effects on NK and NKT cells. J 

Immunol, 172: 3808-3813. 096572

Finnerty K; Choi J-E; Lau A; Davis-Gorman G; Diven C; Seaver N; Linak William P; Witten M; McDonagh Paul F (2007). 

Instillation of coarse ash particulate matter and lipopolysaccharide produces a systemic inflammatory response in 

mice. J Toxicol Environ Health A Curr Iss, 70: 1957-1966. 156434

December 2009 D-177 
Floyd HS; Chen LC; Vallanat B; Dreher K (2009). Fine ambient air particulate matter exposure induces molecular 

alterations associated with vascular disease progression within plaques of atherosclerotic susceptible mice. Inhal 

Toxicol, 21: 394-403. 190350

Folkmann JK; Risom L; Hansen CS; Loft S; Moller P (2007). Oxidatively damaged DNA and inflammation in the liver of 

dyslipidemic ApoE-/- mice exposed to diesel exhaust particles. Toxicology, 237: 134-44. 097344

Fritsch S; Diabate S; Krug HF (2006). Incinerator fly ash provokes alteration of redox equilibrium and liberation of 

arachidonic acid in vitro. Biol Chem, 387: 1421-1428. 156452

Fujii T; Hayashi S; Hogg JC; Mukae H; Suwa T; Goto Y; Vincent R; Van Eeden SF (2002). Interaction of alveolar 

macrophages and airway epithelial cells following exposure to particulate matter produces mediators that stimulate 

the bone marrow. Am J Respir Cell Mol Biol, 27: 34-41. 036478

Fujii T; Hayashi S; Hogg JC; Vincent R; Van Eeden SF (2001). Particulate matter induces cytokine expression in human 

bronchial epithelial cells. Am J Respir Cell Mol Biol, 25: 265-271. 156455

Fujimaki H; Kurokawa Y (2004). Diesel exhaust-associated gas components enhance chemokine production by cervical 

lymph-node cells from mice immunized with sugi basic proteins. , 16: 61-65. 096575

Fujimaki H; Kurokawa Y; Yamamoto S; Satoh M (2006). Distinct requirements for interleukin-6 in airway inflammation 

induced by diesel exhaust in mice. Immunopharmacol Immunotoxicol, 28: 703-714. 096601

Fujimaki H; Yamamoto S; Kurokawa Y (2005). Effect of diesel exhaust on immune responses in C57BL/6 mice 

intranasally immunized with pollen antigen. J UOEH, 27: 11-24. 156456

Fujimoto A; Tsukue N; Watanabe M; Sugawara I; Yanagisawa R; Takano H; Yoshida S; Takeda K (2005). Diesel exhaust 

affects immunological action in the placentas of mice. Environ Toxicol, 20: 431-440. 096556

Furuyama A; Hirano S; Koike E; Kobayashi T (2006). Induction of oxidative stress and inhibition of plasminogen activator 

inhibitor-1 production in endothelial cells following exposure to organic extracts of diesel exhaust particles and 

urban fine particles. Arch Toxicol, 80: 154-162. 097056

Gabelová A; Valovicova Z; Bacova G; Labaj J; Binkova B; Topinka J; Sevastyanova O; Sram RJ; Kalina I; Habalova V; 

Popov TA; Panev T; Farmer PB (2007). Sensitivity of different endpoints for in vitro measurement of genotoxicity 

of extractable organic matter associated with ambient airborne particles (PM10). Mutat Res Fund Mol Mech 

Mutagen, 620: 103-113. 156458

Gabelová A; Valovicova Z; Labaj J; Bacova G; Binkova B; Farmer Peter B (2007). Assessment of oxidative DNA damage 

formation by organic complex mixtures from airborne particles PM(10). Mutat Res, 620: 135-144. 156457

Gao F; Barchowsky A; Nemec AA; Fabisiak JP (2004). Microbial stimulation by Mycoplasma fermentans synergistically 

amplifies IL-6 release by human lung fibroblasts in response to residual oil fly ash (ROFA) and nickel. Toxicol Sci, 

81: 467-479. 087950

Garcon G; Dagher Z; Zerimech F; Ledoux F; Courcot D; Aboukais A; Puskaric E; Shirali P (2006). Dunkerque City air 

pollution particulate matter-induced cytotoxicity, oxidative stress and inflammation in human epithelial lung cells 

(L132) in culture. Toxicol In Vitro, 20: 519-528. 096633

Gavett SH; Haykal-Coates N; Copeland L B; Heinrich J; Gilmour MI (2003). Metal composition of ambient PM2.5 

influences severity of allergic airways disease in mice. Environ Health Perspect, 111: 1471-1477. 053153

Geng H; Meng Z; Zhang Q (2005). Effects of blowing sand fine particles on plasma membrane permeability and fluidity, 

and intracellular calcium levels of rat alveolar macrophages. Toxicol Lett, 157: 129-137. 096689

Geng H; Meng Z; Zhang Q (2006). In vitro responses of rat alveolar macrophages to particle suspensions and water-soluble 

components of dust storm PM(2.5). Toxicol In Vitro, 20: 575-584. 097026

Gerlofs-Nijland ME; Rummelhard M; Boere AJF; Leseman DLAC; Duffin R; Schins RPF; Borm PJA; Sillanpaa M; 

Salonen RO; Cassee FR (2009). Particle induced toxicity in relation to transition metal and polycyclic aromatic 

hydrocarbon contents . Environ Sci Technol, 43: 4729-4736. 190353

Gerlofs-Nijland ME; Boere AJ; Leseman DL; Dormans JA; Sandstrom T; Salonen RO; van Bree L; Cassee FR (2005). 

Effects of particulate matter on the pulmonary and vascular system: time course in spontaneously hypertensive rats. 

Part Fibre Toxicol, 2: 2. 088652

December 2009 D-178 
Gerlofs-Nijland ME; Dormans JA; Bloemen HJ; Leseman DL; John A; Boere F; Kelly FJ; Mudway IS; Jimenez AA; 

Donaldson K; Guastadisegni C; Janssen NA; Brunekreef B; Sandstrom T; van Bree L; Cassee FR (2007). Toxicity 

of coarse and fine particulate matter from sites with contrasting traffic profiles. Inhal Toxicol, 19: 1055-1069. 

097840

Ghelfi E; Rhoden CR; Wellenius GA; Lawrence J; Gonzalez-Flecha B (2008). Cardiac oxidative stress and 

electrophysiological changes in rats exposed to concentrated air particles are mediated by TRP-dependent 

pulmonary reflexes. Toxicol Sci, 102: 328-336. 156468

Ghio AJ; Cohen MD (2005). Disruption of iron homeostasis as a mechanism of biologic effect by ambient air pollution 

particles. Inhal Toxicol, 17: 709-716. 088272

Ghio AJ; Piantadosi CA; Wang X; Dailey LA; Stonehuerner JD; Madden MC; Yang F; Dolan KG; Garrick MD; Garrick 

LM (2005). Divalent metal transporter-1 decreases metal-related injury in the lung. Am J Physiol, 289: L460-L467. 

088275

Gilmour MI; McGee J; Duvall Rachelle M; Dailey L; Daniels M; Boykin E; Cho S-H; Doerfler D; Gordon T; Devlin 

Robert B (2007). Comparative toxicity of size-fractionated airborne particulate matter obtained from different cities 

in the United States. Inhal Toxicol, 19 Suppl 1: 7-16. 096433

Gilmour MI; O'Connor S; Dick CAJ; Miller CA; Linak WP (2004). Differential pulmonary inflammation and in vitro 

cytotoxicity of size-fractionated fly ash particles from pulverized coal combustion. J Air Waste Manag Assoc, 54: 

286-295. 057420

Gilmour PS; Morrison ER; Vickers MA; Ford I; Ludlam CA; Greaves M; Donaldson K; MacNee W (2005). The 

procoagulant potential of environmental particles (PM10). Occup Environ Med, 62: 164-171. 087410

Gilmour PS; Rahman I; Donaldson K; MacNee W (2003). Histone acetylation regulates epithelial IL-8 release mediated by 

oxidative stress from environmental particles. Am J Physiol Lung Cell Mol Physiol, 284: L533-L540. 096959

Gilmour PS; Schladweiler MC; Nyska A; McGee JK; Thomas R; Jaskot RH; Schmid J; Kodavanti UP (2006). Systemic 

imbalance of essential metals and cardiac gene expression in rats following acute pulmonary zinc exposure. J 

Toxicol Environ Health A Curr Iss, 69: 2011-2032. 156472

Gilmour PS; Schladweiler MC; Richards JH; Ledbetter AD; Kodavanti UP (2004). Hypertensive rats are susceptible to 

TLR4-mediated signaling following exposure to combustion source particulate matter. Inhal Toxicol, 16 Suppl 1: 5-

18. 087948

Gilmour PS; Ziesenis A; Morrison ER; Vickers MA; Drost EM; Ford I; Karg E; Mossa C; Schroeppel A; Ferron GA; 

Heyder J; Greaves M; MacNee W; Donaldson K (2004). Pulmonary and systemic effects of short-term inhalation 

exposure to ultrafine carbon black particles. Toxicol Appl Pharmacol, 195: 35-44. 054175

Godleski JJ; Clarke RW; Coull BA; Saldiva PHN; Jiang NF; Lawrence J; Koutrakis P (2002). Composition of inhaled 

urban air particles determines acute pulmonary responses. Ann Occup Hyg, 46: 419-424. 156478

Gong KW; Zhao W; Li N; Barajas B; Kleinman M; Sioutas C; Horvath S; Lusis AJ; Nel A; Araujo JA (2007). Air pollutant 

chemicals and oxidized lipids exhibit genome wide synergistic effects on endothelial cells. Genome Biol, 8: R149. 

091155

Goto Y; Hogg JC; Shih CH; Ishii H; Vincent R; van Eeden SF (2004). Exposure to ambient particles accelerates monocyte 

release from bone marrow in atherosclerotic rabbits. Am J Physiol, 287: L79-L85. 088100

Gottipolu RR; Wallenborn JG; Karoly ED; Schladweiler MC; Ledbetter AD; Krantz T; Linak WP; Nyska A; Johnson JA; 

Thomas R; Richards JE; Jaskot RH; Kodavanti UP (2009). One-month diesel exhaust inhalation produces 

hypertensive gene expression pattern in healthy rats. Environ Health Perspect, 117: 38-46. 190360

Gowdy K; Krantz QT; Daniels M; Linak WP; Jaspers I; Gilmour MI (2008). Modulation of pulmonary inflammatory 

responses and antimicrobial defenses in mice exposed to diesel exhaust. Toxicol Appl Pharmacol, 229: 310-319. 

097226

Graff DW; Schmitt MT; Dailey LA; Duvall RM; Karoly ED; Devlin RB (2007). Assessing the role of particulate matter 

size and composition on gene expression in pulmonary cells. Inhal Toxicol, 19 Suppl 1: 23-28. 156488

Greenwell LL; Moreno T; Richards RJ (2003). Pulmonary antioxidants exert differential protective effects against urban 

and industrial particulate matter. J Biosci, 28: 101-107. 097478

December 2009 D-179 
Gu Z-W; Keane MJ; Ong T; Wallac WE (2005). Diesel exhaust particulate matter dispersed in a phospholipid surfactant 

induces chromosomal aberrations and micronuclei but not 6-thioguanine-resistant gene mutation in V79 cells. J 

Toxicol Environ Health A Curr Iss, 68: 431-444. 195923

Gualtieri M; Rigamonti L; Galeotti V; Camatini M (2005). Toxicity of tire debris extracts on human lung cell line A549. 

Toxicol In Vitro, 19: 1001-1008. 097841

Gunnison A; Chen LC (2005). Effects of subchronic exposures to concentrated ambient particles (CAPs) in mice VI Gene 

expression in heart and lung tissue. Inhal Toxicol, 17: 225-233. 087956

Gurgueira SA; Lawrence J; Coull B; Murthy GGK; Gonzalez-Flecha B (2002). Rapid increases in the steady-state 

concentration of reactive oxygen species in the lungs and heart after particulate air pollution inhalation. Environ 

Health Perspect, 110: 749-755. 036535

Gutierrez-Castillo ME; Roubicek DA; Cebrian-Garcia ME; De Vizcaya-Ruiz A; Sordo-Cedeno M; Ostrosky-Wegman P 

(2006). Effect of chemical composition on the induction of DNA damage by urban airborne particulate matter. 

Environ Mol Mutagen, 47: 199-211. 089030

Hamada K; Suzaki Y; Leme A; Ito T; Miyamoto K; Kobzik L; Kimura H (2007). Exposure of pregnant mice to an air 

pollutant aerosol increases asthma susceptibility in offspring. J Toxicol Environ Health A Curr Iss, 70: 688-695. 

091235

Hamoir J; Nemmar A; Halloy D; Wirth D; Vincke G; Vanderplasschen A; Nemery B; Gustin P (2003). Effect of 

polystyrene particles on lung microvascular permeability in isolated perfused rabbit lungs: role of size and surface 

properties. Toxicol Appl Pharmacol, 190: 278-285. 096664

Hansen C; Neller A; Williams G; Simpson R (2007). Low levels of ambient air pollution during pregnancy and fetal 

growth among term neonates in Brisbane, Australia. Environ Res, 103: 383-389. 090703

Hao M; Comier S; Wang M; Lee James J; Nel A (2003). Diesel exhaust particles exert acute effects on airway 

inflammation and function in murine allergen provocation models. J Allergy Clin Immunol, 112: 905-914. 096565

Happo MS; Salonen RO; Halinen AI; Jalava PI; Pennanen AS; Kosma VM; Sillanpaa M; Hillamo R; Brunekreef B; 

Katsouyanni K; Sunyer J; Hirvonen MR (2007). Dose and time dependency of inflammatory responses in the 

mouse lung to urban air coarse, fine, and ultrafine particles from six European cities. Inhal Toxicol, 19: 227-246. 

096630

Harder V; Gilmour P; Lentner B; Karg E; Takenaka S; Ziesenis A; Stampfl A; Kodavanti U; Heyder J; Schulz H (2005). 

Cardiovascular responses in unrestrained WKY rats to inhaled ultrafine carbon particles. Inhal Toxicol, 17: 29-42. 

087371

Harkema JR; Keeler G; Wagner J; Morishita M; Timm E; Hotchkiss J; Marsik F; Dvonch T; Kaminski N; Barr E (2004). 

Effects of concentrated ambient particles on normal and hypersecretory airways in rats. Health Effects Institute. 

Boston, MA. 056842

Harrod KS; Jaramillo RJ; Berger JA; Gigliotti AP; Seilkop SK; Reed MD (2005). Inhaled diesel engine emissions reduce 

bacterial clearance and exacerbate lung disease to Pseudomonas aeruginosa infection in vivo. Toxicol Sci, 83: 155-

165. 088144

Harrod KS; Jaramillo RJ; Rosenberger CL; Wang S-Z; Berger JA; McDonald JD; Reed MD (2003). Increased 

susceptibility to RSV infection by exposure to inhaled diesel engine emissions. Am J Respir Cell Mol Biol, 28: 451-

463. 097046

Heidenfelder BL; Reif DM; Harkema JR; Cohen Hubal EA; Hudgens EE; Bramble LA; Wagner JG; Morishita M; Keeler 

GJ; Edwards SW; Gallagher JE (2009). Comparative microarray analysis and pulmonary changes in brown Norway 

rats exposed to ovalbumin and concentrated air particulates. Toxicol Sci, 108: 207-221. 190026

Hetland RB; Cassee FR; Lag M; Refsnes M; Dybing E; Schwarze PE (2005). Cytokine release from alveolar macrophages 

exposed to ambient particulate matter: heterogeneity in relation to size, city and season. Part Fibre Toxicol, 2: 1-15. 

087887

Hetland RB; Cassee FR; Refsnes M; Schwarze PE; Lag M; Boere AJF; Dybing E (2004). Release of inflammatory 

cytokines, cell toxicity and apoptosis in epithelial lung cells after exposure to ambient air particles of different size 

fractions. Toxicol In Vitro, 18: 203-212. 097535

December 2009 D-180 
Hiramatsu K; Azuma A; Kudoh S; Desaki M; Takizawa H; Sugawara I (2003). Inhalation of diesel exhaust for three 

months affects major cytokine expression and induces bronchus-associated lymphoid tissue formation in murine 

lungs. Exp Lung Res, 29: 607-622. 155846

Hiramatsu K; Saito Y; Sakakibara K; Azuma A; Kudoh S; Takizawa H; Sugawara I (2005). The effects of inhalation of 

diesel exhaust on murine mycobacterial infection. Exp Lung Res, 31: 405-415. 088285

Hirano S; Furuyama A; Koike E; Kobayashi T (2003). Oxidative-stress potency of organic extracts of diesel exhaust and 

urban fine particles in rat heart microvessel endothelial cells. Toxicology, 187: 161-170. 097345

Holder AL; Lucas D; Goth-Goldstein R; Koshland CP (2008). Cellular Response to Diesel Exhaust Particles Strongly 

Depends on the Exposure Method. Toxicol Sci, 103: 108-115. 093322

Hollingsworth JW; Cook DN; Brass DM; Walker JKL; Morgan DL; Foster WM; Schwartz DA (2004). The role of Toll-like 

receptor 4 in environmental airway injury in mice. Am J Respir Crit Care Med, 170: 126-132. 097816

Hougaard KS; Jensen KA; Nordly P; Taxvig C; Vogel U; Saber AT; Wallin H (2008). Effects of prenatal exposure to diesel 

exhaust particles on postnatal development, behavior, genotoxicity and inflammation in mice. Part Fibre Toxicol, 5: 

3. 156570

Huang JY; Liao JW; Liu YC; Lu SY; Chou CP; Chan WH; Chen SU; Ueng TH (2008). Motorcycle exhaust induces 

reproductive toxicity and testicular interleukin-6 in male rats. Toxicol Sci, 103: 137-148. 156574

Huang SL; Hsu MK; Chan CC (2003). Effects of submicrometer particle compositions on cytokine production and lipid 

peroxidation of human bronchial epithelial cells. Environ Health Perspect, 111: 478-482. 087376

Hutchison GR; Brown DM; Hibbs LR; Heal MR; Donaldson K; Maynard RL; Monaghan M; Nicholl A; Stone V (2005). 

The effect of refurbishing a UK steel plant on PM10 metal composition and ability to induce inflammation. Respir 

Res, 6: 43. 097750

Hwang B-F; Lee Y-L; Lin Y-C; Jaakkola JJK; Guo YL (2005). Traffic related air pollution as a determinant of asthma 

among Taiwanese school children. Thorax, 60: 467-473. 089454

Iba MM; Fung J; Chung L; Zhao J; Winnik B; Buckley BT; Chen LC; Zelikoff JT; Kou YR (2006). Differential inducibility 

of rat pulmonary CYP1A1 by cigarette smoke and wood smoke. Mutat Res Fund Mol Mech Mutagen, 606: 1-11. 

156582

Ichinose T; Takano H; Sadakane K; Yanagisawa R; Kawazato H; Sagai M; Shibamoto T (2003). Differences in airwayinflammation development by house dust mite and diesel exhaust inhalation among mouse strains. Toxicol Appl 

Pharmacol, 187: 29-37. 041525

Ichinose T; Takano H; Sadakane K; Yanagisawa R; Yoshikawa T; Sagai M; Shibamoto T (2004). Mouse Strain Differences 

in Eosinophilic Airway Inflammation Caused by Intratracheal Instillation of Mite Allergen and Diesel Exhaust 

Particles. J Appl Toxicol, 24: 69-76. 180367

Imrich A; Ning Y; Lawrence J; Coull B; Gitin E; Knutson M; Kobzik L (2007). Alveolar macrophage cytokine response to 

air pollution particles: oxidant mechanisms. Toxicol Appl Pharmacol, 218: 256-264. 155859

Inoue K-I; Takano H; Yanagisawa R; Hirano S; Ichinose T; Shimada A; Yoshikawa T (2006). The role of toll-like receptor 4 

in airway inflammation induced by diesel exhaust particles. Arch Toxicol, 80: 275-279. 097815

Inoue K-i; Takano H; Yanagisawa R; Hirano S; Kobayashi T; Ichinose T; Yoshikawa T (2007). Effects of organic chemicals 

derived from ambient particulate matter on lung inflammation related to lipopolysaccharide. Arch Toxicol, 80: 833-

838. 096724

Inoue K-I; Takano H; Yanagisawa R; Ichinose T; Sakurai M; Yoshikawa T (2006). Effects of nano particles on cytokine 

expression in murine lung in the absence or presence of allergen. Arch Toxicol, 80: 614-619. 096720

Inoue K-i; Takano H; Yanagisawa R; Ichinose T; Shimada A; Yoshikawa T (2005). Pulmonary exposure to diesel exhaust 

particles induces airway inflammation and cytokine expression in NC/Nga mice. Arch Toxicol, 79: 595-599. 

097481

Inoue K-I; Takano H; Yanagisawa R; Sakurai M; Abe S; Yoshino S; Yamaki K; Yoshikawa T (2007). Effects of components 

derived from diesel exhaust particles on lung physiology related to antigen. Immunopharmacol Immunotoxicol, 29: 

403-412. 096692

December 2009 D-181 
Inoue K-i; Takano H; Yanagisawa R; Sakurai M; Ueki N; Yoshikawa T (2007). Effects of diesel exhaust particles on 

cytokine production by splenocytes stimulated with lipopolysaccharide. J Appl Toxicol, 27: 95-100. 096702

Inoue K; Takano H; Yanagisawa R; Hirano S; Sakurai M; Shimada A; Yoshikawa T (2006). Effects of airway exposure to 

nanoparticles on lung inflammation induced by bacterial endotoxin in mice. Environ Health Perspect, 114: 1325-

1330. 090951

Inoue K; Takano H; Yanagisawa R; Ichinose T; Sadakane K; Yoshino S; Yamaki K; Uchiyama K; Yoshikawa T (2004). 

Components of diesel exhaust particles differentially affect lung expression of cyclooxygenase-2 related to bacterial 

endotoxin. J Appl Toxicol, 24: 415-418. 087984

Inoue K; Takano H; Yanagisawa R; Sakurai M; Abe S; Yoshino S; Yamaki K; Yoshikawa T (2007). Effects of nanoparticles 

on lung physiology in the presence or absence of antigen. Int J Immunopathol Pharmacol, 20: 737-744. 198885

Inoue K; Takano H; Yanagisawa R; Sakurai M; Ichinose T; Sadakane K; Yoshikawa T (2005). Effects of nano particles on 

antigen-related airway inflammation in mice. Respir Res, 6: 106. 088625

Inoue K; Takano H; Yanagisawa R; Sakurai M; Ueki N; Yoshikawa T (2006). Effects of diesel exhaust on lung 

inflammation related to bacterial endotoxin in mice. Basic Clin Pharmacol Toxicol, 99: 346-352. 190142

Ishihara Y; Kagawa J (2003). Chronic diesel exhaust exposures of rats demonstrate concentration and time-dependent 

effects on pulmonary inflammation. Inhal Toxicol, 15: 473-492. 096404

Ishii H; Fujii T; Hogg JC; Hayashi S; Mukae H; Vincent R; van Eeden SF (2004). Contribution of IL-1 beta and TNF-alpha 

to the initiation of the peripheral lung response to atmospheric particulates (PM10). Am J Physiol, 287: L176-L183. 

088103

Ishii H; Hayashi S; Hogg JC; Fujii T; Goto Y; Sakamoto N; Mukae H; Vincent R; van Eeden SF (2005). Alveolar 

macrophage-epithelial cell interaction following exposure to atmospheric particles induces the release of mediators 

involved in monocyte mobilization and recruitment. Respir Res, 6: 87. 096138

Ito K; Christensen WF; Eatough DJ; Henry RC; Kim E; Laden F; Lall R; Larson TV; Neas L; Hopke PK; Thurston GD 

(2006). PM source apportionment and health effects: 2 An investigation of intermethod variability in associations 

between source-apportioned fine particle mass and daily mortality in Washington, DC. J Expo Sci Environ 

Epidemiol, 16: 300-310. 088391

Ito T; Suzuki T; Tamura K; Nezu T; Honda K; Kobayashi T (2008). Examination of mRNA expression in rat hearts and 

lungs for analysis of effects of exposure to concentrated ambient particles on cardiovascular function. Toxicol Sci, 

243: 271-283. 096823

Izawa H; Watanabe G; Taya K; Sagai M (2007). Inhibitory effects of foods and polyphenols on activation of aryl 

hydrocarbon receptor induced by diesel exhaust particles. Environ Sci J Integr Environ Res, 14: 149-156. 190387

Jacobsen NR; Mrller P; Cohn CA; Loft S; Vogel U; Wallin H (2008). Diesel exhaust particles are mutagenic in FE1-

MutaTM Mouse lung epithelial cells. Mutat Res Fund Mol Mech Mutagen, 641: 54-57. 156597

Jalava P; Salonen RO; Halinen AI; Sillanpaa M; Sandell E; Hirvonen MR (2005). Effects of sample preparation on 

chemistry, cytotoxicity, and inflammatory responses induced by air particulate matter. Inhal Toxicol, 17: 107-117. 

088648

Jalava PI; Salonen RO; Halinen AI; Penttinen P; Pennanen AS; Sillanpaa M; Sandell E; Hillamo R; Hirvonen M-R (2006). 

In vitro inflammatory and cytotoxic effects of size-segregated particulate samples collected during long-range 

transport of wildfire smoke to Helsinki. Toxicol Appl Pharmacol, 215: 341-353. 155872

Jalava PI; Salonen RO; Pennanen AS; Sillanpaa M; Halinen AI; Happo MS; Hillamo R; Brunekreef B; Katsouyanni K; 

Sunyer J; Hirvonen M-R (2007). Heterogeneities in inflammatory and cytotoxic responses of RAW 264.7 

macrophage cell line to urban air coarse, fine, and ultrafine particles from six European sampling campaigns. Inhal 

Toxicol, 19: 213-225. 096950

Jang A-S; Choi Inseon S; Takizawa H; Rhim T; Lee J-H; Park S-W; Park C-S (2005). Additive effect of diesel exhaust 

particulates and ozone on airway hyperresponsiveness and inflammation in a mouse model of asthma. J Korean 

Med Sci, 20: 759-763. 155313

Jaspers I; Ciencewicki JM; Zhang W; Brighton LE; Carson JL; Beck MA; Madden MC (2005). Diesel exhaust enhances 

influenza virus infections in respiratory epithelial cells. Toxicol Sci, 85: 990-1002. 088115

December 2009 D-182 
Jimenez LA; Drost EM; Gilmour PS; Rahman I; Antonicelli F; Ritchie H; MacNee W; Donaldson K (2002). PM10-

exposed macrophages stimulate a proinflammatory response in lung epithelial cells via TNF-alpha. Am J Physiol 

Lung Cell Mol Physiol, 282: L237-L248. 156610

Jones HA; Hamacher K; Clark JC; Schofield JB; Krausz T; Haslett C; Boobis AR (2005). Positron emission tomography in 

the quantification of cellular and biochemical responses to intrapulmonary particulates. Toxicol Appl Pharmacol, 

207: 230-236. 198883

Jung H; Guo B; Anastasio C; Kennedy IM (2006). Quantitative measurements of the generation of hydroxyl radicals by 

soot particles in a surrogate lung fluid. Atmos Environ, 40: 1043-1052. 132421

Kaan PM; Hegele RG (2003). Interaction between respiratory syncytial virus and particulate matter in guinea pig alveolar 

macrophages. Am J Respir Cell Mol Biol, 28: 697-704. 095753

Kafoury RM; Madden MC (2005). Diesel exhaust particles induce the over expression of tumor necrosis factor-alpha 

(TNF-alpha) gene in alveolar macrophages and failed to induce apoptosis through activation of nuclear factorkappaB (NF-kappaB). Int J Environ Health Res, 2: 107-113. 156617

Karlsson HL; Ljungman AG; Lindbom J; Moller L (2006). Comparison of genotoxic and inflammatory effects of particles 

generated by wood combustion, a road simulator and collected from street and subway. Toxicol Lett, 165: 203-211. 

156625

Karlsson HL; Nilsson L; Moller L (2005). Subway particles are more genotoxic than street particles and induce oxidative 

stress in cultured human lung cells. Chem Res Toxicol, 18: 19-23. 086392

Karlsson HL; Nygren J; Möller L (2004). Genotoxicity of airborne particulate matter: the role of cell-particle interaction 

and of substances with adduct-forming and oxidizing capacity. Mutat Res, 565: 1-10. 198976

Kato A; Kagawa J (2003). Morphological effects in rat lungs exposed to urban roadside air. Inhal Toxicol, 15: 799-818. 

089563

Kato T; Yashiro T; Murata Y; Herbert DC; Oshikawa K; Bando M; Ohno S; Sugiyama Y (2003). Evidence that exogenous 

substances can be phagocytized by alveolar epithelial cells and transported into blood capillaries. Cell Tissue Res, 

311: 47-51. 198882

Katterman ME; Birchard S; Seraphin S; Riley Mark R (2007). Cellular evaluation of the toxicity of combustion derived 

particulate matter: influence of particle grinding and washing on cellular response. Chemosphere, 66: 567-573. 

096358

Kendall M; Guntern J; Lockyer NP; Jones FH; Hutton BM; Lippmann M; Tetley TD (2004). Urban PM2.5 surface 

chemistry and interactions with bronchoalveolar lavage fluid. Inhal Toxicol, 16 Suppl 1: 115-129. 156634

Khandoga A; Stampfl A; Takenaka S; Schulz H; Radykewicz R; Kreyling W; Krombach F (2004). Ultrafine particles exert 

prothrombotic but not inflammatory effects on the hepatic microcirculation in healthy mice in vivo. Circulation, 

109: 1320-1325. 087928

Kim YM; Reed W; Wu W; Bromberg PA; Graves LM; Samet JM (2005). Zn2+-induced IL-8 expression involves AP-1, 

JNK, and ERK activities in human airway epithelial cells. Am J Physiol, 290: L1028-L1035. 088454

Klein-Patel ME; Diamond G; Boniotto M; Saad S; Ryan LK (2006). Inhibition of beta-defensin gene expression in airway 

epithelial cells by low doses of residual oil fly ash is mediated by vanadium. Toxicol Sci, 92: 115-125. 097092

Kleinman M; Phalen R (2006). Toxicological interactions in the respiratory system after inhalation of ozone and sulfuric 

acid aerosol mixtures. Inhal Toxicol, 18: 295-303. 088596

Kleinman M; Sioutas C; Stram D; Froines J; Cho A; Chakrabarti B; Hamade A; Meacher D; Oldham M (2005). Inhalation 

of concentrated ambient particulate matter near a heavily trafficked road stimulates antigen-induced airway 

responses in mice. J Air Waste Manag Assoc, 55: 1277-1288. 087880

Kleinman MT Sioutas C Chang MC Boere AJ Cassee FR (2003). Ambient fine and coarse particle suppression of alveolar 

macrophage functions. Toxicol Lett, 137: 151-158. 087938

Kleinman MT; Araujo JA; Nel A; Sioutas C; Campbell A; Cong PQ; Li H; Bondy SC (2008). Inhaled ultrafine particulate 

matter affects CNS inflammatory processes and may act via MAP kinase signaling pathways. Toxicol Lett, 178: 

127-130. 190074

December 2009 D-183 
Kleinman MT; Hyde DM; Bufalino C; Basbaum C; Bhalla DK; Mautz WJ (2003). Toxicity of chemical components of fine 

particles inhaled by aged rats: effects of concentration. J Air Waste Manag Assoc, 53: 1080-1087. 053535

Kleinman MT; Sioutas C; Froines JR; Fanning E; Hamade A; Mendez L; Meacher D; Oldham M (2007). Inhalation of 

concentrated ambient particulate matter near a heavily trafficked road stimulates antigen-induced airway responses 

in mice. Inhal Toxicol, 19 Suppl 1: 117-126. 097082

Knuckles TL; Dreher KL (2007). Fine oil combustion particle bioavailable constituents induce molecular profiles of 

oxidative stress, altered function, and cellular injury in cardiomyocytes. J Toxicol Environ Health A Curr Iss, 70: 

1824-1837. 156652

Knuckles TL; Lund AK; Lucas SN; Campen MJ (2008). Diesel exhaust exposure enhances venoconstriction via uncoupling 

of eNOS. Toxicol Appl Pharmacol, 230: 346-351. 191987

Kocbach A; Namork E; Schwarze P (2008). Pro-inflammatory potential of wood smoke and traffic-derived particles in a 

monocytic cell line. Toxicology, 247: 123-132. 198874

Kodavanti UP; Schladweiler MC; Gilmour PS; Wallenborn JG; Mandavilli BS; Ledbetter AD; Christiani DC; Runge MS; 

Karoly ED; Costa DL; Peddada S; Jaskot R; Richards JH; Thomas R; Madamanchi NR; Nyska A (2008). The role 

of particulate matter-associated zinc in cardiac injury in rats. Environ Health Perspect, 116: 13-20. 155907

Kodavanti UP; Schladweiler MC; Ledbetter AD; McGee JK; Walsh L; Gilmour PS; Highfill JW; Davies D; Pinkerton KE; 

Richards JH; Crissman K; Andrews D; Costa DL (2005). Consistent pulmonary and systemic responses from 

inhalation of fine concentrated ambient particles: roles of rat strains used and physicochemical properties. Environ 

Health Perspect, 113: 1561-1568. 087946

Koike E; Kobayashi T (2005). Organic extract of diesel exhaust particles stimulates expression of Ia and costimulatory 

molecules associated with antigen presentation in rat peripheral blood monocytes but not in alveolar macrophages. 

Toxicol Appl Pharmacol, 209: 277-285. 088303

Kooter IM; Boere AJ; Fokkens PH; Leseman DL; Dormans JA; Cassee FR (2006). Response of spontaneously 

hypertensive rats to inhalation of fine and ultrafine particles from traffic: experimental controlled study. Part Fibre 

Toxicol, 15: 3-7. 097547

Kristovich R; Knight DA; Long JF; Williams MV; Dutta PK; Waldman WJ (2004). Macrophage-mediated endothelial 

inflammatory responses to airborne particulates: impact of particulate physicochemical properties. Chem Res 

Toxicol, 17: 1303-1312. 087963

Kubatova A; Dronen LC; Picklo MJ Sr; Hawthorne SB (2006). Midpolarity and nonpolar wood smoke particulate matter 

fractions deplete glutathione in RAW 264.7 macrophages. Chem Res Toxicol, 19: 255-261. 198835

Kubatova A; Steckler TS; Gallagher JR; Hawthorne SB; Picklo MJSr (2004). Toxicity of wide-range polarity fractions 

from wood smoke and diesel exhaust particulate obtained using hot pressurized water. Environ Toxicol Chem, 23: 

2243-2250. 087986

Kumar VS; Mani U; Prasad AK; Lal K; Gowri V; Gupta A (2004). Effect of fly ash inhalation on biochemical and 

histomorphological changes in rat lungs. Indian J Exp Biol, 42: 964-968. 096655

Kyoso M; Narisawa M; Ito E; Ishijima M; Yana K; Kato A; Ito T; Ishihara Y (2005). Influence of Exposure to Diesel 

Emissions in Rats and Distribution Profile for R-R Interval. Proc Inst Mech Eng H, 5: 5544-5547. 186998

Landvik NE; Gorria M; Arlt VM; Asare N; Solhaug A; Lagadic-Gossmann D; Holme JA (2007). Effects of nitratedpolycyclic aromatic hydrocarbons and diesel exhaust particle extracts on cell signalling related to apoptosis: 

possible implications for their mutagenic and carcinogenic effects. Toxicology, 231: 159-174. 096722

Last JA; Ward R; Temple L; Pinkerton KE; Kenyon NJ (2004). Ovalbumin-induced airway inflammation and fibrosis in 

mice also exposed to ultrafine particles. Inhal Toxicol, 16: 93-102. 097334

Lee CC; Cheng YW; Kang JJ (2005). Motorcycle exhaust particles induce IL-8 production through NF-kappaB activation 

in human airway epithelial cells. J Toxicol Environ Health A Curr Iss, 68: 1537-1555. 156682

Lee CC; Kang JJ (2002). Extract of motorcycle exhaust particles induced macrophages apoptosis by calcium-dependent 

manner. Chem Res Toxicol, 15: 1534-1542. 198864

Lei Y-C; Chan C-C; Wang P-Y; Lee C-T; Cheng T-J (2004). Effects of Asian dust event particles on inflammation markers 

in peripheral blood and bronchoalveolar lavage in pulmonary hypertensive rats. Environ Res, 95: 71-76. 087884

December 2009 D-184 
Lei YC; Chen MC; Chan CC; Wang PY; Lee CT; Cheng TJ (2004). Effects of concentrated ambient particles on airway 

responsiveness and pulmonary inflammation in pulmonary hypertensive rats. Inhal Toxicol, 16: 785-792. 087999

Lei YC; Hwang JS; Chan CC; Lee CT; Cheng TJ (2005). Enhanced oxidative stress and endothelial dysfunction in 

streptozotocin-diabetic rats exposed to fine particles. Environ Res, 99: 335-343. 088660

Lemos M; Mohallen S; Macchione M; Dolhnikoff M; Assun■ao J; Godleski J; Saldiva P (2006). Chronic exposure to urban 

air Pollution induces structural alterations in murine pulmonary and coronary arteries. Inhal Toxicol, 18: 247-253. 

088594

Li J; Li Q; Xu J; Li J; Cai X; Liu R; Li Y; Ma J; Li W (2007). Comparative study on the acute pulmonary toxicity induced 

by 3 and 20 nm TiO2 primary particles in mice. Environ Toxicol Pharmacol, 24: 239-244. 093156

Li N; Kim S; Wang M; Froines JR; Sioutas (2002). Use of a stratified oxidative stress model to study the biological effects 

of ambient concentrated and diesel exhaust particulate matter. Inhal Toxicol, 14: 459-486. 042080

Li N; Wang M; Bramble LA; Schmitz DA; Schauer JJ; Sioutas C; Harkema JR; Nel AE (2009). The adjuvant effect of 

ambient particulate matter is closely reflected by the particulate oxidant potential . Environ Health Perspect, 117: 

1116-1123. 190457

Li N; Wang M; Oberley TD; Sempf JM; Ne (2002). Comparison of the pro-oxidative and proinflammatory effects of 

organic diesel exhaust particle chemicals in bronchial epithelial cells and macrophages. J Immunol, 169: 4531-

4541. 087451

Li Y-J; Kawada T; Matsumoto A; Azuma A; Kudoh S; Takizawa H; Sugawara I (2007). Airway inflammatory responses to 

oxidative stress induced by low-dose diesel exhaust particle exposure differ between mouse strains. Exp Lung Res, 

33: 227-244. 155929

Li Z; Carter JD; Dailey LA; Huang YC (2005). Pollutant particles produce vasoconstriction and enhance MAPK signaling 

via angiotensin type I receptor. Environ Health Perspect, 11: 1009-1014. 088647

Li Z; Hyseni X; Carter JD; Soukup JM; Dailey LA; Huang Y-CT (2006). Pollutant particles enhanced H2O2 production 

from NAD(P)H oxidase and mitochondria in human pulmonary artery endothelial cells. Am J Physiol Lung Cell 

Mol Physiol, 291: C357-C365. 156693

Lichtenfels AJFC; Gomes JB; Pieri PC; Miraglia SGEK; Hallak J; Saldiva PHN (2007). Increased levels of air pollution 

and a decrease in the human and mouse male-to-female ration in Sao Paulo, Brazil. Fertil Steril, 87: 230-232. 

097041

Lijinsky W; Reuber MD (1987). Chronic carcinogenesis studies of acrolein and related compounds. Toxicol Ind Health, 3: 

337-345. 007583

Lindbom J; Gustafsson M; Blomqvist G; Dahl A; Gudmundsson A; Swietlicki E; Ljungman AG (2007). Wear particles 

generated from studded tires and pavement induces inflammatory reactions in mouse macrophage cells. Chem Res 

Toxicol, 20: 937-946. 155934

Lippmann M; Hwang J; Maclejczyk P; Chen L (2005). PM source apportionment for short-term cardiac function changes 

in ApoE-/- mice. Environ Health Perspect, 113: 1575-1579. 087453

Lippmann M; Ito K; Hwang JS; Maciejczyk P; Chen LC (2006). Cardiovascular effects of nickel in ambient air. Environ 

Health Perspect, 114: 1662-1669. 091165

Liu J; Ballaney M; Al-Alem U; Quan C; Jin X; Perera F; Chen LC; Miller RL (2008). Combined Inhaled Diesel Exhaust 

Particles and Allergen Exposure Alter Methylation of T Helper Genes and IgE Production In Vivo. Toxicol Sci, 102: 

76-81. 156709

Liu L-JS; Curjuric I; Keidel D; Heldstab J; Kunzli N; Bayer-Oglesby L; Ackermann-Liebrich U; Schindler C; SAPALDIA 

team (2007). Characterization of source-specific air pollution exposure for a large population-based Swiss cohort 

(SAPALDIA). Environ Health Perspect, 115: 1638-1645. 093093

Liu P-L; Chen Y-L; Chen Y-H; Lin S-J; Kou Y-R (2005). Wood smoke extract induces oxidative stress-mediated caspaseindependent apoptosis in human lung endothelial cells: role of AIF and EndoG. Am J Physiol, 289: L739-L749. 

088304

Liu X; Meng Z (2005). Effects of airborne fine particulate matter on antioxidant capacity and lipid peroxidation in multiple 

organs of rats. Inhal Toxicol, 17: 467-473. 088650

December 2009 D-185 
Liu Y-Q; Keane M; Ensell M; Miller W; Kashon M; Ong T-m; Mauderly J; Lawson D; Gautam M; Zielinska B; Whitney 

K; Eberhardt J; Wallace W (2005). In vitro genotoxicity of exhaust emissions of diesel and gasoline engine vehicles 

operated on a unified driving cycle. J Environ Monit, 7: 60-66. 097019

Long JF; Waldman WJ; Kristovich R; Williams M; Knight D; Dutta PK (2005). Comparison of ultrastructural cytotoxic 

effects of carbon and carbon/iron particulates on human monocyte-derived macrophages. Environ Health Perspect, 

113: 170-174. 087454

Lopes FD; Pinto TS; Arantes-Costa FM; Moriya HT; Biselli PJ; Ferraz LF; Lichtenfels AJ; Saldiva PH; Mauad T; Martins 

MA (2009). Exposure to ambient levels of particles emitted by traffic worsens emphysema in mice. Environ Res, 

109: 544-551. 190430

Lund AK; Knuckles TL; Obot Akata C; Shohet R; McDonald JD; Gigliotti A; Seagrave JC; Campen MJ (2007). Gasoline 

exhaust emissions induce vascular remodeling pathways involved in atherosclerosis. Toxicol Sci, 95: 485-94. 

125741

Lundborg M; Bouhafs R; Gerde P; Ewing P; Camner P; Dahlen SE; Jarstrand C (2007). Aggregates of ultrafine particles 

modulate lipid peroxidation and bacterial killing by alveolar macrophages. Environ Res, 104: 250-257. 096040

Ma C; Wang J; Luo J (2004). Activation of nuclear factor kappa B by diesel exhaust particles in mouse epidermal cells 

through phosphatidylinositol 3-kinase/Akt signaling pathway. Biochem Pharmacol, 67: 1975-1983. 088417

Maciejczyk P; Chen LC (2005). Effects of subchronic exposures to concentrated ambient particles (CAPs) in mice: VIII 

source-related daily variations in in vitro responses to CAPs. Inhal Toxicol, 17: 243-253. 087456

Madden MC; Dailey LA; Stonehuerner JG; Harris BD (2003). Responses of cultured human airway epithelial cells treated 

with diesel exhaust extracts will vary with the engine load. J Toxicol Environ Health A Curr Iss, 66: 2281-2297. 

198877

Mangum JB; Bermudez E; Sar M; Everitt JI (2004). Osteopontin expression in particle-induced lung disease. Exp Lung 

Res, 30: 585-598. 097326

Martin S; Dawidowski L; Mandalunis P; Cereceda-Balic F; Tasat DR (2007). Characterization and biological effect of 

Buenos Aires urban air particles on mice lungs. Environ Res, 105: 340-349. 096366

Mason CE (2002). Gasoline ETBE vapor condensate rat micronucleus test (satellite procedure). Princeton Research Center 

. East Millstone, NJ. 087645

Matsumoto A; Hiramatsu K; Li Y; Azuma A; Kudoh S; Takizawa H; Sugawara I (2006). Repeated exposure to low-dose 

diesel exhaust after allergen challenge exaggerates asthmatic responses in mice. Clin Immunol, 121: 227-235. 

098017

Matsumoto S; Ishii H; Tanabe T; Kawai J (2007). Chemical state analysis of fine particles using XAFS. Tetsu-To-Hagane, 

93: 132-137. 187020

Matsuo M; Shimada T; Uenishi R; Sasaki N; Sagai M (2003). Diesel exhaust particle-induced cell death of cultured normal 

human bronchial epithelial cells. Biol Pharm Bull, 26: 438-447. 198879

Matsuzaki T; Amakawa K; Yamaguchi K; Ishizaka A; Terashima T; Matsumaru A; Morishita T (2006). Effects of diesel 

exhaust particles on human neutrophil activation. Exp Lung Res, 32: 427-439. 199517

Mauad T; Rivero DH; de Oliveira RC; Lichtenfels AJ; Guimaraes ET; de Andre PA; Kasahara DI; Bueno HM; Saldiva PH 

(2008). Chronic exposure to ambient levels of urban particles affects mouse lung development. Am J Respir Crit 

Care Med, 178: 721-728. 156743

McDonald JD; Harrod KS; Seagrave J; Seikop SK; Mauderly JL (2004). Effects of low sulfur fuel composition and a 

catalyzed particle trap on the composition and toxicity of diesel emissions. Environ Health Perspect, 112: 1307-

1312. 087459

McQueen DS; Donaldson K; Bond SM; McNeilly JD; Newman S; Barton NJ; Duffin R (2007). Bilateral vagotomy or 

atropine pre-treatment reduces experimental diesel-soot induced lung inflammation. Toxicol Appl Pharmacol, 219: 

62-71. 096266

Medeiros N Jr; Rivero DH; Kasahara DI; Saiki M; Godleski JJ; Koutrakis P; Capelozzi VL; Saldiva PH; Antonangelo L 

(2004). Acute pulmonary and hematological effects of two types of particle surrogates are influenced by their 

elemental composition. Environ Res, 95: 62-70. 096012

December 2009 D-186 
Mehta M; Chen LC; Gordon T; Rom W; Tang MS (2008). Particulate matter inhibits DNA repair and enhances 

mutagenesis. Mutat Res Genet Toxicol Environ Mutagen, 657: 116-121. 190440

Meng Z; Zhang Q (2007). Damage effects of dust storm PM2.5 on DNA in alveolar macrophages and lung cells of rats. 

Food Chem Toxicol, 45: 1368-1374. 198963

Mohallem SV; de Araujo Lobo DJ; Pesquero CR; Assuncao JV; de Andre PA; Saldiva PH; Dolhnikoff M (2005). Decreased 

fertility in mice exposed to environmental air pollution in the city of Sao Paulo. Environ Res, 98: 196-202. 088657

Molinelli AR; Santacana GE; Madden MC; Jiménez BD (2006). Toxicity and metal content of organic solvent extracts 

from airborne particulate matter in Puerto Rico. Environ Res, 102: 314-325. 198949

Moller W; Hofer T; Ziesenis A; Karg E; Heyder J (2002). Ultrafine particles cause cytoskeletal dysfunctions in 

macrophages. Toxicol Appl Pharmacol, 182: 197-207. 036589

Montiel-Davalos A; Alfaro-Moreno E; Lopez-Marure R (2007). PM2.5 and PM10 induce the expression of adhesion 

molecules and the adhesion of monocytic cells to human umbilical vein endothelial cells. Inhal Toxicol, 19 Suppl 1: 

91-98. 156778

Mori T; Watanuki T; Kashiwagura T (2007). Diesel exhaust particles disturb gene expression in mouse testis. Environ 

Toxicol, 22: 58-63. 096564

Morishita M; Keeler G; Wagner J; Marsik F; Timm E; Dvonch J; Harkema J (2004). Pulmonary retention of particulate 

matter is associated with airway inflammation in allergic rats exposed to air pollution in urban Detroit. Inhal 

Toxicol, 16: 663-674. 087979

Motta S; Federico C; Saccone S; Librando V; Mosesso P (2004). Cytogenetic evaluation of extractable agents from 

airborne particulate matter generated in the city of Catania (Italy). Mutat Res, 561: 45-52. 198953

Moyer CF; Kodavanti UP; Haseman JK; Costa DL; Nyska A (2002). Systemic vascular disease in male B6C3F1 mice 

exposed to particulate matter by inhalation: studies conducted by the national toxicology program. Toxicol Pathol, 

30: 427-434. 052222

Mutlu GM; Green D; Bellmeyer A; Baker CM; Burgess Z; Rajamannan N; Christman JW; Foiles N; Kamp DW; Ghio AJ; 

Chandel NS; Dean DA; Sznajder JI; Budinger GR (2007). Ambient particulate matter accelerates coagulation via an 

IL-6-dependent pathway. J Clin Invest, 117: 2952-2961. 121441

Mutlu GM; Snyder C; Bellmeyer A; Wang H; Hawkins K; Soberanes S; Welch LC; Ghio AJ; Chandel NS; Kamp D; 

Sznajder Jacob I; Budinger GRS (2006). Airborne particulate matter inhibits alveolar fluid reabsorption in mice via 

oxidant generation. Am J Respir Cell Mol Biol, 34: 670-676. 155994

Nadziejko C; Fang K; Nadziejko E; Narciso SP; Zhong M; Chen LC (2002). Immediate effects of particulate air pollutants 

on heart rate and respiratory rate in hypertensive rats. Cardiovasc Toxicol, 2: 245-252. 087460

Nadziejko C; Fang K; Narciso S; Zhong M; Su WC; Gordon T; Nadas A; Chen LC (2004). Effect of particulate and 

gaseous pollutants on spontaneous arrhythmias in aged rats. Inhal Toxicol, 16: 373-380. 055632

Nam HY; Choi BH; Lee JY; Lee SG; Kim YH; Lee KH; Yoon HK; Song JS; Kim HJ; Lim Y (2004). The role of nitric 

oxide in the particulate matter (PM2.5)-induced NFkappaB activation in lung epithelial cells. Toxicol Lett, 148: 95-

102. 198887

Nemmar A; Al-Maskari S; Ali Badreldin H; Al-Amri Issa S (2007). Cardiovascular and lung inflammatory effects induced 

by systemically administered diesel exhaust particles in rats. Am J Physiol Lung Cell Mol Physiol, 292: L664-

L670. 156800

Nemmar A; Hoet PH; Dinsdale D; Vermylen J; Hoylaerts MF; Nemery B (2003). Diesel exhaust particles in lung acutely 

enhance experimental peripheral thrombosis. Circulation, 107: 1202-1208. 096567

Nemmar A; Hoet PH; Vermylen J; Nemery B; Hoylaerts MF (2004). Pharmacological stabilization of mast cells abrogates 

late thrombotic events induced by diesel exhaust particles in hamsters. Circulation, 110: 1670-1677. 087959

Nemmar A; Hoylaerts MF; Hoet PHM; Vermylen J; Nemery B (2003). Size effect of intratracheally instilled particles on 

pulmonary inflammation and vascular thrombosis. Toxicol Appl Pharmacol, 186: 38-45. 087931

Nemmar A; Inuwa IM (2008). Diesel exhaust particles in blood trigger systemic and pulmonary morphological alterations. 

Toxicol Lett, 176: 20-30. 096566

December 2009 D-187 
Nemmar A; Nemery B; Hoet PHM; Vermylen J; Hoylaerts MF (2003). Pulmonary inflammation and thrombogenicity 

caused by diesel particles in hamsters: role of histamine. Am J Respir Crit Care Med, 168: 1366-1372. 097487

Niwa Y; Hiura Y; Murayama T; Yokode M; Iwai N (2007). Nano-sized carbon black exposure exacerbates atherosclerosis 

in LDL-receptor knockout mice. Circ J, 71: 1157-1161. 091309

Niwa Y; Hiura Y; Sawamura H; Iwai N (2008). Inhalation exposure to carbon black induces inflammatory response in rats. 

Circ J, 72: 144-149. 156812

Nozaki JI; Yamamoto R; Ma L; Shima M (2007). Trial to evaluate effects of ambient particulate matter on health: A 

preliminary study using two-dimensional gel electrophoresis. Environ Health Prev Med, 12: 138-142. 097862

Nurkiewicz TR; Porter DW; Barger M; Castranova V; Boegehold MA (2004). Particulate matter exposure impairs systemic 

microvascular endothelium-dependent dilation. Environ Health Perspect, 112: 1299-1306. 087968

Nurkiewicz TR; Porter DW; Barger M; Millecchia L; Rao KMK; Marvar PJ; Hubbs AF; Castranova V; Boegehold MA 

(2006). Systemic microvascular dysfunction and inflammation after pulmonary particulate matter exposure. 

Environ Health Perspect, 114: 412-419. 088611

Nurkiewicz TR; Porter DW; Hubbs AF; Cumpston JL; Chen BT; Frazer DG; Castranova V (2008). Nanoparticle inhalation 

augments particle-dependent systemic microvascular dysfunction. Part Fibre Toxicol, 5: 1. 156816

Nurkiewicz TR; Porter DW; Hubbs AF; Stone S; Chen BT; Frazer DG; Boegehold MA; Castranova V (2009). Pulmonary 

nanoparticle exposure disrupts systemic microvascular nitric oxide signaling. Toxicol Sci, 110: 191-203. 191961

Nygaard UC; Alberg T; Bleumink R; Aase A; Dybing E; Pieters R; Lovik M (2005). Ambient air particles from four 

European cities increase the primary cellular response to allergen in the draining lymph node. Toxicology, 207: 241-

254. 088655

Nygaard UC; Ormstad H; Aase A; Lovik M (2005). The IgE adjuvant effect of particles: characterisation of the primary 

cellular response in the draining lymph node. Toxicology, 206: 181-193. 087980

Nygaard UC; Samuelsen M; Aase A; Lovik M (2004). The capacity of particles to increase allergic sensitization is 

predicted by particle number and surface area, not by particle mass. Toxicol Sci, 82: 515-524. 058558

Obot CJ; Morandi MT; Beebe TP; Hamilton RF; Holian A (2002). Surface components of airborne particulate matter 

induce macrophage apoptosis through scavenger receptors. Toxicol Appl Pharmacol, 184: 98-106. 042370

Obot CJ; Morandi MT; Hamilton RF; Holian A (2004). A comparison of murine and human alveolar macrophage responses 

to urban particulate matter. Inhal Toxicol, 16: 69-76. 095938

Oh S-M; Chung K-H (2006). Identification of mammalian cell genotoxins in respirable diesel exhaust particles by 

bioassay-directed chemical analysis. Toxicol Lett, 161: 226-235. 088296

Okayama Y; Kuwahara M; Suzuki AK; Tsubone H (2006). Role of reactive oxygen species on diesel exhaust particleinduced cytotoxicity in rat cardiac myocytes. J Toxicol Environ Health A Curr Iss, 69: 1699-1710. 156824

Okeson CD; Riley MR; Fernandez A; Wendt JOL (2003). Impact of the composition of combustion generated fine particles 

on epithelial cell toxicity: influences of metals on metabolism. Chemosphere, 51: 1121-1128. 042292

Okeson CD; Riley MR; Riley-Saxton E (2004). In vitro alveolar cytotoxicity of soluble components of airborne particulate 

matter: effects of serum on toxicity of transition metals. Toxicol In Vitro, 18: 673-680. 087961

Ono N; Oshio S; Niwata Y; Yoshida S; Tsukue N; Sugawara I; Takano H; Takeda K (2007). Prenatal exposure to diesel 

exhaust impairs mouse spermatogenesis. Inhal Toxicol, 19: 275-281. 156007

Osornio-Vargas AR; Bonner JC; Alfaro-Moreno E; Martinez L; Garcia-Cuellar C; Rosales SP; Miranda J; Rosas I (2003). 

Proinflammatory and cytotoxic effects of Mexico City air pollution particulate matter in vitro are dependent on 

particle size and composition. Environ Health Perspect, 111: 1289-1293. 052417

Pastorkova A; Cerna M; Smid J; Vrbikova V (2004). Mutagenicity of airborne particulate matter PM10. Cent Eur J Public 

Health, 12: S72-S75. 087431

Penn A; Murphy G; Barker S; Henk W; Penn L (2005). Combustion-derived ultrafine particles transport organic toxicants 

to target respiratory cells. Environ Health Perspect, 113: 956-963. 088257

Pereira CEL; Heck TG; Saldiva PHN; Rhoden CR (2007). Ambient particulate air pollution from vehicles promotes lipid 

peroxidation and inflammatory responses in rat lung. Braz J Med Biol Res, 40: 1353-1359. 156019

December 2009 D-188 
Pinkerton KE; Zhou Y; Teague SV; Peake JL; Walther RC; Kennedy IM; Leppert VJ; Aust AE (2004). Reduced lung cell 

proliferation following short-term exposure to ultrafine soot and iron particles in neonatal rats: key to impaired lung 

growth? Inhal Toxicol, 1: 73-81. 087465

Pires-Neto RC; Lichtenfels AJ; Soares SR; Macchione M; Saldiva PHN; Dolhnikoff M (2006). Effects of Sao Paulo air 

pollution on the upper airways of mice. Environ Res, 101: 356-361. 096734

Poma A; Limongi T; Pisani C; Granato V; Picozzi P (2006). Genotoxicity induced by fine urban air particulate matter in the 

macrophages cell line RAW 264.7. Toxicol In Vitro, 20: 1023-1029. 096903

Pourazar J; Mudway IS; Samet JM; Helleday R; Blomberg A; Wilson SJ; Frew AJ; Kelly FJ; Sandstrom T (2005). Diesel 

exhaust activates redox-sensitive transcription factors and kinases in human airways. Am J Physiol, 289: L724-

L730. 088305

Pozzi R; De Berardis B; Paoletti L; Guastadisegni C (2005). Winter urban air particles from Rome (Italy): effects on the 

monocytic-macrophagic RAW 2647 cell line. Environ Res, 99: 344-354. 088610

Pradhan A; Waseem M; Dogra S; Khanna AK; Kaw JL (2005). Alterations in bronchoalveolar lavage constituents, 

oxidant/antioxidant status, and lung histology following intratracheal instillation of respirable suspended particulate 

matter. J Environ Pathol Toxicol Oncol, 24: 19-32. 096128

Proctor SD; Dreher KL; Kelly SE; Russell JC (2006). Hypersensitivity of prediabetic JCR: LA-cp rats to fine airborne 

combustion particle-induced direct and noradrenergic-mediated vascular contraction. Toxicol Sci, 90: 385-391. 

088480

Prophete C; Maciejczyk P; Salnikow K; Gould T; Larson T; Koenig J; Jaques P; Sioutas C; Lippmann M; Cohen M (2006). 

Effects of select PM-associated metals on alveolar macrophage phosphorylated ERK1 and -2 and iNOS expression 

during ongoing alteration in iron homeostasis. J Toxicol Environ Health A Curr Iss, 69: 935-951. 156888

Radomski A; Jurasz P; Alonso-Escalano D; Drews M; Morandi M; Malinski T; Radomski MW (2005). Nanoparticleinduced platelet aggregation and vascular thrombosis. Br J Pharmacol, 146: 882-893. 091377

Ramage L; Guy K (2004). Expression of C-reactive protein and heat-shock protein-70 in the lung epithelial cell line A549, 

in response to PM10 exposure. Inhal Toxicol, 16: 447-452. 055640

Ramos C; Cisneros J; Gonzalez-Avila G; Becerril C; Ruiz V; Montaño M (2009). Increase of matrix metalloproteinases in 

woodsmoke-induced lung emphysema in guinea pigs. Inhal Toxicol, 21: 119-132. 190116

Rao KM; Ma JY; Meighan T; Barger MW; Pack D; Vallyathan V (2005). Time course of gene expression of inflammatory 

mediators in rat lung after diesel exhaust particle exposure. Environ Health Perspect, 113: 612-617. 095756

Reed MD; Barrett EG; Campen MJ; Divine KK; Gigliotti AP; McDonald JD; Seagrave JC; Mauderly JL; Seilkop SK; 

Swenberg JA (2008). Health effects of subchronic inhalation exposure to gasoline engine exhaust. Inhal Toxicol, 

20: 1125-1143. 156903

Reed MD; Campen MJ; Gigliotti AP; Harrod KS; McDonald JD; Seagrave JC; Mauderly JL; Seilkop SK (2006). Health 

effects of subchronic exposure to environmental levels of hardwood smoke. Inhal Toxicol, 18: 523-539. 156043

Reed MD; Gigliotti AP; McDonald JD; Seagrave JC; Seilkop SK; Mauderly JL (2004). Health effects of subchronic 

exposure to environmental levels of diesel exhaust. Inhal Toxicol, 16: 177-193. 055625

Reibman J; Hsu Y; Chen LC; Bleck B; Gordon T (2003). Airway epithelial cells release MIP-3alpha/CCL20 in response to 

cytokines and ambient particulate matter. Am J Respir Cell Mol Biol, 28: 648-654. 156905

Rengasamy A; Barger MW; Kane E; Ma JKH; Castranova V; Ma JYC (2003). Diesel exhaust particle-induced alterations 

of pulmonary phase I and phase II enzymes of rats. J Toxicol Environ Health A Curr Iss, 66: 153-167. 156907

Renwick LC; Brown D; Clouter A; Donaldson K (2004). Increased inflammation and altered macrophage chemotactic 

responses caused by two ultrafine particle types. Occup Environ Med, 61: 442-447. 056067

Rhoden CR; Ghelfi E; González-Flecha B (2008). Pulmonary inflammation by ambient air particles is mediated by 

superoxide anion. Inhal Toxicol, 20: 11-15. 190475

Rhoden CR; Lawrence J; Godleski JJ; Gonzalez-Flecha B (2004). N-acetylcysteine prevents lung inflammation after shortterm inhalation exposure to concentrated ambient particles. Toxicol Sci, 79: 296-303. 087969

Rhoden CR; Wellenius GA; Ghelfi E; Lawrence J; Gonzalez-Flecha B (2005). PM-induced cardiac oxidative stress and 

dysfunction are mediated by autonomic stimulation. Biochim Biophys Acta, 1725: 305-313. 087878

December 2009 D-189 
Riley MR; Boesewetter DE; Kim AM; Sirvent FP (2003). Effects of metals Cu, Fe, Ni, V, and Zn on rat lung epithelial 

cells. Toxicology, 190: 171-184. 053237

Riley MR; Boesewetter DE; Turner RA; Kim AM; Collier JM; Hamilton A (2005). Comparison of the sensitivity of three 

lung derived cell lines to metals from combustion derived particulate matter. Toxicol In Vitro, 19: 411-419. 096452

Ritz SA; Wan J; Diaz-Sanchez D (2007). Sulforaphane-stimulated phase II enzyme induction inhibits cytokine production 

by airway epithelial cells stimulated with diesel extract. Am J Physiol Lung Cell Mol Physiol, 292: L33-39. 198901

Rivedal E; Myhre O; Sanner T; Eide I (2003). Supplemental role of the Ames mutation assay and gap junction intercellular 

communication in studies of possible carcinogenic compounds from diesel exhaust particles. Arch Toxicol, 77: 533-

542. 097684

Rivero DH; Soares SR; Lorenzi-Filho G; Saiki M; Godleski JJ; Antonangelo L; Dolhnikoff M; Saldiva PH (2005). Acute 

cardiopulmonary alterations induced by fine particulate matter of Sao Paulo, Brazil. Toxicol Sci, 85: 898-905. 

088653

Roberts E; Charboneau L; Espina V; Liotta L; Petricoin E; Dreher K (2004). Application of laser capture microdissection 

and protein microarray technologies in the molecular analysis of airway injury following pollution particle 

exposure. J Toxicol Environ Health A Curr Iss, 67: 851-861. 198903

Roberts JR; Young S-H; Castranova V; Antonini JM (2007). Soluble metals in residual oil fly ash alter innate and adaptive 

pulmonary immune responses to bacterial infection in rats. Toxicol Appl Pharmacol, 221: 306-319. 097623

Rodriguez Ferreira Rivero DH; Sassaki C; Lorenzi-Filho G; Nascimento Saldiva PH (2005). PM2.5 induces acute 

electrocardiographic alterations in healthy rats. Environ Res, 99: 262-266. 088659

Rosas Perez I; Serrano J; Alfaro-Moreno E; Baumgardner D; Garcia-Cuellar C; Martin Del Campo JM; Raga GB; 

Castillejos M; Colin RD; Osornio Vargas AR (2007). Relations between PM10 composition and cell toxicity: a 

multivariate and graphical approach. Chemosphere, 67: 1218-28. 097967

Roubicek DA; Gutierrez-Castillo ME; Sordo M; Cebrian-Garcia ME; Ostrosky-Wegman P (2007). Micronuclei induced by 

airborne particulate matter from Mexico City. Mutat Res Fund Mol Mech Mutagen, 631: 9-15. 156929

Saber AT; Bornholdt J; Dybdahl M; Sharma AK; Loft S; Vogel U; Wallin H (2005). Tumor necrosis factor is not required 

for particle-induced genotoxicity and pulmonary inflammation. Arch Toxicol, 79: 177-182. 097865

Sakamoto N; Hayashi S; Gosselink J; Ishii H; Ishimatsu Y; Mukae H; Hogg JC; van Eeden SF (2007). Calcium dependent 

and independent cytokine synthesis by air pollution particle-exposed human bronchial epithelial cells. Toxicol Appl 

Pharmacol, 225: 134-141. 096282

Salnikow K; Li X; Lippmann M (2004). Effect of nickel and iron co-exposure on human lung cells. Toxicol Appl 

Pharmacol, 196: 258-265. 087469

Salonen R; Halinen A; Pennanen A; Hirvonen M; Sillanpaa M; Hillamo R; Shi R; Borm P; Sandell E; Koskentalo T; 

Aarnio P (2004). Chemical and in vitro toxicologic characterization of wintertime and springtime urban-air particles 

with an aerodynamic diameter below 10 μm in Helsinki. Scand J Work Environ Health, 30: 80-90. 187053

Samet JM; Dewar BJ; Wu W; Graves LM (2003). Mechanisms of Zn2+-induced signal initiation through the epidermal 

growth factor receptor. Toxicol Appl Pharmacol, 191: 86-93. 113782

Santini MT; Rainaldi G; Ferrante A; Romano R; Clemente S; Motta A; De Berardis B; Balduzzi M; Paoletti L; Indovina PL 

(2004). Environmental fine particulate matter (PM25) activates the RAW 2647 macrophage cell line even at very 

low concentrations as revealed by 1H NMR. Chem Res Toxicol, 17: 63-74. 087879

Saxena QB; Saxena RK; Siegel PD; Lewis DM (2003). Identification of organic fractions of diesel exhaust particulate 

(DEP) which inhibit nitric oxide (NO) production from a murine macrophage cell line. Toxicol Lett, 143: 317-322. 

096986

Saxena RK; Saxena QB; Weissman DN; Simpson JP; Bledsoe TA; Lewis DM (2003). Effect of diesel exhaust particulate 

on bacillus Calmette-Guerin lung infection in mice and attendant changes in lung interstitial lymphoid 

subpopulations and IFN gamma response. Toxicol Sci, 73: 66-71. 054395

Schins RPF; Lightbody JH; Borm PJA; Shi T; Donaldson K; Stone V (2004). Inflammatory effects of coarse and fine 

particulate matter in relation to chemical and biological constituents. Toxicol Appl Pharmacol, 195: 1-11. 054173

December 2009 D-190 
Schneider J; Hock N; Weimer S; Borrmann S; Kirchner U; Vogt R; Scheer V (2005). Nucleation particles in diesel exhaust: 

composition inferred from in situ mas spectrometric analysis. Environ Sci Technol, 39: 6153-6161. 088368

Schober W; Belloni B; Lubitz S; Eberlein-Konig B; Bohn P; Saritas Y; Lintelmann J; Matuschek G; Behrendt H; Buters J 

(2006). Organic extracts of urban aerosol (< or =PM25) enhance rBet v 1-induced upregulation of CD63 in 

basophils from birch pollen-allergic individuals. Toxicol Sci, 90: 377-384. 097321

Seagrave J; Campen M; McDonald J; Mauderly J; Rohr A (2008). Oxidative stress, inflammation, and pulmonary function 

assessment in rats exposed to laboratory-generated pollutant mixtures. J Toxicol Environ Health A Curr Iss, 71: 

1352-1362. 191990

Seagrave J; Dunaway S; McDonald JD; Mauderly JL; Hayden P; Stidley C (2007). Responses of differentiated primary 

human lung epithelial cells to exposure to diesel exhaust at an air-liquid interface. Exp Lung Res, 33: 27-51. 

097549

Seagrave J; Knall C; McDonald JD; Mauderly JL (2004). Diesel particulate material binds and concentrates a 

proinflammatory cytokine that causes neutrophil migration. Inhal Toxicol, 1: 93-98. 087470

Seagrave J; Mauderly JL; Seilkop SK (2003). In vitro relative toxicity screening of combined particulate and semivolatile 

organic fractions of gasoline and diesel engine emissions. J Toxicol Environ Health A Curr Iss, 66: 1113-1132. 

054979

Seagrave J; McDonald JD; Reed MD; Seilkop SK; Mauderly JL (2005). Responses to subchronic inhalation of low 

concentrations of diesel exhaust and hardwood smoke measured in rat bronchoalveolar lavage fluid. Inhal Toxicol, 

17: 657-670. 088000

Seagrave JC; McDonald JD; Bedrick E; Edgerton ES; Gigliotti AP; Jansen JJ; Ke L; Naeher LP; Seilkop SK; Zheng M; 

Mauderley JL (2006). Lung toxicity of ambient particulate matter from southeastern US sites with different 

contributing sources: relationships between composition and effects. Environ Health Perspect, 114: 1387-93. 

091291

Seaton A; Cherrie J; Dennekamp M; Donaldson K; Hurley JF; Tran CL (2005). The London Underground: Dust and 

hazards to health. Occup Environ Med, 62: 355-362. 198904

Sevastyanova O; Binkova B; Topinka J; Sram RJ; Kalina I; Popov T; Novakova Z; Farmer PB (2007). In vitro genotoxicity 

of PAH mixtures and organic extract from urban air particles part II: human cell lines. Mutat Res Fund Mol Mech 

Mutagen, 620: 123-134. 156969

Sharma AK; Jensen KA; Rank J; White PA; Lundstedt S; Gagne R; Jacobsen NR; Kristiansen J; Vogel U; Wallin H (2007). 

Genotoxicity, inflammation and physico-chemical properties of fine particle samples from an incineration energy 

plant and urban air. Mutat Res Fund Mol Mech Mutagen, 633: 95-111. 156975

Shi T; Knaapen AM; Begerow J; Birmili W; Borm PJA; Schins RPF (2003). Temporal variation of hydroxyl radical 

generation and 8-hydroxy-2'-deoxyguanosine formation by coarse and fine particulate matter. Occup Environ Med, 

60: 315-321. 088248

Shwe T-T-W; Yamamoto S; Kakeyama M; Kobayashi T; Fujimaki H (2005). Effect of intratracheal instillation of ultrafine 

carbon black on proinflammatory cytokine and chemokine release and mRNA expression in lung and lymph nodes 

of mice. Toxicol Appl Pharmacol, 209: 51-61. 111553

Sigaud S; Goldsmith Carroll-Ann W; Zhou H; Yang Z; Fedulov A; Imrich A; Kobzik L (2007). Air pollution particles 

diminish bacterial clearance in the primed lungs of mice. Toxicol Appl Pharmacol, 223: 1-9. 096100

Silva PJ; Erupe ME; Price D; Elias J; Malloy QG; Li Q; Warren B; Cocker DR 3rd (2008). Trimethylamine as precursor to 

secondary organic aerosol formation via nitrate radical reaction in the atmosphere. Environ Sci Technol, 42: 4689-

4696. 156981

Simkhovich BZ; Marjoram P; Kleinman MT; Kloner RA (2007). Direct and acute cardiotoxicity of ultrafine particles in 

young adult and old rat hearts. Basic Res Cardiol, 102: 467-475. 096594

Singal M; Finkelstein JN (2005). Use of indicator cell lines for determining inflammatory gene changes and screening the 

inflammatory potential of particulate and non-particulate stimuli. Inhal Toxicol, 17: 415-425. 198905

Singh P; DeMarini DM; Dick CA; Tabor DG; Ryan JV; Linak WP; Kobayashi T; Gilmour MI (2004). Sample 

characterization of automobile and forklift diesel exhaust particles and comparative pulmonary toxicity in mice. 

Environ Health Perspect, 112: 820-825. 087472

December 2009 D-191 
Sirivelu MP; MohanKumar SMJ; Wagner JG; Harkema JR; MohanKumar PS (2006). Activation of the stress axis and 

neurochemical alterations in specific brain areas by concentrated ambient particle exposure with concomitant 

allergic airway disease. Environ Health Perspect, 114: 870-874. 111151

Skarek M; Janosek J; Cupr P; Kohoutek J; Novotna-Rychetska A; Holoubek I (2007). Evaluation of genotoxic and nongenotoxic effects of organic air pollution using in vitro bioassays. Environ Int, 33: 859-66. 096814

Smith KR; Kim S; Recendez JJ; Teague SV; Menache MG; Grubbs DE; Sioutas C; Pinkerton KE (2003). Airborne particles 

of the California Central Valley alter the lungs of healthy adult rats. Environ Health Perspect, 111: 902-908. 042107

Smith KR; Veranth JM; Kodavanti UP; Aust AE; Pinkerton KE (2006). Acute pulmonary and systemic effects of inhaled 

coal fly ash in rats: comparison to ambient environmental particles. Toxicol Sci, 93: 390-399. 110864

Somers CM; McCarry BE; Malek F; Quinn JS (2004). Reduction of particulate air pollution lowers the risk of heritable 

mutations in mice. Science, 304: 1008-1010. 078098

Somers CM; Yauk CL; White PA; Parfett CLJ; Quinn JS (2002). Air pollution induces heritable DNA mutations. PNAS, 

99: 15904-15907. 078100

Song CL; Zhou YC; Huang RJ; Wang YQ; Huang QF; Lu G; Liu KM (2007). Influence of ethanol-diesel blended fuels on 

diesel exhaust emissions and mutagenic and genotoxic activities of particulate extracts. J Hazard Mater, 149: 355-

363. 155306

Song H-M; Jang A-S; Ahn M-H; Takizawa H; Lee S-H; Kwon J-H; Lee Y-M; Rhim T; Park C-S (2008). Ym1 and Ym2 

expression in a mouse model exposed to diesel exhaust particles. Environ Toxicol, 23: 110-116. 156093

Steerenberg P; Verlaan A; De Klerk A; Boere A; Loveren H; Cassee F (2004). Sensitivity to ozone, diesel exhaust particles, 

and standardized ambient particulate matter in rats with a listeria monocytogenes-induced respiratory infection. 

Inhal Toxicol, 16: 311-317. 087474

Steerenberg PA; Van Amelsvoort L; Lovik M; Hetland RB; Alberg T; Halatek T; Bloemen HJT; Rydzynski K; Swaen G; 

Schwarze P; Dybing E; Cassee FR (2006). Relation between sources of particulate air pollution and biological 

effect parameters in samples from four European cities: an exploratory study. Inhal Toxicol, 18: 333-346. 088249

Steerenberg PA; Withagen CE; van Dalen WJ; Dormans JA; Heisterkamp SH; van Loveren H; Cassee FR (2005). Dose 

dependency of adjuvant activity of particulate matter from five European sites in three seasons in an ovalbuminmouse model. Inhal Toxicol, 17: 133-145. 088649

Steerenberg PA; Withagen CET; van Dalen WJ; Dormans JAMA; van Loveren H (2004). Adjuvant Activity of Ambient 

Particulate Matter in Macrophage Activity-Suppressed, N-Acetylcysteine-Treated, iNOS- and IL-4-Deficient Mice. 

Inhal Toxicol, 16: 835-843. 087981

Stevens JP; Zahardis J; MacPherson M; Mossman BT; Petrucci GA (2008). A new method for quantifiable and controlled 

dosage of particulate matter for in vitro studies: the Electrostatic Particulate Dosage and Exposure System 

(EPDExS). Toxicol In Vitro, 22(7): 1768-1774. 155363

Stinn W; Teredesai A; Anskeit E; Rustemeier K; Schepers G; Schnell P; Haussmann H-J; Carchman RA; Coggins CR; 

Reininghaus W (2005). Chronic nose-only inhalation study in rats, comparing room-aged sidestream cigarette 

smoke and diesel engine exhaust. Inhal Toxicol, 17: 549-576. 088307

Sugamata M; Ihara T; Sugamata M; Takeda K (2006). Maternal Exposure to Diesel Exhaust Leads to Pathological 

Similarity to Autism in Newborns. Eisei Kagaku, 52: 486-488. 157025

Sun Q; Wang A; Jin X; Natanzon A; Duquaine D; Brook RD; Aguinaldo JG; Fayad ZA; Fuster V; Lippmann M; Chen LC; 

Rajagopalan S (2005). Long-term air pollution exposure and acceleration of atherosclerosis and vascular 

inflammation in an animal model. JAMA, 294: 3003-3010. 087952

Sun Q; Yue P; Deiuliis JA; Lumeng CN; Kampfrath T; Mikolaj MB; Cai Y; Ostrowski MC; Lu B; Parthasarathy S; Brook 

RD; Moffatt-Bruce SD; Chen LC; Rajagopalan S (2009). Ambient air pollution exaggerates adipose inflammation 

and insulin resistance in a mouse model of diet-induced obesity. Circulation, 119: 538-546. 190487

Sun Q; Yue P; Kirk RI; Wang A; Moatti D; Jin X; Lu B; Schecter AD; Lippmann M; Gordon T; Chen LC; Rajagopalan S 

(2008). Ambient air particulate matter exposure and tissue factor expression in atherosclerosis. Inhal Toxicol, 20: 

127-137. 157033

December 2009 D-192 
Sun Q; Yue P; Ying Z; Cardounel AJ; Brook RD; Devlin R; Hwang JS; Zweier JL; Chen LC; Rajagopalan S (2008). Air 

Pollution Exposure Potentiates Hypertension Through Reactive Oxygen Species-Mediated Activation of 

Rho/ROCK. Arterioscler Thromb Vasc Biol, 28: 1760-1766. 157032

Sureshkumar V; Paul B; Uthirappan M; Pandey R; Sahu AP; Lal K; Prasad AK; Srivastava S; Saxena A; Mathur N; Gupta 

YK (2005). Proinflammatory and anti-inflammatory cytokine balance in gasoline exhaust induced pulmonary injury 

in mice. Inhal Toxicol, 17: 161-168. 088306

Takizawa H; Abe S; Okazaki H; Kohyama T; Sugawara I; Saito Y; Ohtoshi T; Kawasaki S; Desaki M; Nakahara K; 

Yamamoto K; Matsushima K; Tanaka M; Sagai M; Kudoh S (2003). Diesel exhaust particles upregulate eotaxin 

gene expression in human bronchial epithelial cells via nuclear factor-kappa B-dependent pathway. Am J Physiol 

Lung Cell Mol Physiol, 284: L1055-L1062. 157039

Tal TL; Graves LM; Silbajoris R; Bromberg PA; Wu W; Samet JM (2006). Inhibition of protein tyrosine phosphatase 

activity mediates epidermal growth factor receptor signaling in human airway epithelial cells exposed to Zn2+. 

Toxicol Appl Pharmacol, 214: 16-23. 108588

Tamagawa E; Bai N; Morimoto K; Gray C; Mui T; Yatera K; Zhang X; Xing L; Li Y; Laher I (2008). Particulate matter 

exposure induces persistent lung inflammation and endothelial dysfunction. Am J Physiol Lung Cell Mol Physiol, 

295: L79-L85. 191988

Tamaoki J; Isono K; Takeyama K; Tagaya E; Nakata J; Nagai A (2004). Ultrafine carbon black particles stimulate 

proliferation of human airway epithelium via EGF receptor-mediated signaling pathway. Am J Physiol Lung Cell 

Mol Physiol, 287: L1127-L1133. 157040

Tankersley CG; Bierman A; Rabold R (2007). Variation in heart rate regulation and the effects of particle exposure in 

inbred mice. Inhal Toxicol, 19: 621-629. 097910

Tankersley CG; Campen M; Bierman A; Flanders SE; Broman KW; Rabold R (2004). Particle effects on heart-rate 

regulation in senescent mice. Inhal Toxicol, 16: 381-390. 094378

Tankersley CG; Champion HC; Takimoto E; Gabrielson K; Bedja D; Misra V; El-Haddad H; Rabold R; Mitzner W (2008). 

Exposure to inhaled particulate matter impairs cardiac function in senescent mice. Am J Physiol Regul Integr Comp 

Physiol, 295: R252-R263. 157043

Tao F; Kobzik L (2002). Lung macrophage-epithelial cell interactions amplify particle-mediated cytokine release. Am J 

Respir Cell Mol Biol, 26: 499-505. 157044

Tesfaigzi Y; McDonald JD; Reed MD; Singh SP; De Sanctis GT; Eynott PR; Hahn FF; Campen MJ; Mauderly JL (2005). 

Low-level subchronic exposure to wood smoke exacerbates inflammatory responses in allergic rats. Toxicol Sci, 

88: 505-513. 156116

Tesfaigzi Y; Singh SP; Foster JE; Kubatko J; Barr EB; Fine PM; McDonald JD; Hahn FF; Mauderly JL (2002). Health 

effects of subchronic exposure to low levels of wood smoke in rats. Toxicol Sci, 65: 115-125. 025575

Thomson E; Kumarathasan P; Goegan P; Aubin RA; Vincent R (2005). Differential regulation of the lung endothelin 

system by urban particulate matter and ozone. Toxicol Sci, 88: 103-113. 087554

Thomson E; Kumarathasan P; Vincent R (2006). Pulmonary expression of preproET-1 and preproET-3 mRNAs is altered 

reciprocally in rats after inhalation of air pollutants. Exp Biol Med, 231: 979-984. 097483

Tomita S; Maekawa S-I; Rahman M; Saito F; Kizu R; Tohi K; Ueno T; Nakase H; Gonzalez FJ; Hayakawa K; Korenaga T; 

Takahama Y (2006). Thymic involution produced by diesel exhaust particles and their constituents in mice. Toxicol 

Environ Chem, 88: 113-124. 097827

Tong Y; Zhang G; Li Y; Tan M; Wang W; Chen J; Hwu Y; Hsu P-C; Je JH; Margaritondo G; Song W; Jiang R; Jiang Z 

(2006). Synchrotron microradiography study on acute lung injury of mouse caused by PM(25) aerosols. Eur J 

Radiol, 58: 266-272. 097699

Totlandsdal AI; Refsnes M; Skomedal T; Osnes JB; Schwarze PE; Lag M (2008). Particle-induced cytokine responses in 

cardiac cell cultures--the effect of particles versus soluble mediators released by particle-exposed lung cells. 

Toxicol Sci, 106: 233-241. 157056

Tozuka Y; Watanabe N; Ohsawa M; Toriba A; Kizu R; Hayakawa K (2004). Transfer of polycyclic aromatic hydrocarbons 

to fetuses and breast milk of rats exposed to diesel exhaust. Eisei Kagaku, 50: 497-502. 090864

December 2009 D-193 
Tsukue N; Tsubone H; Suzuki AK (2002). Diesel exhaust affects the abnormal delivery in pregnant mice and the growth of 

their young. Inhal Toxicol, 14: 635-651. 030593

Tsukue N; Yoshida S; Sugawara I; Taked K (2004). Effect of diesel exhaust on development of fetal reproductive function 

in ICR female mice. Eisei Kagaku, 50: 174-80. 096643

Tzeng H-P; Yang R-S; Ueng T-H; Lin-Shiau S-Y; Liu S-H (2003). Motorcycle exhaust particulates enhance 

vasoconstriction in organ culture of rat aortas and involve reactive oxygen species. Toxicol Sci, 75: 66-73. 097247

Tzeng H-P; Yang Rong S; Ueng T-H; Liu S-H (2007). Upregulation of cyclooxygenase-2 by motorcycle exhaust 

particulate-induced reactive oxygen species enhances rat vascular smooth muscle cell proliferation. Chem Res 

Toxicol, 20: 1170-1176. 097883

U.S. EPA (1976). Air quality data - 1970 annual statistics. U.S. Environmental Protection Agency. Research Triangle Park, 

NC. EPA-450/2-76-019. 015607

Ueng T-H; Hung C-C; Kuo M-L; Chan P-K; Hu S-H; Yang P-C; Chang LW (2005). Induction of fibroblast growth factor-9 

and interleukin-1alpha gene expression by motorcycle exhaust particulate extracts and benzo(a)pyrene in human 

lung adenocarcinoma cells. Toxicol Sci, 87: 483-496. 097054

Ueng T-H; Wang H-W; Huang Y-P; Hung C-C (2004). Antiestrogenic effects of motorcycle exhaust particulate in MCF-7 

human breast cancer cells and immature female rats. Arch Environ Contam Toxicol, 46: 454-462. 096199

Umbuzeiro GA; Franco A; Martins MH; Kummrow F; Carvalho L; Schmeiser HH; Leykauf J; Stiborova M; Claxton LD 

(2008). Mutagenicity and DNA adduct formation of PAH, nitro-PAH, and oxy-PAH fractions of atmospheric 

particulate matter from Sao Paulo, Brazil. Mutat Res Genet Toxicol Environ Mutagen, 652: 72-80. 190491

Upadhyay D; Panduri V; Ghio A; Kamp DW (2003). Particulate matter induces alveolar epithelial cell DNA damage and 

apoptosis: role of free radicals and the mitochondria. Am J Respir Cell Mol Biol, 29: 180-187. 097370

Upadhyay S; Stoeger T; Harder V; Thomas RF; Schladweiler MC; Semmler-Behnke M; Takenaka S; Karg E; Reitmeir P; 

Bader M; Stampfl A; Kodovanti U; Schulz H (2008). Exposure to ultrafine carbon particles at levels below 

detectable pulmonary inflammation affects cardiovascular performance in spontaneously hypertensive rats. Part 

Fibre Toxicol, 5: 19. 159345

Valavanidis A; Vlahoyianni T; Fiotakis K (2005). Comparative study of the formation of oxidative damage marker 8-

hydroxy-2'-deoxyguanosine (8-OHdG) adduct from the nucleoside 2'-deoxyguanosine by transition metals and 

suspensions of particulate matter in relation to metal content and redox reactivity. Free Radic Res, 39: 1071-1081. 

096432

Veranth J; Kaser E; Veranth M; Koch M; Yost G (2007). Cytokine responses of human lung cells (BEAS-2B) treated with 

micron-sized and nanoparticles of metal oxides compared to soil dusts. Part Fibre Toxicol, 4: 2. 090346

Veranth JM; Moss TA; Chow JC; Labban R; Nichols WK; Walton JC; Walton JG; Yost GS (2006). Correlation of in vitro 

cytokine responses with the chemical composition of soil-derived particulate matter. Environ Health Perspect, 114: 

341-349. 087479

Veranth JM; Reilly CA; Veranth MM; Moss TA; Langelier CR; Lanza DL; Yost GS (2004). Inflammatory cytokines and 

cell death in BEAS-2B lung cells treated with soil dust, lipopolysaccharide, and surface-modified particles. Toxicol 

Sci, 82: 88-96. 087480

Veras MM; Damaceno-Rodrigues NR; Caldini EG; Maciel Ribeiro AA; Mayhew TM; Saldiva PH; Dolhnikoff M (2008). 

Particulate urban air pollution affects the functional morphology of mouse placenta. Biol Reprod, 79: 578-584. 

190493

Veras MM; Damaceno-Rodrigues NR; Guimarães Silva RM; Scoriza JN; Saldiva PH; Caldini EG; Dolhnikoff M (2009). 

Chronic exposure to fine particulate matter emitted by traffic affects reproductive and fetal outcomes in mice. 

Environ Res, 109: 536-543. 190496

Veronesi B; De Haar C; Lee L; Oortgiesen M (2002). The surface charge of visible particulate matter predicts biological 

activation in human bronchial epithelial cells. Toxicol Appl Pharmacol, 178: 144-154. 024599

Veronesi B; Makwana O; Pooler M; Chen LC (2005). Effects of subchronic exposures to concentrated ambient particles: 

VII. Degeneration of dopaminergic neurons in Apo E-/- mice. Inhal Toxicol, 17: 235-241. 087481

December 2009 D-194 
Verstraelen S; Van Den Heuvel R; Nelissen I; Witters H; Verheyen G; Schoeters G (2005). Flow cytometric characterisation 

of antigen presenting dendritic cells after in vitro exposure to diesel exhaust particles. Toxicol In Vitro, 19: 903-

907. 096872

Vogel CFA; Sciullo E; Wong P; Kuzmicky P; Kado N; Matsumura F (2005). Induction of proinflammatory cytokines and 

C-reactive protein in human macrophage cell line U937 exposed to air pollution particulates. Environ Health 

Perspect, 113: 1536-1541. 087891

Walczak-Drzewiecka A; Wyczolkowska J; Dastych J (2003). Environmentally relevant metal and transition metal ions 

enhance Fc epsilon RI-mediated mast cell activation. Environ Health Perspect, 111: 708-713. 188803

Wallenborn JG; Evansky P; Shannahan JH; Vallanat B; Ledbetter AD; Schladweiler MC; Richards JH; Gottipolu RR; 

Nyska A; Kodavanti UP (2008). Subchronic inhalation of zinc sulfate induces cardiac changes in healthy rats. 

Toxicol Appl Pharmacol, 232: 69-77. 191171

Wallenborn JG; McKee JK; Schladweiler MC; Ledbetter AD; Kodavanti UP (2007). Systemic translocation of particulate 

matter-associated metals following a single intratracheal instillation in rats. J Toxicol Sci, 98: 231-239. 156144

Wan ECH; Yu JZ (2006). Determination of sugar compounds in atmospheric aerosols by liquid chromatography combined 

with positive electrospray ionization mass spectrometry. J Chromatogr A, 1107: 175-181. 157104

Wang Y-Z; Ingram JL; Walters DM; Rice AB; Santos JH; Van Houten B; Bonner JC (2003). Vanadium-induced STAT-1 

activation in lung myofibroblasts requires H2O2 and P38 MAP kinase. Free Radic Biol Med, 35: 845-855. 157106

Watanabe N (2005). Decreased number of sperms and Sertoli cells in mature rats exposed to diesel exhaust as fetuses. 

Toxicol Lett, 155: 51-58. 087985

Wegesser TC; Last JA (2008). Lung response to coarse PM: bioassay in mice. Toxicol Appl Pharmacol, 230: 159-166. 

190506

Wellenius GA; Batalha JRF; Diaz EA; Lawrence J; Coull BA; Katz T; Verrier RL; Godleski JJ (2004). Cardiac effects of 

carbon monoxide and ambient particles in a rat model of myocardial infarction. Toxicol Sci, 80: 367-376. 087874

Wellenius GA; Coull BA; Batalha JRF; Diaz EA; Lawrence J; Godleski JJ (2006). Effects of ambient particles and carbon 

monoxide on supraventricular arrhythmias in a rat model of myocardial infarction. Inhal Toxicol, 18: 1077-1082. 

156152

Wellenius GA; Coull BA; Godleski JJ; Koutrakis P; Okabe K; Savage ST (2003). Inhalation of concentrated ambient air 

particles exacerbates myocardial ischemia in conscious dogs. Environ Health Perspect, 111: 402-408. 055691

Whitekus MJ; Li N; Zhang M; Wang M; Horwitz MA; Nelson SK; Horwitz LD; Brechun N; Diaz-Sanchez D; Nel AE 

(2002). Thiol antioxidants inhibit the adjuvant effects of aerosolized diesel exhaust particles in a murine model for 

ovalbumin sensitization. J Immigr Minor Health, 168: 2560-2567. 157142

Wichers LB; Nolan JP; Winsett DW; Ledbetter AD; Kodavanti UP; Schladweiler MCJ; Costa DL; Watkinson WP (2004). 

Effects of instilled combustion-derived particles in spontaneously hypertensive rats Part II: pulmonary responses. 

Inhal Toxicol, 16: 407-419. 055636

Wichers LB; Rowan WH, 3rd; Nolan JP; Ledbetter AD; McGee JK; Costa DL; Watkinson WP (2006). Particle deposition 

in spontaneously hypertensive rats exposed via whole-body inhalation: measured and estimated dose. Toxicol Sci, 

93: 400-410. 103806

Wilson MR; Foucaud L; Barlow PG; Hutchison GR; Sales J; Simpson RJ; Stone V (2007). Nanoparticle interactions with 

zinc and iron: Implications for toxicology and inflammation. Toxicol Appl Pharmacol, 225: 80-89. 097268

Win-Shwe TT; Yamamoto S; Ahmed S; Kakeyama M; Kobayashi T; Fujimaki H (2006). Brain cytokine and chemokine 

mRNA expression in mice induced by intranasal instillation with ultrafine carbon black. Toxicol Lett, 63: 153-160. 

088415

Win-Shwe TT; Yamamoto S; Fujitani Y; Hirano S; Fujimaki H (2008). Spatial learning and memory function-related gene 

expression in the hippocampus of mouse exposed to nanoparticle-rich diesel exhaust. Neurotoxicology, 29: 940-

947. 190146

Witten ML; Wong SS ; Sun NN; Keith I; Kweon C; Foster DE; Schauer JJ; Sherrill DL (2005). Neurogenic responses in rat 

lungs after nose-only exposure to diesel exhaust. Health Effects Institute. Boston, MA. 087485

December 2009 D-195 
Wold LE; Simkhovich BZ; Kleinman MT; Nordlie MA; Dow JS; Sioutas C; Kloner RA (2006). In vivo and in vitro models 

to test the hypothesis of particle-induced effects on cardiac function and arrhythmias. Cardiovasc Toxicol, 6: 69-78. 

097028

Wong SS; Sun NN; Keith I; Kweon C-B; Foster DE; Schauer James J; Witten ML (2003). Tachykinin substance P signaling 

involved in diesel exhaust-induced bronchopulmonary neurogenic inflammation in rats. Arch Toxicol, 77: 638-650. 

097707

Wottrich R; Diabate S; Krug HF (2004). Biological effects of ultrafine model particles in human macrophages and 

epithelial cells in mono- and co-culture. Int J Hyg Environ Health, 207: 353-361. 094518

Wu W; Samet JM; Silbajoris R; Dailey LA; Sheppard D; Bromberg PA; Graves LM (2004). Heparin-binding epidermal 

growth factor cleavage mediates zinc-induced epidermal growth factor receptor phosphorylation. Am J Respir Cell 

Mol Biol, 30: 540-547. 096949

Wu W; Silbajoris RA; Whang YE; Graves LM; Bromberg PA; Samet JM (2005). p38 and EGF receptor kinase-mediated 

activation of the phosphatidylinositol 3-kinase/Akt pathway is required for Zn2+-induced cyclooxygenase-2 

expression. Am J Physiol Lung Cell Mol Physiol, 289: L883-L889. 097350

Wu W; Wang X; Zhang W; Reed W; Samet JM; Whang YE; Ghio AJ (2003). Zinc-induced PTEN protein degradation 

through the proteasome pathway in human airway epithelial cells. J Biol Chem, 278: 28258-28263. 199749

Wu YH; Vincent JH (2007). A modified Marple-type cascade impactor for assessing aerosol particle size distributions in 

workplaces. J Occup Environ Hyg, 4: 798-807. 098412

Xu D-Q; Zhang W-L (2004). Monitoring of pollution of air fine particles (PM25) and study on their genetic toxicity. 

Biomed Environ Sci, 17: 452-458. 097231

Yacobi NR; Phuleria HC; Demaio L; Liang CH; Peng CA; Sioutas C; Borok Z; Kim KJ; Crandall ED (2007). Nanoparticle 

effects on rat alveolar epithelial cell monolayer barrier properties. Toxicol In Vitro, 21: 1373-1381. 156166

Yamamoto S; Tin Tin Win S; Ahmed S; Kobayashi T; Fujimaki H (2006). Effect of ultrafine carbon black particles on 

lipoteichoic acid-induced early pulmonary inflammation in BALB/c mice. Toxicol Appl Pharmacol, 213: 256-266. 

096671

Yanagisawa R; Takano H; Inoue K; Ichinose T; Sadakane K; Yoshino S; Yamaki K; Kumagai Y; Uchiyama K; Yoshikawa 

T; Morita M (2003). Enhancement of acute lung injury related to bacterial endotoxin by components of diesel 

exhaust particles. Thorax, 58: 605-612. 087487

Yanagisawa R; Takano H; Inoue KI; Ichinose T; Sadakane K; Yoshino S; Yamaki K; Yoshikawa T; Hayakawa K (2006). 

Components of diesel exhaust particles differentially affect Th1/Th2 response in a murine model of allergic airway 

inflammation. Clin Exp Allergy, 36: 386-395. 096458

Yang C-Y; Tseng Y-T; Chang C-C (2003). Effects of air pollution on birthweight among children born between 1995 and 

1997 in Kaohsiung, Taiwan. J Toxicol Environ Health A Curr Iss, 66: 807-816. 087886

Yatera K; Hsieh J; Hogg James C; Tranfield E; Suzuki H; Shih C-H; Behzad Ali R; Vincent R; van Eeden Stephan F 

(2008). Particulate matter air pollution exposure promotes recruitment of monocytes into atherosclerotic plaques. 

Am J Physiol Heart Circ Physiol, 294: H944-H953. 157162

Yauk C; Polyzos A; Rowan-Carroll A; Somers CM; Godschalk RW; Van Schooten FJ; Berndt ML; Pogribny IP; Koturbash 

I; Williams A; Douglas GR; Kovalchuk O (2008). Germ-line mutations, DNA damage, and global hypermethylation 

in mice exposed to particulate air pollution in an urban/industrial location. PNAS, 105: 605-610. 157164

Yin H; Too HP; Chow GM (2005). The effects of particle size and surface coating on the cytotoxicity of nickel ferrite. 

Biomaterials, 26: 5818-5826. 088133

Yin X-J; Schafer R; Ma JYC; Antonini JM; Roberts JR; Weissman DN; Siegel PD; Ma JKH (2003). Alteration of 

pulmonary immunity to Listeria monocytogenes by diesel exhaust particles (DEPs) II Effects of DEPs on T-cellmediated immune responses in rats. Environ Health Perspect, 111: 524-530. 096127

Yin XJ; Dong CC; Ma JY; Roberts JR; Antonini JM; Ma JK (2007). Suppression of phagocytic and bactericidal functions 

of rat alveolar macrophages by the organic component of diesel exhaust particles. J Toxicol Environ Health A Curr 

Iss, 70: 820-828. 198980

December 2009 D-196 
Yin XJ; Dong CC; Ma JYC; Antonini JM; Roberts JR; Stanley CF; Schafer R; Ma JKH (2004). Suppression of cellmediated immune responses to listeria infection by repeated exposure to diesel exhaust particles in brown Norway 

rats. Toxicol Sci, 77: 263-271. 097685

Yin XJ; Ma JYC; Antonini JM; Castranova V; Ma JKH (2004). Roles of reactive oxygen species and heme oxygenase-1 in 

modulation of alveolar macrophage-mediated pulmonary immune responses to listeria monocytogenes by diesel 

exhaust particles. Toxicol Sci, 82: 143-153. 087983

Ying Z; Kampfrath T; Thurston G; Farrar B; Lippmann M; Wang A; Sun Q; Chen LC; Rajagopalan S (2009). Ambient 

particulates alter vascular function through induction of reactive oxygen and nitrogen species. Toxicol Sci, 111: 80-

88. 190111

Yokohira M; Takeuchi H; Yamakawa K; Saoo K; Matsuda Y; Zeng Y; Hosokawa K; Imaida K (2007). Bioassay by 

intratracheal instillation for detection of lung toxicity due to fine particles in F344 male rats. Exp Toxicol Pathol, 

58: 211-221. 097976

Yokota S; Furuya M; Seki T; Marumo H; Ohara N; Kato A (2004). Delayed exacerbation of acute myocardial 

ischemia/reperfusion-induced arrhythmia by tracheal instillation of diesel exhaust particles. Inhal Toxicol, 16: 319-

331. 096516

Yokota S; Mizuo K; Moriya N; Oshio S; Sugawara I; Takeda K (2009). Effect of prenatal exposure to diesel exhaust on 

dopaminergic system in mice. Neurosci Lett, 449: 38-41. 190518

Yokota S; Seki T; Furuya M; Ohara N (2005). Acute functional enhancement of circulatory neutrophils after intratracheal 

instillation with diesel exhaust particles in rats. Inhal Toxicol, 17: 671-679. 096003

Yokota S; Seki T; Naito Y; Tachibana S; Hirabayashi N; Nakasaka T; Ohara N; Kobayashi H (2008). Tracheal instillation of 

diesel exhaust particles component causes blood and pulmonary neutrophilia and enhances myocardial oxidative 

stress in mice. J Toxicol Sci, 33: 609-620. 190109

Yoshida S; Ono N; Tsukue N; Oshio S; Umeda T; Takano H; Takeda K (2006). In utero exposure to diesel exhaust 

increased accessory reproductive gland weight and serum testosterone concentration in male mice. Environ Sci J 

Integr Environ Res, 13: 139-147. 097015

Yoshida S; Takedab K (2004). The effects of diesel exhaust on murine male reproductive function. Eisei Kagaku, 50: 210-

214. 097760

Yoshida S; Yoshida M; Sugawara I; Takeda K (2006). Mice strain differences in effects of fetal exposure to diesel exhaust 

gas on male gonadal differentiation. Environ Sci J Integr Environ Res, 13: 117-123. 156170

Yun Y-P; Joo J-D; Lee J-Y; Nam H-Y; Kim Y-H; Lee K-H; Lim C-S; Kim H-J; Lim Y-G; Lim Y (2005). Induction of 

nuclear factor-"kappa"B activation through TAK1 and NIK by diesel exhaust particles in L2 cell lines. Toxicol Lett, 

155: 337-342. 088302

Zanchi AC; Venturini CD; Saiki M; Nascimento Saldiva PH; Tannhauser Barros HM; Rhoden CR (2008). Chronic nasal 

instillation of residual-oil fly ash (ROFA) induces brain lipid peroxidation and behavioral changes in rats. Inhal 

Toxicol, 20: 795-800. 157173

Zelikoff JT; Chen LC; Cohen MD; Fang K; Gordon T; Li Y; Nadziejko C; Schlesinger RB (2003). Effects of inhaled 

ambient particulate matter on pulmonary antimicrobial immune defense. Inhal Toxicol, 15: 131-150. 039009

Zelikoff JT; Schermerhorn KR; Fang K; Cohen MD; Schlesinger RB (2002). A role for associated transition metals in the 

immunotoxicity of inhaled ambient particulate matter. Environ Health Perspect, 5: 871-875. 037797

Zhang J; Ghio AJ; Chang W; Kamdar O; Rosen GD; Upadhyay D (2007). Bim mediates mitochondria-regulated particulate 

matter-induced apoptosis in alveolar epithelial cells. FEBS Lett, 581: 4148-4152. 156179

Zhang Q; Kleeberger SR; Reddy SP (2004). DEP-induced fra-1 expression correlates with a distinct activation of AP-1-

dependent gene transcription in the lung. Am J Physiol Lung Cell Mol Physiol, 286: L427-L436. 157183

Zhang Z; Che W; Liang Y; Wu M; Li N; Shu Y; Liu F; Wu D (2007). Comparison of cytotoxicity and genotoxicity induced 

by the extracts of methanol and gasoline engine exhausts. Toxicol In Vitro, 21: 1058-1065. 157186

Zhao H; Barger MW; Ma JKH; Castranova V; Ma JYC (2006). Cooperation of the inducible nitric oxide synthase and 

cytochrome P450 1A1 in mediating lung inflammation and mutagenicity induced by diesel exhaust particles. 

Environ Health Perspect, 114: 1253-1258. 100996

December 2009 D-197 
December 2009 D-198 

Zhao L; Wang X; He Q; Wang H; Sheng G; Chan LY; Fu J; Blake DR (2004). Exposure to hazardous volatile organic 

compounds, PM sub(10) and CO while walking along streets in urban Guangzhou, China. Atmos Environ, 38: 

6177-6184. 100972

Zhong W; Levin L; Reponen T; Hershey GK; Adhikari A; Shukla R; LeMasters G (2006). Analysis of short-term influences 

of ambient aeroallergens on pediatric asthma hospital visits. Sci Total Environ, 370: 330-336. 093264

Zhou Y-M; Zhong C-Y; Kennedy IM; Pinkerton KE (2003). Pulmonary responses of acute exposure to ultrafine iron 

particles in healthy adult rats. Environ Toxicol, 18: 227-235. 087940