# Annex D. T oxicological 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/m 3 ; Other emissions measured: non-methane hydrocarbons (7.7 ppm), NO 2 (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 doc ument 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)
Zn 2+ and Cu 2+ 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; CuSO 4 /ZnSO 4 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’swas observed, and the activity of the particle suspension was stronger than the filtrate, with the difference being significant starting at 30 μg/mL. Both Zn 2+ and Cu 2+ in sulfate salts (10-100 μmol) induced relaxation in pre-contracted aortic rings, with Cu 2+ having a greater effect than Zn 2+ 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)
Cu 2+ and Zn 2+ solutions
Particle Size: PM: 4.6 μm (GSD = 3.2) Route: In Vitro
Dose/Concentration: PM and PMF Suspensions: 10-100 μg/mL; CuSO 4 or ZnSO 4 :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: AfterPM 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 duringthe 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,
ZnSO 4 Particle Size: PM: 4.6 μm (GSD = 3.2); Carbon Particles: 44 nm Route: In Vitro
Dose/Concentration: PM: 1, 50, 100 μg/mL; ZnSO 4 : 50 μmol Time to Analysis: 30 min incubation Effect of EHC-93 filtrate on Ca 2+ Uptake in Cardiomyocytes: Both PMF and Zn 2+ inhibited ATP or ionophore-stimulated Ca 2+ 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/m 3

, BC: 1.3-32(7.5 ± 6.1) μg/m 3 , Particle count: 3000-69300(18230 ± 13.151) particles/cm 3 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/m 3

, BC: 1.3-32 (7.5 ± 5.6) μg/m 3 , Particle number: 3000-69300 (20381 ± 13075) particles/cm 3 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/m 3 ; NO X = 102 ppm LWDE: PM = 0.512 mg/m 3 ; NO X = 19 ppm; PM = 0.770 mg/m 3 ; NO X = 105 ppm LPMF: PM = 0.006 mg/m 3 ; NO X = 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-induceddilation 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/m 3 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 prolongedamong 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/m 3 Gasoline engine emissions: 5 to 60 μg/m 3 (at dilutions of 10:1, 15:1, and 90:1) Mean concentrations of PM: 61 μg/m 3 ; NO X : 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 upregulatedET-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 ischemicoxidant 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 Fe 2 (SO 4 ) 3 Nickel sulfate NiSO 4 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 smallinitial increases. Minor effects observed after lowdose Fe and Ni instillation; biphasic changes occurred after high-dos e 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: PM 2.5 Route: Nose-only Inhalation
Dose/Concentration: 315.55 μg/m 3 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: PM 2.5 Route: Nose-only Inhalation
Dose/Concentration: Spring exposure: 202.0 ± 68.8 μg/m 3 ; Mean number concentration: 2.30 × 10 5 particles/cm 3 (range: 7.12 ×10 3 - 8.26 ×10 5 ) Summer exposure: 141.0 ± 54.9 μg/m 3 ; Mean number concentration: 2.78 ×10 5 particles/cm 3 (range: 7.76 ×10 3 - 8.87 × 10 5 ) 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: PM 2.5 (0.1-2.5 μm) Route: Nose-only Inhalation
Dose/Concentration: 202.0 ± 68.8 μg/m 3 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 (SiO 2 ) and SRM-154b (TiO 2 ) from the NIST EHC-93 from Ontario, Canada (EHCsol, EHCinsol)
Particle Size: EHC-93 median physical diameter: 0.4 μm; TiO 2 and SiO 2 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 levelsECE-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 upregulate 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: PM 2.5 Route: Whole-body Inhalation
Dose/Concentration: 19.7 μg/m 3 averageconcentration over 5 mo (daily average exposure concentration was 110 μg/m 3 ) 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: PM 2.5 Route: Whole-body Inhalation
Dose/Concentration: Mean exposure concentration: 110 μg/m 3 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: PM 2.5 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 doubledthe 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 thelevel 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: PM 2.5 Route: Whole-body Inhalation
Dose/Concentration: Average concentration 354 μg/m 3 Time to Analysis: 8 h/days for 3 consecutivedays; 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/m 3 ; 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: (PM 2.5 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: PM 2.5 concentrated from Tuxedo, NY (April-Sept 2003)
Particle Size: NR Route: Whole-body Inhalation
Dose/Concentration: Avg 120 μg/m 3 (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 ofthrombomodulin, 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- β 1 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 aMotorway 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: PM 2.5 Route: CAPs: Whole-body Inhalation; CPZ: IP Injection or Aerosol
Dose/Concentration: CAPs: mean mass concentration: 218 ± 23 μg/m 3 ; 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 incardiac 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/m 3 ; fCB: 1.40 mg/m 3 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 PM 10 : (Carbon Black from Degussa Ltd, Frankfurt, Germany)
Particle Size: PM 10 Route: Cell Culture
Dose/Concentration: PM 10 : 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 PM 10 had an enhanced ability to cause clotting. H 2 O 2 also increased clotting activity. Apoptosis was significantly increased in MPs exposed to PM 10 and LPS as shown by annexin V binding. TF gene expression was enhanced in MPs exposed to PM 10 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 (ZnSO 4 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 4×10 5 /cm 3 ; fp: 2.7 ×10 5 /cm 3 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 largenumber 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: PM 10 ; 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 PM 10 : 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 instilla tion. 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-1 β , TNF- α , IL-8 and MCP-1. No effect on m-CSF and MIP-1 β . 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) (O 2 - 20%, CO- 1.3-4.8 ppm, NO- <2.5-5.9 ppm, NO 2 - <0.25-1.2ppm, SO 2 - 0.2-0.3 ppm, OC/EC- 0.3 ± 0.03)
Particle Size: Number Median Diameter: Low- 83 ± 2 nm, High- 88. 2nm; Volume Median Diameter: Low207 ± 2 nm, High- 225 ± 2 nm Route: Inhalation
Dose/Concentration: Low: 507 ± 4 μg/m 3 ; High: 2201 ± 14 μg/m 3 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, Zninduced significant changes in the gene expression of Kv4.2 and KvQLt, α -1 subunit of Ltype Ca channel, Cx43, IL-6, and IL-1 α . 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: O 3 and NO 2 . Particle Size: 389 ± 2 nm Route: Whole-body Inhalation
Dose/Concentration: CAPs: 131 ± 99 μg/m 3 (range 13-441 μg/m 3 )
O 3 : 10 ppb
NO 2 : 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 (PM 2.5 ) Route: Whole-body Inhalation
Dose/Concentration: CAPs: average mass concentration: 300 ± 60 μg/m 3 ; ROFA: 1.7 mg/m 3 ; CB: 170 μg/m 3 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 ashincreased the expression of collagen 1 and TGFβ 1, 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 + orphenylephrine 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/m 3 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 UFPexposed 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/m 3 . Mean CAPs Concentration: 133μg/m 3

. Mean Concentrations of O 3 and NO 2 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 CH 2 Cl 2 ); Washed DEP+LPS and DEP-OC+LPS
Particle Size: PM 2.5 Route: IT Instillation
Dose/Concentration: Washed DEP: 4 mg/kg. DEP-OC: 4 mg/kg. LPS: 2.5 mg/kg. WashedDEP+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 vascularpermeability. 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/m 3 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. Theup-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: 1×10 7 and 5 ×10 7 total particles infused
300 m 2 /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 ofrolling/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 wasaltered 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-Laltered 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: PM 2.5 Route: Whole-body Inhalation. Ex Vivo.
Dose/Concentration: In vivo: 350 μg/m 3 ; Ex vivo: PM 2.5 concentration 2-3 mg/m 3 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 SaintHelen’s ash (SH); G3: whole suspension of oil combustion PM at high concentration (PM-HD); G4: whole suspension of oil combustion PM atlow concentration (PM-LD); G5: salineleachable fraction of PM highconcentration suspension; G6: zinc sulfate
Particle Size: PM 2.5 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 receivingsoluble 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/m 3 CAP-UF 269-556 μg/m 3 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 NO X exposures Particle Size: NR Route: Whole-body Inhalation
Dose/Concentration: PM (mg/m 3 ): 0.01, 0.109, 0.54, 1.09, 0.01 (from 1.09 concentration w/o PM)
NO X (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, SO 2 , NO 2 , nitrate, sulfate
Particle Size: 0.01- 2.5 μm Route: Nose-only Inhalation
Dose/Concentration: 315.6 μg/m 3 (Low) or 684.5 μg/m 3 (High) Time to Analysis: Low: Exposed for 6 h. Sacrificed 36 h post-exposureHigh: Exposed for 4.5 h. Sacrificed 36 h postexposurePulmonary 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 bufferadministered to 8 nondiabetic rats CAPs: Hsin-Chuang, Taipei
Particle Size: PM: 0.01-2.5 μm Route: IT Instillation
Dose/Concentration: PM 2.5 : 200 μg in 0.5 mL saline. Components (μg/m 3 ): (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) ofdiabetic (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 inplasma 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 PM 10 , CO, NO 2 , and SO 2 from Universidade de Sao Paulo, Brazil.
Particle Size: PM 10 Route: Whole-body Inhalation
Dose/Concentration: Mean (± SD) concentrations were: CO 2 : 2.06 ± 0.08 ppm (8h mean); NO 2 : 104.75 ± 42.62 μg/m 3 (24 h mean); SO 2 : 11.07 ± 5.32 μg/m 3 (24 h mean); PM 10 : 35.52 ± 12.84 μg/m 3 (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 HPAEC s 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 H 2 0 2 Release: Within minutes after UPs treatment, HPAEC increased H 2 O 2 production that could be inhibited by DPI, APO, and NaN 3 . The water soluble fraction of UPs as well as its two transition metal components Cu and V, also stimulated H 2 O 2 production. NaN 3 inhibited H 2 O 2 production stimulated by Cu andV, whereas DPI and APO inhibited only Cu-stimulated H 2 O 2 production. Inhibitors of other H 2 O 2 -producing enzymes, including N-methyl-Larginine, indomethacin, allopurinol, cimetidine, rotenone, and antimycin, had no effects.
Effects of UP-induced H 2 0 2 on MAPK Activation: DPI but not NaN 3 attenuated UPsinduced pulmonary vasoconstriction and phosphorylation of ERK1/2 and p38 MAPKs. Knockdown of p47phox gene expression by small interfering RNA attenuated UPs-induced H 2 O 2 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: PM 2.5 Route: Whole-body Inhalation
Dose/Concentration: PM 2.5 concentrated tenfold, producing an average of 113 μg/m 3 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 thethree intervals. There were significant associations between PM 2.5 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 PM 2.5 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 PM 2.5 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: PM 2.5 Route: Inhalation
Dose/Concentration: PM 2.5 average concentration: 110 μg/m 3 , Long-term average: 19.7 μg/m 3 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- κ B. 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: PM 2.5 Route: Whole-body Inhalation
Dose/Concentration: Average daily CAPs: 85.6 μg/ m3; Average daily Ni: 43 ng/m 3 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/m 3 andusually 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, NO X , CO, and Total Hydrocarbons (THC)
Particle Size: NR Route: Whole-body Inhalation
Dose/Concentration: FA: PM (2 μg/m 3 ), NO X (0 ppm), CO (0.1 ppm), HC (0.1 ppm); Low (1: 90 dilution of exhaust): PM (8 μg/m 3 ), NO X (2 ppm), CO (9 ppm), HC (0.9 ppm); Mid (1: 20): PM (39 μg/m 3 ), NO X (12 ppm), CO (50 ppm), HC (8.4 ppm); High (1: 12): PM (61 μg/m 3 ), NO X (19 ppm), CO (80 ppm), HC (12 ppm); High-filtered (1:12): PM (2 μg/m 3 ), NO X (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/m 3 and 40 μg/m 3 ; PM Whole Exhaust: 60 μg/m 3 ; or Filtered Exhaust w/ gases matching the 60 μg/m 3 concentration. Inhalation exposure to gasoline engine emissions resulted in increased aortic mRNA expression of matrix metalloproteinase-3 (MMP-3), MMP-7, andMMP-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/m 3 , NO 2 : 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 7days. 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 vascularET-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 O 2 increased after DEP, but not when compared for each animal. CO 2 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 precip itator, 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/m ouse; 0.005, 0.05, 0.5 mg/kg PSB: 0.1, 1 or 10 μg/m ouse; 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 hi gher 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. PM 2.5 and PM 10 from Mexico City
Particle Size: PM 2.5 , PM 10 Route: In Vitro
Dose/Concentration: HUVEC TNF- α (10 ng/mL), and a PM range of 5, 10, 20, and 40 μg/cm 2 concentrations. Time to Analysis: 6 or 24 h (early and late adhesion molecules respectively) Results showed that both PM 2.5 and PM 10 induced the adhesion of U937 cells to HUVEC, and their maximal effect was observed at 20 μg/cm 2 . This adhesion was associated with an increase in the expression of all adhesion molecules evaluated for PM 10 , and E-selectin, Pselectin, and ICAM-1 for PM 2.5 . In general themaximum expression of adhesion molecules induced by PM 2.5 and PM 10 was obtained with 20 μg/cm 2 ; however PM 10 -induced expression was observed from 5 μg/cm 2 . 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 (CoSO 4 ·7H 2 O), Vanadium pentoxide(V 2 O 5 ) Gallium arsenide (GaAs), Ni oxide (NiO), Ni subsulfide (Ni 3 S 2 ), Ni sulfate hexahydrate (NiSO 4 · 6H 2 O), talc, and Mo trioxide (MoO 3 )
Particle Size: MMAD particle size (μm): InP (1.1-1.3), CoSO 4 ·7H 2 O (1.5- 1.8), V 2 O 5 : (1.0), GaAs: (1.0) Route: Inhalation
Dose/Concentration: High-Dose Concentration in Chronic Studies, Male (μg/m 3 ): InP: 0.3, CoSO 4 ·7H 2 O: 3.0, V 2 O 5 : 4.0, GaAs: 1.0 High-Dose Concentration in Sub-Chronic Studies, Male or Female (μg/m 3 ): InP: 100, CoSO 4 ·7H 2 O: 30, V 2 O 5 : 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 fromthe 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 CoSO 4 ·7H 2 O), while marginal increases of arteritis were detected in 2 additional studies (V 2 O 5 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 normalvascular 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 PM 10 from ambient air in Düsseldorf, Germany
Particle Size: PM 10 Route: IT Instillation
Dose/Concentration: PM 10 : 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 PM 10 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 (PM 2.5 ) from Tuxedo, NY. (SO 2 , NO 2 O 3 and NH 3 were removed prior to exposure).
H 2 SO 4 (fine and ultrafine)
Particle Size: Ultrafine H 2 SO 4 50-75 nm (MMAD) Route: Nose-only Inhalation
Dose/Concentration: CAPs: 80 and 66 μg/m 3 (avg 73); Fine H 2 SO 4 : 299, 280, 119, and 203 μg/m 3 (avg 225); Ultrafine H 2 SO 4 : 140, 565, 416, 750 μg/m 3 (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 H 2 SO 4 aerosol also caused a significant decrease in respiratory rate similar to the effect of CAPs. Ultrafine H 2 SO 4 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)
SO 2 Particle Size: PM (Size Range): 0.5- 2.5μm; UFC (MMAD): 30-50 nm Route: Nose-only Inhalation
Dose/Concentration: PM (μg/m 3 ): 161-200, avg. 180; UFC (μg/m 3 ): 500-1280, avg. 890; SO 2 (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 SO 2 . 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, SO 2 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 SO 2 .



### 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 werealso 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 minpost-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: UnmodifiedPolystyrene 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 (C 60 (OH) 24 ); Fluoresbrite Carboxylate Microspheres; Ox-LDL; Acetylated-LDL
Particle Size: Carbon Black and C 60 (OH) 24 : 7.1 nm (SD 2.4); Fluoresbrite Carboxylate Microspheres: 6 nm Route: Cell Culture
Dose/Concentration: CB: 1, 10, 100 μg/mL; C 60 (OH) 24 : 20, 100ng/mL Time to Analysis: RAW264.7+CB for 24 h, 13 days, and 50 days;
RAW264.7+ C 60 (OH) 24 for 24 h or 10 days;
RAW264.7+ C 60 (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. C 60 (OH) 24 alone or CB and C 60 (OH) 24 together w/ Ox-LDL induced cytotoxicmorphological changes, such as Ox-LDL uptakeinduced foam cell-like formation and decreased cell growth, in a dose-dependent manner. C 60 (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 C 60 (OH) 24 alone did not induce platelet aggregation, C 60 (OH) 24 facilitated ADP-induced platelet aggregation. C 60 (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/m 3 Time to Analysis: 6 h/day, 5 days/wk, for atotal 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: TiO 2 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 TiO 2 Exposed Rats: A23187-induced dilation was significantly attenuated. Sensitivity of Arteriol ar 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; TiO 2 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: TiO 2 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 TiO 2 Exposure and Arteriolar Dilation: Exposure caused a dose-dependent impairment of endothelium-dependent arteriolar dilation.
ROFA or TiO 2 Exposure and Arteriolar Constriction: Exposure did not affect microvascular constriction in response to PHE.
ROFA and TiO 2 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 TiO 2 and MPO: MPO was found inPMN 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 TiO 2 (DeGussa, Sigma-Aldrich)
Particle Size: Fine- 1 μm, UF- 21 nm Route: Whole-body Inhalation
Dose/Concentration: Concentrations: Fine- 3- 16 mg/m 3 ; UF- 1.5-12 mg/m 3 ; 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 TiO 2 under different conditions.

Reference: Nurkiewicz et al. (2009, 191961 ) Species: Rat
Gender: Male
Strain: SD Age: 7-8 wk Weight: NR Fine TiO 2 (Sigma-Aldrich, St. Louis, MO) (~99% rutile)
TiO 2 nanoparticles (DeGussaAeroxide TiO 2 P25, Parsippany, NJ) (80% anatase, 20% rutile)
Particle Size: Fine TiO 2 - MMAD: 402 nm, Primary size: <5 μm, ,CMD: 710 nm; Nano-TiO 2 - MMAD: 138 nm, Primary size: 21 nm, , CMD: 100 nm Route: Aerosol Inhalation
Dose/Concentration: 1.5-16mg/m 3 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-TiO 2 significantly impaired endothelium-dependent arteriolar dilation. Equivalent levels of arteriolar dysfunction were found in fine and nano-TiO 2 . 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-TiO 2 significantly increased the area of tissue containing nitrotyrosine in the lung and spinotrapezius microcirculation.
NO: Fine and nano-TiO 2 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 TiO 2 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 damagedcardiac 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 withSOD 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 FeCl 3 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: FeCl 3 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 (2.5×10 8 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, matrixmetalloproteinase- 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, NO 3 , SO 4 , NH 4 , metals
Particle Size: ~0.25 μm Route: Whole-body Inhalation
Dose/Concentration: Low: 30 μg/m 3 Mid-low: 100 μg/m 3 Mid-high: 300 μg/m 3 High: 1000 μg/m 3 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 ofboth 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/m 3 Mid-low: 100 μg/m 3 Mid-high: 300 μg/m 3 High: 1000 μg/m 3 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-LV-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/m 3 , Medium- 30.3 ± 11.8 μg/m 3 , High- 59.1 ± 28.3 μg/m 3 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 dataobtained 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/m 3 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 weretreated with 50 mg/kg NAC 1 h prior to UAPs instillation or CAPs inhal ation. NAC preventedchanges 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 PM 2.5 , collected from heavy traffic area in Sao Paulo, Brazil. PM 2.5 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: PM 2.5 Route: IT Instillation
Dose/Concentration: 100 or 500 μg of PM 2.5 . Time to Analysis: 24 h post-instillation Hematology: Total reticulocytes significantly increased at both PM 2.5 doses, while hematocritlevels 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 PM 2.5 . Pulmonary Vasculature: Significant dose-dependent decrease of intra-acinar pulmonary arteriole lumen/wall ratio was observed in both PM 2.5 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 PM 2.5 , collected from heavy traffic area in Sao Paulo, Brazil. PM 2.5 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: PM 2.5 Route: IT Instillation
Dose/Concentration: 50 and 100 μg of PM 2.5 . Time to Analysis: HR and SDNN were assessed immedi ately before in stillation, 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 PM 2.5 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, NO 2 , SO 2 , THC) (PM 2.5 composition- EC, OC, SO 4 , NH 4 , NO 3 )
Simulated downwind coal emissionatmospheres (SDCAs) (fly ash, gasphase pollutants, sulfate aerosols, NO, NO 2 , SO 2 ) 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/m 3 , SDCAs: 317-1072 μg/m 3 , RD: 306-954 μg/m 3 ; GEE: CO104 ppm, NO- 16.7 ppm, NO 2 - 1.1 ppm, SO 2 - 1.0 ppm, THC- 12ppm; SDCAs: CO- <1 ppm, NO- 0.19-0.62 ppm, NO 2 - 0.10-0.37 ppm, SO 2 - 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/m 3 PM 2.5 including 600 μg/m 3 PM 1 Time to Analysis: 4 h/day for 3 consecutivedays. 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 CO 2 : 2170, 6540 ppm NO: 7.0, 22.8 ppm NO X : 8.6, 28.3 ppm SO 2 : 0.83, 3.09 ppm NH 4 : ND Measured Major Components: NO, SO 2 , 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/m 3 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 30mo. 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: PM 2.5 from Tuxedo, NY. HFCD: High Fat Chow Diet
NCD: Normal Chow Diet
Particle Size: PM 2.5 Route: Whole-body Inhalation
Dose/Concentration: PM 2.5 : 85 μg/m 3 ; Daily concentration: 10.6 (SD 3.4) μg/m 3 (mean) Average exposure over 6 mo period: 15.2 μg/m 3

. Time to Analysis: Study diets fed for at least 10 wk prior to exposure to PM 2.5 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 PM 2.5 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 PM 2.5 : 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 PM 2.5 and fed HFCD vs. mice exposed to FA and fed HFCD were 33 (10) vs. 27 (13) units, respectively.
PM 2.5 and Vascular Inflammation: A 2.6-fold higher inducible NOS content was apparent in the mice exposed to PM 2.5 and fed HFCD compared with the mice exposed to FA and fed HFCD chow and a 4-fold increase in the mice exposed to PM 2.5 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 PM 2.5 Collected from Sterling Forest State Park, Tuxedo NY (40 miles NW of Manhattan)
Particle Size: PM 2.5 Route: Whole-body Inhalation
Dose/Concentration: Average Concentration of: 85 μg/m 3 CAPs in chamber. Average exposure over 6 mo was 15.2 μg/m 3

. 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 anincrease 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 increasedrapidly within the first hour in response to SRM1694a PM. The lowest dose of SRM PM 10 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 PM 2.5 or UFP
Particle Size: PM 2.5 ; UFP: <0.1 μm Route: Whole-body Inhalation
Dose/Concentration: Mean PM 2.5 concentration: 79.1 ± 7.4 μg/m 3 . Normalized PM 2.5 over 10wk period: 14.1 μg/m 3

. Time to Analysis: 6 h/day, 5 day/wk random exposure to PM 2.5 , 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. PM 2.5 , UFP, or FA, continued during AII infusion period. All = angiotensin II Mean Arterial Pressure (MAP): After AII infusion, MAP was significantly higher in PM 2.5 - 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 PM 2.5 - AII group. Superoxide production in the aorta was increased in the PM 2.5 . 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) PM 2.5 or UFP
Particle Size: PM 2.5 ; UFP: <0.1 μm Route: Cell Culture
Dose/Concentration: UFP, PM 2.5 : 10 or 50 μg/mL; All: 100 nmol/L Time to Analysis: Exposed to UFP or PM 2.5 and parameters measured at 0, 1, 3, 6, and 15 min. All = angiotensin II Exposure to UFPs and PM 2.5 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 MLCand MYPT-1 phosphorylation by UFPs suggesting a superoxide-mediated mechanism for PM 2.5 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/m 3 , 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- β 11 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 andadipocyte 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 PM 10 (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: PM 10 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: PM 10 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 PM 2.5 concentration- 401 ± 46 μg/m 3 , Average PM 10 concentration- 553 ± 49 μg/m 3 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/m 3 ; 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/m 3 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; O 3 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/m 3 ; O 3 : 0, 0.4, 0.8 ppm Time to Analysis: 4 h to particles, O 3 , or combination of particles and O 3 . 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 O 3 . Both pollutants transiently increased ET-B receptor mRNA expression, while O 3 decreased ET-A receptor mRNA levels. Coexposure to particles plus O 3 increased lung preproET-1 mRNA but notplasma 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; O 3 Particle Size: NR Route: Nose-only Inhalation
Dose/Concentration: EHC-93: 0, 50 mg/m 3 ; O 3 : 0, 0.8 ppm Time to Analysis: 4 h to particles, O 3 , or combination of particles and O 3 . 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 O 3 . 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 O 3 and particles, while altering lung preproET-1 and preproET-3 mRNA levels in a fashion similar to O 3 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 cm 3 two-stroke engine using 95% octane unleaded gasoline.
Particle Size: PM 1 , PM 2.5 , PM 10 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 unchang ed. MEPE increased theproduction 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- κ B-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 cm 3 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-Dain 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 m 2 particles/m 3

, Mass specific surface area- 807m 2 /g Route: Whole-body Inhalation
Dose/Concentration: 172 μg/m 3 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 (ZnSO 4 , aerosolized)
Particle Size: NR Route: Nose-only Inhalation
Dose/Concentration: 9.0 ± 2.1 μg/m 3 , 35 ± 8.1 μg /m 3 , 123.2 ± 29.6 μg /m 3 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/m 3 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/m 3

, Range: 161.3-957.3 μg/m 3 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 MyocardialInfarction (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: PM 2.5 Route: Whole-body Inhalation
Dose/Concentration: CO: 35ppm; CAPs (median concentration): 350.5 μg.m 3 ; CAPs+CO: (CAPs median concentration): 318.2 μg/m 3 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 MyocardialInfarction (AMI): Leftventricular MI induced. Animals allowed to recover for at least 12 h after surgery. CAPs: (Boston, MA)
Particle Size: PM 2.5 Route: Whole-body Inhalation
Dose/Concentration: CO: 35 ppm; CAPs (median concentration): 645.7 μg.m 3 ; 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 ofobserving 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: PM 10 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 PM 10 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 PM 10increased 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: PM 2.5 Route: Whole-body Inhalation
Dose/Concentration: 138.4 ± 83.7 μg/m 3 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 aortacompared 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 atthe 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 radicalproduction, 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-inst illation, 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 approximately0.4-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 markedelevation 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 thisdose 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-1 β

. 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 effe cts: 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 3.4x10 9 particles/mL PC10 = 0.8 g/mL or 3x10 6 particles/mL Time to Analysis: PC2 = 12, 24, 8 h PC10 = 2, 6, 12, 24 h Calcium Imaging: PC10 induced increase of Ca 2+ 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 Ca 2+ . Levels remained elevated as long as PM bound to plasma membrane. Washing and disjoining PM from membrane caused Ca 2+ to slowly decline to baseline. CPZ (or CPZ and amiloride) reversibly inhibited PM-induced rises in Ca 2+

. Calcium Imaging in TRPV1(+/+) and (-/-) mice sensory neurons: All sensitive neurons in TRPV1(+/+) increased Ca 2+ in response to ROFA. No effect of ROFA in TRPV1(-/-).
cAMP: ROFA and MSHA induced increases in Ca 2+ in NHBE and SAEC cells, wh ich 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 Ca 2+

. 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 Ca 2+ 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-929cells; 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/mLPretreatment: 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 inL929-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) PM 10 : Collected from 3 zones in Mexico City: North (industrial), Center (business) and South (residential)
Particle Size: PM 10 Route: Cell Culture
15000 cells/cm 2 except: Cytotoxicity: Confluent Cultures 180,000 cells/cm 2

. DNA Breakage: 20,000 cells/well. Cytokine Assays: 180,000 cells/cm 2
Dose/Concentration: Cytotoxicity: 10, 20, 40, 80, 160 μg/cm 2 Apoptosis: 160 μg/cm 2 DNA Breakage: 2.5, 5, 10, 20, 40 μg/cm 2 Cytokine Assays: 10, 20, 40, 80 μg/cm 2 E-Selectin Expression: 40 μg/cm 2 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 variat ion 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/cm 2

. 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: PM 10 from all zones induced DNA breakage. A dose-dependent relationship was established with PM 2.5 particles at concentrations of 10 μg/cm 2 . The Southern zone required a higher dose of PM (10 μg/cm 2 ) to produce the same effect as other zones (2.5 μg/cm 2 ). 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/cm 2 from all three PM zones. E-Selectin Expression: HUVEC cells showed a 25% increase in E-selectin expression after exposure to 40 μg/cm 2 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: 5×10 5 cells/mL; Human: 3×10 5 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/cm 2 CB = 10 μg/cm 2 CSC = 10 μg/cm 2 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 (3X10 5 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
PM 2.5 : obtained from a highway in Paris, France
Particle Size: DEP: 400 nm (mean diameter); PM 2.5 Route: Cell Culture (2-3.5x10 4 cells/cm 2 )
Dose/Concentration: 10-80 μg/cm 2 Time to Analysis: Cells treated on apical side. Parameters measured 24 h following treatment. Cytotoxicity (LDH): No cytotoxicity for DEP or PM 2.5 (80 μg/cm 2 ). 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 PM 2.5 induced IL-8 and amphiregulin release in a dose-dependent manner through the basolateral surface. PM 2.5 stimulated IL-6 and GM-CSF release through the apical surface. ICAM-1 expression: No effect from DEP or PM 2.5 . ROS: DEP and PM 2.5 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 PM 10 from two Paris, France subway sites: RER and Metro CB (Frankfurt, Germany)
TiO 2 (Calais, France) DEP: SRM1650 (NIST)
Particle Size: CB: 95 nm; TiO 2 :150 nm; DEP: NR
RER PM 10 : 79% <0.5 μm, 20% 0.5-1 μm; Metro PM 10 : 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/cm 2 Time to Analysis: 3, 8, 24 h Cell Viability: No effects from any particulate at concentrations up to 10 μg/cm 2 for 24 h. Inflammatory Effect: Exposure of cells to 10 μg/cm 2 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 PM 10 . No effect of CB, TiO 2 or DEP was observed. GM-CSF or KC production: RER and Metro PM 10 did not induce any effect at any concentration. Effect on Protease mRNA Expression: Exposure of cells to 10 μg/cm 2 RER or Metro PM 10 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 PM 10 for 8 h. Effects on HO-1 Protein Expression: Exposure to 10 μg/cm 2 of RER or Metro PM 10 for 24 h induced positive cytoplasmic staining for HO-1.
Reference: Baulig et al. (2007, 151733 ) Species: Human
Cell Line: 16-HBE14oWUB: Winter Urban BackgroundParticles (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: PM 2.5 ; WC, SC, DEP: NR Route: Cell Culture (20,000 cells/cm 2 )
Dose/Concentration: 10 μg/cm 2 Time to Analysis: 18 or 24 h EGF: All native PM 2.5 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-1 α increased significantly with WUB, WC,SC, DEP, DPL (in decreasing order). No data was available for SUB. Exposure to WUB caused IL-1 β 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 JNKDEP-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.
SP 600125 : 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 (1.5×10 6 cells/well AM; 6×10 6 cells/well Type II)
Dose: 200 μg/mL = 20 μg/cm 2 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: PM 2.5 ; PMF: PM 0.1 ; PM UF: <0.1μm Route: Cell Culture (0.5-1×10 5 cells/well NHBE; 2-3×10 5 /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 Endoto xin 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 (3-5×10 5 cells/well NHBE; 2-3×10 5 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 (1×10 6 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 (3×10 7 cells)
Dose/Concentration: 44 ± 4 ng/cm 2 ; 87 ± 23 ng/cm 2 ; 230 ± 70 ng/cm 2 Time to Analysis: 6 h Cell Viability: Exposure to clean air resulted in a 93.7 ± 9.1% viability. Expos ure 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 PM 2.5 (Vitry-sur-Seine, Paris, France)
DEP = SRM 1650a CB = Carbon Black (Degussa)
TiO 2 (Huntsman)
Particle Size: CB: 95 nm; TiO 2 : 150 nm Route: Cell Culture (45,000 cells/cm 2 )
Dose/Concentration: All particles: 0.1, 1, 10, 30 μg/cm 2 Time to Analysis: 6, 18, 24, 30 h Amphiregulin Expression: DEP and PM 2.5 both increased AR mRNA expression from 6 to 30 h, with PM 2.5 inducing higher expression levels than DEP. Both DEP and PM 2.5 increased AR protein secretion. No observed effect for CB and TiO 2 . PM 2.5 induced protein secretion dose-dependently. Signal Pathways in AR Secretion: MAP kinase and tyrosine kinase inhibitors reduced effects of DEP and PM 2.5 but p38MAP kinase inhibitor did not.
Role of Oxidative Stress: N-Acetylcysteine blocked AR secretion following PM 2.5 . Antioxidant enzyme catalase had no effect. Cytokines: DEP induced a significantly high release of GM-CSF, higher than PM 2.5 . 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 (3×10 6 cells)
Dose/Concentration: DEP, sDEP, nDEP and CB = 10 μg/cm 2 OE-DEP = 15 μg/mL BaP = 0.25, 50 and 250 μg/mL Time to Analysis: 24 h Proinflammatory Response: At 10 μg/cm 2 , 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- κ B Activation: nDEP and OE-DEP induced enhanced degradation of I κ B at 2-4 h and 1 h respectively. NF- κ B DNA binding was enhanced by OE-DEP (15 μg/mL, peak <1 h) and nDEP (10 μg/cm 2 , 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- κ B 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) PM 10 (London, England)
CM from PM 10 -treated human monocytes
Particle Size: PM 10 Route: Cell Culture (1×10 6 cells/mL J774A.1; 5×10 6 cells/mL PBMC; 5×10 5 cells/well A549)
Dose/Concentration: PM 10 : 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: PM 10 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 PM 2.5 (Rome, Italy)
Particle Size: PM 2.5 Route: Cell Culture (5×10 4 cells/well)
Dose/Concentration: 30, 60 μg/cm 2 (aliquot of 0.1 μg/μl) Time to Analysis: 5, 24, 48, 72 h Particle Characterization: Components measured include C-rich particles, Ca sulfates, silic a, 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 (5×10 5 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 H 2 O 2 . Reaction induced by H 2 O 2 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 (7×10 5 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 PM 2.5 (Vermillion, Ohio) Cristobalite: SRM 1879 (NIST)
TiO 2 : SRM 154b (NIST)
Particle Size: EHC-93: 0.5 μm (median diameter); Cristobalite, SRM 1648, SRM 1649, TiO 2 ,: NR; VERP: PM 2.5 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 TiO 2 . iNOS: EHC-I, EHC-T, Crisobalite and SRM1648 inhibited iNOS expression. TiO 2 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 (SiO 2 ): SRM-1879
TiO 2 : SRM-154b
Particle Size: EHC-93: 0.4 μm (median physical diameter); TiO 2 , SiO 2 : 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 TiO 2 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. TiO 2 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-1 β 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 highdose. ECE-1 mRNA expression increased with EHCT and EHC-I. Other particles had no effect. ETaR mRNA increased with EHC-T, EHC-S, and TiO 2 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. TiO 2 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 (Exhaustflow-through cell culture with air-cellinterface, exhaust diluted 10-15x with 8×10 5 cells/mL)
Dose/Concentration: DEP (total): 1.5- 3.5×10 6 particles/cm 3 ; GEP (total): 1-2×10 6 particles/cm 3 ; 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 TiO 2 (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/cm 2 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- κ B: Both particle types increased nuclear translocation of NF- κ B. TiFe and EHC-93 increased NF- κ B 1.5 fold at 1 h. TiFe increased NFκ B 3.5 fold at 2 h. EHC-93 increased NF- κ B more than 2 fold. TiO 2 by itself did not increase NF- κ B at any exposure duration. Morphological changes in tracheal epithelial cells: No evidence of dust particles was observed (EHC-93 or TiO 2 ) 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- κ B 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- κ B activation in both EHC93 and TiFe exposed-cells. Iron-containing citrate extract of both dusts increased NF- κ B 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 = PM 2.5 (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 (3×10 6 , 1.5×10 6

, 0.75×10 6 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.
I κ B α Protein: Phosphorylated I κ B α 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/cm 2 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.
TGF β 1: Treatment of explant with EHC93 approximately doubled gene expression for TGF β 1. Treatment with SN50, TMTU and fetuin (TGF β antagonist) blocked increase. DFX, MAP kinase inhibitors (SB203580 and PD98059) had no effect. DEP roughly doubled TGF β 1 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 (NaVO 3 ) Al = aluminum chloride hexahydrate (AlCl 3 ) Mn = manganese chloride tetrahydrate (MnCl 2 ) Fe = ferric chloride hexahydrate (FeCl 3 )
Ratios based on PM 2.5 measurements from NYC, LA and Seattle
Particle Size: Metals from PM 2.5 samples Route: Cell Culture (2×10 5 cells/mL)
Dose/Concentration: Fe = 16 μmol (equivalent to urban NYC 500 μg PM 2.5 ); 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 ( α 3) and CD29 ( β 1) integrinsubunits 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-1 β : Increased levels of IL-1 β 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/cm 2 )
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 isassociated 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 (10 5 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-1 α , 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 MAFO 2 : 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 (1×10 6 cells/well)
Dose/Concentration: 6.3-188 μg/cm 2 for Toxicity; 2.6, 6.5, 13.2 μg/cm 2 for Arachidonic Acid; 13.2 μg/cm 2 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/cm 2 . Lower doses had no effect.
Arachidonic Acid: At 2.5 h, AA level increased 2 fold for 6.5 μg/cm 2 and 6 fold for 13.2 μg/cm 2 . No increase was observed after 5 h.
MAPKs: Cells pretreated with PD98059, an inhibitor of MEK-1, inhibited AA liberation due to MAFO 2 treatment of 13.2 μg/cm 2
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/cm 2 after 3 h.
GSH: There was an observed increase of production at 20 h. Doses greater than 60 μg/cm 2 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 currentsmokers), AMs, CoCulture: AMs+HBEC Age: HBEC: 48-70 yr PM 10 : EHC-93 (Ottawa, Canada)
Particle Size: PM 10 Route: Cell Culture (HBEC: 2.5-3× 10 6 cells/well); (AMs: 1.0×10 7 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-1 β , 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-1 β , IL-6 mRNA expression increased with 100 μg/mL at 2 h. IN HBECs, IL-1 β and LIF increased with 100 μg/mL at 2 h. HBECs added to AMs exposed to PM 10 , further increase in mRNA of IL-1 β
, LIF and IL-8. Cytokine Protein: In co-culture and AMs, significantincrease in protein production of GM-CSF, IL-8, IL1 β , 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 PM 10 :EHC93 (Ottawa, Canada)99% <3.0um
Particle Size: PM 10 ( 99% < 3.0 μm) Route: Cell Culture (2.5-3×10 6 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-1 α 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-1 β and IL-8 increased dose-dependently. Soluble fraction of 100 μg/mL PM 10 did not affect cytokine production.



### December 2009 D-44

### Study Pollutant Exposure Effects

Reference: Garcon et al. (2006, 096633 ) Species: Human Cell Type: L132 PM 2.5 (collected in Dunkerque, France for 9mo, Jan-Sept)
Particle Size: PM 2.5 - 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: 3×10 6 cells/20ml (24 h); 1.5×10 6 cells/20ml (48h); 0.75×10 6 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 (R 2 =.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 PM 2.5 ; PM collected from Wuwei City, Gansu Province, China (Blowing days correspond to desert storm days) NPM: Non-blowing (normal) PM 2.5 Particle Size: PM 2.5 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 enzymeactivity 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: PM 2.5 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 ce ll 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. Ca 2+ Mg 2+ -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)
VOSO 4 : 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 VOSO 4 - 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. VOSO 4 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 VOSO 4 . Pre-exposure to FAC again increase the uptake of both iron and vandium. VOSO 4 induced opposite effect, decreasing Fe uptake.
ROS: Increased acetaldehyde, indicating increased oxidative stress. ROS decreased with FAC pretreatment. ROS increased with VOSO 4 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 CoarseCoal 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 (2×10 5 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 PM 10 : Collected from the Marylebone and Bloomsbury monitoring sites in London, UK
Particle Size: PM 10 Route: Cell Culture
Dose/Concentration: 50 μg/mL Time to Analysis: 4 h, 6 h, 20 h IL-8 : PM 10 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 : PM 10 induced a significant decrease in macrophage mediated clotting time in 16HBE. Other cell types were unaffected. Annexin V Binding : At 100 μg/mL, PM 10 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 PM 10 : Collected from the Marylebone and Bloomsbury monitoring sites in London, UK
TSA
H 2 O 2 NAC
Mannitol Provided by Sigma Chemical, Poole, UK or GIBCO-BRL, Paisley, UK
Particle Size: PM 10 Route: Cell Culture
Dose/Concentration: PM 10 : 100 μg/mL; TSA: 100 ng/mL; H2O 2 : 200 μM; NAC and Mannitol: 5 mM Time to Analysis: 24 h IL-8: PM 10 , TSA and H2O 2 treatment induced an increase of IL-8. Concomitant exposure of TSA with PM 10 or H 2 O 2 significantly increased IL-8 release when compared to PM 10 or H 2 O 2 alone. IL-8 mRNA expression with PM 10 or H 2 O 2 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, PM 10 and TNF treatment.
H4: PM 10 exposure significantly increased acetylation levels of H4 over controls. Increased acetylated H4 was mediated by PM 10 in a dosedependent manner. Treatment with PM 10 and H 2 O 2 increased HAT activity associated with H4 by 245% and 166% respectively. Significant increases in acetylation of H4 following treatment of cells with TSA, PM 10 and H 2 O 2 for 24 h was observed. PM 10 induced 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 PM 10 . There was a decreasing trend in HDAC2 gene expression following TSA and PM 10 treatment.
NF- κ B: The activation of the transcription factor NFκ B 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 PM 10 (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 blueshowed 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 (1.5×10 6 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: PM 10 , (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 (1×10 6 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 : NO X 7 ppm, CO 2 0.1%
Particle Size: Suspension: 223 nm (mean diameter); ALI: 122 nm (mean diameter) Route: Suspension (1×10 5 cells/cm 2 ), Air Liquid Interface (ALI, 1×10 5 cells/cm 2 )
Dose/Concentration: Suspension: 0.13, 0.24, 1.88, 2.5, and 12.5 μg/cm 2 ; ALI: 1 g/cm 3 (total number of particles: 2.3×10 7 particles/cm 2 ) 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/cm 2 or higher. IL-8 release (corrected for viability) increased at concentrations of 1.88 μg/cm 2 or higher in a dosedependent manner. IL-8 exhibited intermediate levels of secretion between in vitro levels of 0.25 and 1.88 μg/cm 2 . 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 submicronCollected 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 (5×10 5 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 (R 2 = 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 PM 10 : 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: PM 10 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)
TiO 2 : Particle control CAPs (Boston, MA) All cells primed with LPS Coexposure with NAC, dimethylthiourea (DMTU), H2O 2 or catalase
Particle Size: CAPs: ≤ 2.5 μm; UAP: PM 2.5 ; TiO 2 : ~1 μm Route: Cell Culture (2×10 5 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; H 2 O 2 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. H 2 O 2 increased TNF release in CAPs-exposed cells. TiO 2 had no increased ability to induced cytokine release when mixed with H 2 O 2 . Cell Death: Viability decreased substantially when exposed to H 2 O 2 + CAPs. The soluble fraction of CAPs showed to be more effective with H 2 O 2 than the insoluble portion. TiO 2 had no significant effect.
NO: Some CAPs induced slight increases when mixed with H 2 O 2 . No difference was observed between soluble and insoluble portions of CAPs.
DFO: DFO at 0.05 mM completely inhibited oxidation induced with soluble CAPs + H 2 O 2 . Insoluble CAPs + H 2 O 2 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:PM 10 (obtained from Environmental Health Directorate, Ottawa, Ontario, Canada)
Particle Size: PM 10 Route: Cell Culture (1×10 7 cells)
Dose/Concentration: 100 μg/mL Time to Analysis: 3, 6, 24 h Cytokines: TNF- α , IL-1 β , 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 PM 10 exposed AM, levels of RANTES, TNF- α , ICAM-1, IL-1 β , 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-1 β -neutralizing Antibodies: IL-1 β 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 PM 10 -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: PM 10 (obtained from Environmental Health Directorate, Ontario, Canada)
Particle Size: PM 10 Route: Cell Culture (HBEC: 2.5-3.0×10 6 cells; AM: 1×10 7 cells; co-culture of AM/HBEC: 5×10 6 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-1 β , GM-CSF, M-CSF, IL-6, MCP-1 and ICAM-1-mRNA. mRNA Expression After 24 h Exposure: AMsexhibited no effect. HBEC increased levels of GMCSf, LIF and ICAM-1. Co-culture, on the other hand, increased expression of MIP-1 β , GM-CSF, M-CSF and ICAM-1 mRNA.
Protein Levels: AM and HBEC both increased GMCSF, IL-6 and MIP-1 β 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 PM 2.5 sample from Helsinki, Finland
M-UPM: methanol extract of UPM
Particle Size: SRM 1649a, SRM 1650, EHC-93: NR; HFP-00: PM 2.5 Route: Cell Culture (5×10 5 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: PM 10-2.5 ; PM 2.5-1 ; PM 1-0.2 ; PM 0.2 Route: Cell Culture (5×10 6 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 PM 1-0.2 . NO: All 12 samples increased NO production when compared to corresponding unexposed controls. Peaks were observed at 150 μg/mL, except in PM 1- 0.2 . Cytokines: All 12 samples increased TNF- α and IL6 production. PM 10-2.5 and PM 2.5-1 produced a much larger response than PM 1-0.2 and PM 0.2 . IL-6 production for PM 0.2 was not measured. MIP-2 production also increased with similar trends. Cytotoxicity: All 12 samples induced dosedependent decreases in cell viability. PM 10-2.5 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
PM 10 , PM 2.5 , 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: PM 10 : 2.5-10 μm; PM 2.5 : 0.2-2.5 μm; PM 0.2 : <0.2 μm Route: Cell Culture (5×10 5 cells/mL)
Dose/Concentration: 15, 50, 150, 300 μg/mL Time to Analysis: 24 h PM Characterizations: The highest mass concentrations of PM 10 and PM 0.2 were measured in Athens. Prague had the highest PM 2.5 concentrations.
NO: All PM fractions induced statistically significant NO production in macrophages. PM 2.5 -P and PM 2.5 - AT produced significantly larger responses, though all samples at 150 and 200 μg/mL induced statistically significant production. When compared to the other PM 0.2 samples, -P and -HR produced significantly larger responses. Cytokines: PM 10 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 PM 2.5 and PM 0.2 respectively. PM 10 induced statistically significant increases in production of TNF- α , MIP-2 and IL-6. PM 2.5 , with exception of Prague, caused significant increases in cytokines. PM 0.2 -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 PM 10 and slightly with PM 2.5 . In the PM 0.2 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. PM 0.2 effects on IL-6 response were negligible. Cytotoxicity: The average cytotoxicity of PM 10 and PM 2.5 were roughly equal, but PM 0.2 were lesscytotoxic with the exception of -P. The doseresponse trends for most of the samples were linearly declining, with PM 10 and PM 2.5 exhibiting statistically significant declines in viability.
Reference: Jimenez et al. (2002, 156610 ) Species: Human Cell Type/Line: A549, THP-1, Mono Mac 6 (DSMZ) PM 10 : Collected from London and Edinburgh air particulate monitoring stations.
TiO 2 : Tioxide Europe (London, UK) and Degussa-Huls (Cheshire, UK)
UFTiO 2 : Tioxide Europe (London, UK) and Degussa-Huls (Cheshire, UK)
Particle Size: PM 10 , TiO 2 : 200 nm; UFTiO 2 : 20 nm Route: Cell Culture (110,625 cells/well)
Dose/Concentration: PM 10 , TiO 2 , UFTiO 2 : 100 μg/mL; TNF- α : 10 ng/mL Time to Analysis: 4 h NF- κ B and AP-1 DNA Binding : NF- κ B DNA binding increased in PM 10 and TNF- α exposed macrophages by 9.5 and 12 fold. NF- κ B activity remained unaltered in TiO 2 and UFTiO 2 exposed macrophages.
IL-8: Cells treated with PM 10 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 PM 10 . TiO 2 or UFTiO 2 had no effect. Increases observed in IL-8 production with PM 10 . IL-8 Promoter CAT Activity: PM 10 media increased CAT expression by 65% over control. No differences observed with TiO 2 or UFTiO 2 media. Neutrophil Chemotaxis: PM 10 conditioned media induced a 2.3 fold increase compared to control.
TNF- α and IL-1 β Production: PM 10 media increased TNF- α and IL-1 β production. No increases were observed in TiO 2 and UFTiO 2 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)
PM 2.5 : Collected using IMPROVE air pollution samplers
Particle Size: Soot: 185 nm; CB: 25 nm, PM 2.5 Route: Surrogate Lung Fluid
Dose/Concentration: Soot: 0-30 mg; CB: 5- 10 mg; PM 2.5 : 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.
PM 2.5 : A high variability in the increase of OH radicals was observed with PM 2.5 . Pretreatment with DSF partially blocked OH radical production, but a significant level remained. This may be due to PM 2.5 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- κ B inhibitor (coexposure)
IL-1ß: obtained from Santa Cruz Biotechnology (Santa Cruz, CA)
Particle Size: DEP: 0.3 μm (mean diameter) Route: Cell Culture (3-4×10 5 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-1 β : 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-1 β containing PM samples at 100 μg/mL also resulted in a significant release of TNF- α

. NF- κ B Binding Activity: Treatment of RAW 264.7 with BAY11-7082 significantly inhibited IL-1 β

induced 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: PM 10 tire debris with studded tires and ABT pavement; T2a: PM 10 tire debris with studded tires and ABS pavement; T2b: PM 2.5 tire debris with studded tires and ABS pavement; T3: PM 10 tire debris with friction tires and ABS pavement; St: PM 10 from busy street in Stockholm, Sweden; Su: PM 10 from platform of subway station in Stockholm)
Particle Size: W: NR, T1, T2a, T3, St, Su: PM 10 , T2b: PM 2.5 Route: Cell Culture
Dose/Concentration: Suspension: 40 μg/cm 2 ; Culture: 100 μg/cm 2 (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
Al 2 O 3 , Fe 2 O 3 , SiO 2 , TiO 2 , 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; Al 2 O 3 0.5 mg/mL; Fe 2 O 3 0.7 mg/mL; SiO 2 0.7 mg/mL; TiO 2 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 SiO 2 , 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 TiO 2 (increase) and ZnO (decrease).
Reference: Kendall et al. (2004, 156634 ) Species: Human Tissue Type: BALF (obtained by bronchoscope from 6 nonsmokers and 3 smokers) PM 2.5 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 (NH 4 + , N-C, NO 3 2- ), 0.6-1% S, and 0.3-2 % Si. Only NO 3 - higher in roadside samples.
NH 4 and NO 3 - correlated with NO and NO X in air but not NO 2 . Particle Size: PM 2.5 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 NH 4 , NO 3 , O and S relative to C1. BALF treatment (XPS): PM 2.5 surfaces interacted strongly with BALF within hours of contact. Specific surface components of PM 2.5 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 NH 4 , NO 3 , O and S. ToF-SIMS - Organics: Particle loading and surface hydrocarbons showed a linear correlation. Loss of hydrocarbons from PM 2.5 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 PM 2.5 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 NH 4 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 Zn 2+
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, Zn 2+ 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 Zn 2+

. Proteases: With 50 μM Zn 2+ , 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 Zn 2+ 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 Zn 2+

.



### 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 (10 5 cells/well at 10 6 cells/mL)
Dose/Concentration: 1.2 to 1200 ng/10 6 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/10 5 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 particlesdepressed 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/10 6 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 (1×10 6 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-1 β
, and IL-8 with 30 or 70 μg/mL. PMW similarly induced TNF- α and IL-8. DEP induced IL-1 β 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-1 β , 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 cytokinerelease. 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 (4×10 6 cells/well)
Dose/Concentration: CP: 5-50 μg/cm 2 ; CFE: 2.5-25 μg/cm 2 ; CFE+: 2.5-25 μg/cm 2 ; CFA: 10-100 μg/cm 2 ; DEP: 2.5-25 μg/cm 2 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/cm 2 . No toxicity was apparent when treated with CFA (up to 200 μg/ cm 2 ) 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: 10 6 cells/mL; BEAS-2B: 10 5 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/m L 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 (1×10 5 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 si milar 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- κ B: 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 (5×10 5 cells/mL (Cytotoxicity), 3×10 5 cells/mL (Apoptosis), 2×10 6 cells (MMP and ROI), 1×10 7 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 resu lts. 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 Ca 2+ 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 PM 10 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 (2×10 6 cells/well Mouse RAW 264.7 and THP-1; 0.67×10 6 cells/well Murine RAW 264.7)
Dose/Concentration: 10-200 μg/mL
JNK Activation and IL-8 Production: THP-1cells- 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 (10 6 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 β

actin. 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. Co PP 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 PM 10 : -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: PM 10; Bimodal with peaks around 4-5 um and 7-8 um. Route: Cell Culture (130
, 000 cells/cm 2 )
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 combinati on 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 reachsignificance. 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 O 2 - and H2O 2 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 mannerMitochondrial Translocation/ CapsaseIndependent Apoptosis/DNA fragmentation: Exposure for up to 60 min caused an increase in thepercentage 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 H2O 2 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 (5×10 6 cells, 2 mL /well MDMs) Dose/Concentration: 5 μg/cm 2 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- κ B 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- κ B and AP-1: DEP stimulated NF- κ B activity at 5 and 10 μg/mL. At 20 μg/mL, NF- κ B 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 t he 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- κ B 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- κ B activation.
Reference: Maciejczyk and Chen (2005, 087456 ) Species: Human Cell Type: BEAS-2B CAPs: PM 2.5 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: PM 2.5 Route: Cell Exposure (subchronic exposures); Cell Culture (NF- κ B) (9×10 4 cells/well)
Dose/Concentration: CAPS 109 ± 178 μg/m 3 (air exposure); 300 μg PM/ml (culture) Time to Analysis: 24 h NF- κ B: NF- κ B 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 extracthas 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 highestdose 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: 5×10 4 cells/cm 2 ; NHPAE: 3×10 3 cells/cm 2 ; TIG-1 and TIG -7: 3×10 3 cells/cm 2 ; Apoptosis: 2×10 5 cells/cm 2 ; ROS/NO: 2×10 4 cells/cm 2 ; Cytotoxicity Modulating Agent: 3×10 4 cells/cm 2 ; GSH: 3×10 4 cells/cm 2 )
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 (5×10 5 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
H 2 O 2 : 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
H 2 O 2 : 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%.
H 2

O 2 : 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: PM 10 extracts in hexane
PMA = PM 10 extracts in acetone of residue after hexane extraction -G: Guaynabo(Urban) and -F: Fajardo (Preservation Area)
Particle Size: PM 10 Route: Cell Culture (3×10 3 cells/well)
Dose/Concentration: NHBE exposed to 0- 100 μg/mL of PM 10 BEAS-2B exposed to 10,100, 250 μg/mL of PM 10 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 (noanalysis). 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 fTiO 2 (origin NR)
ufTiO 2 (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) TiO 2 : 220 nm; ufTiO 2 : 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. fTiO 2 and ufP90 had no effect at any dose. ufTiO 2 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: ufTiO 2 and fTiO 2 both induced some retarded relaxation and increased stiffening at 100 μg/mL dose. ufTiO 2 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, ufTiO 2 and fTiO 2 significantly reduced phago cytic 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: ufTiO 2 significantly inhibited proliferation compared to the control and fTiO 2 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 ufTiO 2 inducing greater apoptosis than fTiO 2 , ufEC90 than ufP90 and ufEC90 than ufP-G.
Reference: Mutlu et al. (2006, 155994 ) Species: Human, Rat Cell Type: A549 PM 10 (Collected by baghouse from ambient air in Dusseldorf, Germany)
Particle Size: PM 10 Route: Cell Culture
Dose/Concentration: 0.05, 0.5, 5. 50 μg/cm 2 Time to Analysis: 24 h Na, K-ATPase Plasma Membrane Protein: PM 10 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: PM 10 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 PM 2.5 Collected from hospital rooftop, Seoul, South Korea
Particle Size: PM 2.5 Route: Cell Culture
Dose/Concentration: 0.5, 1, 10, 25, 50 μg/cm 2 Time to Analysis: 6, 24 h NF- κ B/I κ B α : 50 μg/cm 2 DEP induced I κ B α degradation which peaked at 2 h and recovered after 4 h. Treatment with increasing amount of PM 2.5 resulted in a dose-dependent decrease in I κ B α

. PM 2.5 increased NF- κ B in a dose-dependent manner up to 10 μg/cm 2 . NF- κ B induction peaked at 12 h.
IL-8: PM 2.5 treatment increased protein level more than 3 fold with 100 μg/cm 2 PM 2.5 . mRNA levels also increased.
iNOS Inhibitor: PM 2.5 induced IL-8 elevation was completely blocked by iNOS inhibitor. iNOS inhibitor also negated PM 2.5 induction of NF- κ B activity. Antioxidants and iNOS inhibitor reduced PM-induced I κ B α 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 (1.4×10 4 cells/cm 2 )
Dose/Concentration: 1.1 μg/cm 2 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 (5×10 5 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, O 2 , N, S, Si, Ca, Al, P, Cl. PM-PH mostly contained O 2 and Si. PM-500 had increased O 2 , 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
PMH 2 O: Water extraction
All of the 6 extract fractions from PM1648
PM 2.5 : Collected in Houston, TX
Particle Size: PM1648: NR; PM 2.5 Route: Cell Culture (5×10 5 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
PM 2.5 = 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 differ ed 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 PM 2.5 : 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 C ontent 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) PM 10 PM 2.5 -N = Northern (industrial) -SE = Southeastern (lake basin dust) sites, both heavy vehicular traffic, Mexico City, Mexico
Particle Size: PM 10 ; PM 2.5 Route: Cell Culture (J774A.1: 15000 cells/cm 2 ; L929: 30,000 cells/well)
Dose/Concentration: 20, 40, 80 μg/cm 2 Time to Analysis: 24-72 h PM Characterization: Elements similar in particle types with elements in PM 10 more abundant. Northern particles contained more Co, Zn, Ni, Pb.
Endotoxin: All PM samples had detectable amounts of endotoxin. PM 2.5 -N had 22 EU/mg. PM 10 -N had 30 EU/mg. PM 2.5 -SE had 12 EU/mg. PM 10 -SE had 59 EU/mg. Cytotoxicity (J774A.1): The two northern samples, PM 2.5 and PM 10 , both induced similar cytotoxic effects at 40% survival. PM 10 -SE and PM 2.5 -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 PM 10 and PM 2.5 induced apoptosis.
TNF- α and IL-6 (J774A.1): TNF- α and IL-6 induced dose-dependent increases. At 80 μg/cm 2 , PM 10 -SE induced the most production of IL-6 followed by PM 2.5 -SE, PM 10 -N , and PM 2.5 -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 (1-1.5×10 6 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 (1.3×10 5 cells/well)
Dose/Concentration: 30 μg/mL; 14 μg/cm 2 120 μg/mL; 54 μg/cm 2 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 PM 2.5 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: Na 3 VO 4 Al: AlCl 3 •6H 2 O
Mn: MnCl 2 •4 h 2 0
Fe: FeCl 3 •6H 2 0
Particle Size: PM 2.5 Route: Cell Culture (2×10 5 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 wi th 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 PM 2.5 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-1 α ): Only V and Al induced HIF-1 α

. 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 PM 10 : Collected in Wolverhampton, UK
ufCB: Ultrafine Carbon Particles (Origin not reported)
Particle Size: PM 10 , 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 PM 10 produced an increase in CRP expression with similar effects noted after 6 h. PM 10 induced greater increases than ufCB. Both the cytoplasm and nucleus contained CRP. Hsp70: PM 10 and ufCB induced increased levels at all time points with ufCB inducing greater levels than PM 10 . 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-1 β 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/cm 2 ; 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.
MIP3 α /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-1 β (10 mg/mL).
MIP3 β /CCL20 protein in HBEC (18 h): TNF- α and IL-1 β induced a dose-dependent increase in MIP3 α /CCL20 protein (0-10 ng/mL), whereas Il-4 and IL-13 induced MIP3 α /CCL20 protein release that reached maximum levels at 1 ng/mL. No release of MIP1 α /CCL3 nor RANTES/CCL-5 was observed upon stimulation with cytokines.
Secretion of MIP3 α /CCL20 in response to PM (18 h): All PM fractions less than 2.5 μm resulted in the release of MIP3 α /CCL20 protein in HBEC roughly equivalent amounts. CB similar in size to UF/fine PM did not result in the release of MIP3 α /CCL20, nor did LPS (0.01-1.0 μg/mL). No release of MIP1 α /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-1 β , IL-4 and IL13 within 15 min and was sustained for at least 60 min. Erk1/2 and p38 inhibitors reduced MIP3 α /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: ZnCl 2 Cu: CuCl 2 Fe: FeCl 2 V: VCl 4 Ni: NiCl 2 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: FeCl 2 Ni: NiCl 2 Cu: CuCl 2 V: VCl 2 Particle Size: NR Route: Cell Culture (5×10 4 cells/well Alveolar Cells; 1.2×10 5 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 expressi on 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 PM 10 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: PM 10 Route: Cell Culture (1.5×10 4 cells/cm 2 )
Dose/Concentration: 20, 40 or 80 μg/cm 2 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/cm 2 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 PM 10 : EHC-93 (Obtained from Health Canada, Canata)
Particle Size: PM 10 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 PM 10 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 PM 10 . This indicates that PM 10 blocks Ca clearance via the calcium pumps. Cytokines: PM 10 induced a dose-dependentincrease in cytokine mRNA levels and cytokines IL1 β , 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-1 β and IL-8 but not LIF or GM-CSF.
Reference: Salnikow et al. (2004, 087469 ) Species: Human
Cell Line: 1 hAEoFeSO 4 FeCl 3 NiSO 4 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. NiSO 4 caused marginal cytotoxicity (75%). Hypoxic Stress: At 20 h, NiSO 4 (at concentrations of 0.25 or 0.5 mM) induc ed NDRG-1/Cap43 protein production indicating hypoxic stress. DFX and DMOG induced a similar effect.
IL-8: NiSO 4 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. NiSO 4 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 PM 10 (urban traffic) Finland Pooled as winter (W), spring I (SI), or spring II (SII) based on component/time considerations
Particle Size: PM 10 : 0.12-10 μm Route: (2×10 6 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 PM 2.5 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 PM 10 . 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/m 3 ) in the calf thymus DNA were observed.
Reference: Samet et al. (2003, 113782 ) Species: Human Cell Type: A431 (Epidermoid Cells) As: NaASO 3 V: VOSO 4 Zn: ZnSO 4 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: PM 2.5 Route: Cell Culture (2.5×10 5 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, α and β gamma- glutamine/glutamate (0.01 >0.1 μg/mL), lactate, and CH 2 , CH 3 moieties of fatty acids. DEP decreased inositol and phosphoreatinine. 700 MHz Results (no 1 μg/mL): DEP induced similar results, except α -, β gamma-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 (2.5×10 4 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 fordays 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: Thecarbonaceous 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/m 3 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-1 α , 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 (1×10 5 cells/well)
Dose/Concentration: 0.03 -1,000 μg/cm 2 Time to Analysis: 0, 18 h IL-8 release: DPM increased semi dosedependently (perhaps steady based on error range) up to 1 μg/cm 2 after which IL-8 declined dose dependently to zero (control = 100%) at 300 and 1000 μg/cm 2 . 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 (1×10 5 cells/well)
Dose/Concentration: 0.03-10,000 μg/cm 2 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 PM 2.5 from London
PM 10 from Manchester (positive control) PM from Holland Park, Hampstead and Oxford Circus stations (HP, HR and OC)
Particle Size: PM 2.5 , PM 10 , 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). PM 10 from Manchester caused a 7% LDH release at the highest dose. The negative control (TiO 2 ) 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 PM 2.5 . HP induced a 3 fold increase. Also, the highest TiO 2 concentration caused the least IL-8 stimulation. Hydroxyl Radical Gene ration/ DNA Plasmid assay: The plasmid assay indicated that the tunnel dusts induce more free radical activity than the Manchester PM 10 and TiO 2 . HP nearly doubled the percentage of DNA damage with intermediate results for HR and OC. Results for PM 10 , TiO 2 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 m 2 /g; CI: ~40 nm Route: Cell Culture (5×10 5 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- κ B 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 (5×10 5 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 (10 4 cells/well)
Dose/Concentration: 6.1, 12.3, 18.4, 24.5, 30.7 μg/cm 2 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/cm 2 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)
TiO 2 SiO 2 ROFA
Particle Size: TiO 2 : ~1 μm; SiO 2 : ~1 μm; ROFA: NR Route: Cell Culture (1×10 5 cells AM
1.4×10 5 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: SiO 2 at 25μg/mL, UAP at 12.5 μg/mL, ROFA at 25 μg/mL, and TiO 2 at 50 μg/mL. Except for SiO 2 , the blocking of effects caused by LPS absorbed on the particles did not affect the cytokine response. For SiO 2 , 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 Al 2 O 3 M-Al: Micro Al 2 O 3 N-Ce: nano CeO 2 M-Ce: micro CeO 2 N-Fe: nano Fe 2 O 3 M-Fe: micro Fe 2 O 3 N-Ni: nano NiO M-Ni: micro NiO N-Si: nano SiO 2 M-Si: micro SiO 2 N-Ti: nano TiO 2 M-Ti: micro TiO 2 KLN: kaolin MUS: Min-U-Sil (ground crystalline silica)
DD: desert rural soil Utah PM 2.5 JE: Juarez, urban street PM 2.5 MNC: Mancos, rural Utah PM 2.5 LPS: lipopolysaccharide
V: VOSO 4 (soluble) (19 μg/mL) Particle Size: ( Surface mean diameter) N-Al: 6 nm (261 m 2 /g) M-Al: 210 nm (7.7 m 2 /g) N-Ce: 14 nm (71 m 2 /g) M-Ce: 1500 nm (0.6 m 2 /g) N-Fe: 5 nm (221 m 2 /g) M-Fe: 100 nm (12 m 2 /g) N-Ni: 6 nm (145 m 2 /g) M-Ni: 16 nm (57 m 2 /g) N-Si: 19 nm (127 m 2 /g) M-Si: 440 nm (5.4 m 2 /g) N-Ti: 6 nm (242 m 2 /g) M-Ti: 410 nm (3.5 m 2 /g) KLN: 100 nm (24.3 m 2 /g) MUS: (NOS <5 μm) DD: 400 nm (6.2 m 2 /g) JE: (NOS <3 μm) MNC: 200 nm (13.0 m 2 /g) Route: Cell Culture (35,000 cells/cm 2 BEAS; 2500 cells/cm 2 NHBE; 20,000 cells/cm 2 A549)
Dose/Concentration: 0.53, 5.3 and 53 μg/cm 2 (= 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 ce lls 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/cm 2 ).
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, PM 2.5 enriched) V: vanadium soluble (prepared from VOSO 4 , Alfa Aesar, Ward Hill, MA) C: Coal fly ash (PM 2.5 enriched and derived from commercial power plant burning Utah bituminous coal) D: Diesel PM (tail-pipe particlescollected 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 (m 2 /g) S: 6.2 (PM 2.5 enriched)
V: NA C: 5.4 (PM 2.5 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/cm 2 V = 100 μg/cm 2 C = 100 μg/cm 2 D = 32 μg/cm 2
L = 1000 EU/mL T = 100 μg/cm 2 K = 100 μg/cm 2 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 ka olin, 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 PM 2.5 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)
TiO 2 kaolin clay
Particle Size: PM 2.5 ; TiO 2 : 1-2 μm Route: Cell Culture (35,000 cells/ cm 2 )
Dose/Concentration: 10, 20, 40, 80 μg/cm 2 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, TiO 2 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/cm 2 : Correlation was medial for OC1(OC) and P at 0.001<p<0.01. IL-6, 80 μg/cm 2 : 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/cm 2 : Correlation was weak for EU (Endotoxin), CO 3 , Si, and Br. IL-8, 80 μg/cm 2 : Correlation was medial for CO 3 , 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 PM 2.5 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/cm 2 )
Dose/Concentration: 10, 20, 40, 80, 160 μg/cm 2 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/cm 2 ) showed dose-dependent responses and induced an 8-fold increase at the highest concentration levels. WM peaked at 40 μg/cm 2 and UN induced similar responses above 10 μg/cm 2

. IL- 8 Release: DD induced a dose-dependent response. WM peaked at 10 μg/cm 2 . 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/cm 2 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/cm 2

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/cm 2 100μg/mL; 60 μg/cm 2 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 Ca 2+ 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 (2×10 5 - 2×10 6 cells/mL)
Dose/Concentration: DEP, UDP: 2.5, 10 or 40 μg/cm 2 (eq to 12.5, 40, 200 μg/mL) DEP-OE, UDP- OE: 10 μg/cm 2 (particle equivalent) Time to Analysis: 24 h Effect On mRNA Expression (COX-2, TNF- α , IL-6, IL-8, C/EBP β , CRP, CYP1a1): All DEP and UDP induced dose-dependent increases. IL-6 tended to plateau at 10 μg/cm 2 . 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/cm 2 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 m RNA Expression: CRP: pretreatment with IgG and wortmannin (Fc γ 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/cm 2 all increased cholesterol accumulation by at least 2 fold
Reference: Wang et al. (2003, 157106 ) Species: Rat Cell Type: Lung Myofibroblasts V 2 O 5 : (Aldrich Chemical Co., Wisconsin)
Particle Size: NR Route: Cell Culture (1×10 5 cells/100 mm dish; 3.2×10 4 cells/cm 2 )
Dose/Concentration: 400 μm Time to Analysis: 0.5, 1, 4, 24 h H 2 O 2

Drives STAT-1 Activation: Pretreatment with NAC or catalase reduced V 2 O 5 -induced STAT activation by more than 90% and completely abolished H 2 O 2 -induced STAT activation. Within 5 min of V 2 O 5 treatment, H 2 O 2 was significantly decreased in the supernatants of cultured myofibroblasts and suppression of H 2 O 2 levels continued for up to 24 h post V 2 O 5 treatment. This supports the findings that myofibroblast-generated H 2 O 2 is required for V 2 O 5 -induced STAT activation. Temporal STAT-1 Activation: H 2 O 2 induced rapid activation within minutes whereas activation by V 2 O 5 occurred more slowly (beginning 8h post treatment). p38, ERK, EGFR: p38 and EGFR are required for H 2 O 2 - or V 2 O 5 -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)
FeCl 3 ZnCl 2 Particle Size: CB: 14 nm Route: Cell Culture (4×10 5 cells/mL at 1mL/well)
Dose/Concentration: CB 1.9 -31 μg/mL; FeCl 3 , ZnCl 2 0.01-100 μmol Time to Analysis: 4 h ROS Production in Cells: CB alone increased ROS. Coexposure with ZnCl 2 did not affect ROS.
ROS Production - Cell Free: CB induced a significant increase in ROS. ZnCl 2 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 -Fe 2 O 3 Si60: silicasol (SiO 2 , 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 (2×10 4 cells/well. Coculture: 2×10 4 A549 and 2×10 3 Macrophages)
Dose/Concentration: A549 light microscopy hematite 100μg/mL (23 μg/cm 2 ) TEM hematite 50 μg/mL (16 μg/cm 2 ) Cytotoxicity 10, 50, 100 and 200 μg/mL (6.1, 30, 61 and 121 μg/cm 2 ) 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) ZnSO 4 (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 5tyrosine 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: Zn 2+
Particle Size: NR Route: Cell Culture
Dose/Concentration: 10, 25, 50 μmol Time to Analysis: 0-8 h Cytotoxicity: Exposure to 50 μmol Zn 2+ for 8 h did not result in significant alterations in cell viability.
PTEN Protein Levels: 50 μmol Zn 2+ 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 Zn 2+

, 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 Zn 2+ 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 Zn 2+ 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 Zn 2+ 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: Zn 2+
Particle Size: NR Route: Cell Culture
Dose/Concentration: 100 μmol Time to Analysis: 2 h Cell Viability: After 2 h of exposure, Zn 2+ induced effects in NHBE cells at 100 and 200 μmol levels (but not 50 μmol). Continuing exposure to 100 μmol Zn 2+ for 4 and 6 h did not significantly alter cell viability. Thus, in all subsequent studies, NHBE cells were treated with 100 μmol Zn 2+

. Induced EGFR Phosphorylation: Exposure to 100μM Zn 2+ 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 Zn 2+ -induced phosphorylation to subside. Zn 2+ activity requires tyrosine kinase activity. EGFR Phosphorylation Pathway: To test whether Zn 2+ exposure results in ligand release, which in turn can activate phosphorylation, NHBE cells were pretreated with LA1 blocking antibody. Results showed significant suppression of Zn 2+ induced phosphorylation, therefore Zn 2+ 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 Zn 2+ 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 Zn 2+ . Previous studies indicate metalloproteinase (MMP) involvement in cleaving ligand precursors. It was found that MMP-3 inhibitor partially blocks Zn 2+ induced HB-EGF release. (MMP-2 and MMP-9 did not show similar inhibition patterns) Zn 2+ 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: Zn 2+
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 Zn 2+ 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 Zn 2+ induced expression, LY-294002 (a P13 inhibitor) and another unnamed P13 inhibitor were used. Exposed cells indicated suppressed levels of Zn 2+ induced COX-2. To determine Akt role, ad-DN-Akt (AAA) was used. Infected cells indicated over-expression of Akt and significant reduction of Zn 2+ induced GSK-3 α / β phosphorylation. Over expression of DN-Akt(AAA) blocked Zn 2+ 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 Zn 2+ -induced mRNA COX-2 expression, suggesting PTEN inhibits PIP3 signal transduction to Akt. Analysis of the Src/EGFR Signaling Pathway: Zn 2+ induced a time-dependent increase in Src and EGFR phosphorylation in cells. Blockage of Src activity via PP2 (Src inhibitor) decreased Zn 2+ induced EGFR phosphorylation. The EGFR tyrosine inhibitor completely blocked Zn 2+ -induced EGFR phosphorylation. EGF (a ligand of EGFR signaling) induced COX-2 expression, suggesting that EGFR regulated Zn 2+ -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 Zn 2+ -induced Akt phosphorylation of Src., EGFR and p38. It is thought that p38 is a critical kinase in regulation of Zn 2+ -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 Am bient 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 (1.2×10 6 cells/cm 2 )
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 (3×10 4 cells/well)
Dose/Concentration: 1, 10, 100, 250, 500 and 1000 μg/mL; main testing 250 μg/mL Time to Analysis: 12 h NF- κ B 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 I κ B α which is an intracellular inhibitor of nuclear translocation of NF- κ B. TAK1 and NIK Required for NF- κ B 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 PM 2.5 : 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: PM 2.5 Route: Cell Culture
Dose/Concentration: 100 μg/cm 2 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 effe cts: in vivo studies.

Reference Pollutant Exposure Effects

Reference: Adamson et al. (2003, 087943 ) Species: Rat
Gender: Male
Strain: SD Weight: 150 g PM 10 : 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: PM 10 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 neut rophilic 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/m 3 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-1 α , 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-1 α 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: PM 10 Paris, France subway
CB TiO 2 DEP
Particle Size: RER: 79% < 0.5 μm; 20%: 0.5-1 μm
CB: 95 nm TiO 2 : 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/m 3 Time to Analysis: CAPs exposure 5 h/day, 3 days (consecutive). SO 2 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 lowerlevels 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 PM 2.5 (Shanghai, China) Components: As, Cd, Cr, Cu, Fe, Ni, Pb, Zn, V, Ba, Se, Mg, Co, Mn
Particle Size: PM 2.5 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 andincreased 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-1 β , 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/m 3 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.
H 2

O 2 : 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 H 2 O 2 . 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: PM 2.5 Netherland suburban, industrial and freeway tunnel site collections Wistar rats pre-exposed to O 3 SO 4 , NO 3 and NH 4 ions: 54 ± 4% suburban, 53 ± 7% industrial and 35 ± 5% freeway site conc. of total CAPS mass
Particle Size: PM 2.5 (0.15<PM<2.5 μm) Route: Nose-only Inhalation
Dose/Concentration: PM 365-3720 μg/m 3 (results from 16 different exposures 2000, 2002); O 3 : 1600 μg/m 3 (0.8 ppm) Time to Analysis: 8 h O 3 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 O 3 , 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 O 3 - only rats. Both O 3 -only and O 3 +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 (R 2 = 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), α 2M 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 α 2M bands in BALF as well as most other proteins. Statistical analysis showed positive correlation between VEGF and albumin (R 2 = 0.796) and VEGF and α 2M (R 2 = 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 OuJmice: 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-1 β , LT- β , IL-1 α , 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 - >1000×10 6 g dry tissue; Mexico City: PM 10 : 66 μg/m 3 , Vancouver: PM 10 : 25 μg, PM 2.5 : 15 μg Time to Analysis: Lung samples taken from deceased lifelong Mexico City residents andVancouver 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 Inhalat ion (IH)
Dose/Concentration: IT instillation = 110 μg/rat IH = 12 mg/m 3 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 (R 2 = 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 TiO 2 Particle Size: PM mean diameter: 0.4 μm; Carbon black: FW- 13 nm, P60- 21 nm; TiO 2 mean diameter: 0.14 μm Route: IT Instillation
Dose/Concentration: 5 mg PM or TiO 2 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/m 3 II: 5, 20 mg/m 3 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/m 3 DEP, significantly higher IL-6 gene level was observed, both 1 and 22 h post exposure. For 20 mg/m 3 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/m 3 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/m 3 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/m 3 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- 3.13×10 5 particles/cm 3

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 orbiochemical 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 surfacearea 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/m 3 HSCb; 50 mg/m 3 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 coalunder conditions simulating fullscale utility boiler conditions) Transition metals of Coal Fly Ash: Fe, Mg, Ti, Mn, V
Particle Size: >PM 2.5 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 groupswere 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/m 3 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/m 3 compared to WT mice. IL-6 production increased in WT mice exposed to 3 mg/m 3

. CCL3 increased in both WT and IL-6(-/-) at high dose. IL-1 β 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 inHendrik-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 groupsat 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 mildlymphocytosis. 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 ofattenuated 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 mutationG185R 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 (VOSO 4 )
Particle Size: NR Route: IT Instillation
Dose/Concentration: 0.5 mL 100 μm FAC/rat; 0.5 mL 10 μm VOSO 4 /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 VOSO 4 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 alveolarmacrophages. Increased metal clearance with preexposure to FAC. Less metal clearance with preexposure to VOSO 4 . 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 mostsignificant 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 va riability. 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- κ B: A significant increase in NF- κ B 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/m 3 fCB: 1.40 mg/m 3

Number concentrations ufCB: 52380 particles/cm 3 fCB: 3800 particles/cm 3 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/m 3 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, SO 4 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) (O 2 - 20%, CO- 1.3-4.8 ppm, NO- <2.5-5.9 ppm, NO 2 - <0.25-1.2 ppm, SO 2 - 0.2-0.3 ppm, OC/EC- 0.3 ± 0.03)
Particle Size: Number MedianDiameter: 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/m 3 , High2201 ± 14 μg/m 3 Time to Analysis: Exposed 4 h/day, 5 days/wk, 4 wk. Necropsied 1 day post-exposure. DE increased neutrophils in a concentration-dependentmanner, 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 O 3 , NO 2 Particle Size: 389 ± 2 nm Route: Whole-body Inhalation
Dose/Concentration: CAPS = 131 ± 99 μg/m 3 including O 3 = 10 ppb and NO 2 = 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% O 2 , 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/m 3 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: PM 10 -2.5; PMF: PM 2.5 -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/m 3 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/m 3 , 5 day concentration: 313 ± 119 μg/m 3

, July concentration: 16-185 μg/m 3 , September concentration: 81-755 μg/m 3 ; 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 PM 2.5 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/m 3 or 3 mg/m 3 ; SO 2 <0.01 ppm; NO 2 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 PM 10 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: PM 10 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-1 β and KC. However, levels of MIP-1x were significantly less in the knockout than the wild type while levels of IL-1 β 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: NO 2 , SO 4 , SO 2 , CO, CO 2 , NO X , NO, HTHC, HCHO, O 2 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/m 3 M: 0.92- 1.18 mg/m 3 MG: 0.01 mg/m 3 HR: 2.57 - 2.94 mg/m 3 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 da ys, 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 Metropolitanexpressway and SatsukibashiMizuecho city road, Japan)
Particle Size: NR Route: Whole-body Inhalation
Dose/Concentration: Exposed group: 62.7 μg/m 3 PM, 55.7 ppb NO 2 ,; Control group: 14.3 μg/m 3 PM, 5.1 ppb NO 2 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 O 3 CCL: O 3 + Ammonium bisulfate (ABS) + Elemental Carbon (EC)
CCH: O 3 + 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: O 3 : 0.2 ppm CCL: 50 μg/m 3 EC + 70 μg/m 3 ABS + 0.2 ppm O 3 CCH: 100 μg/m 3 EC + 140 μg/m 3 ABS + 0.2 ppm O 3 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 O 3 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 O 3 was observed. DNA Replication: O 3 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 LO 3 : Low O 3 HO 3 : High O 3 LS: Low H 2 SO 4 HS: High H 2 SO 4 LOLS: Low O 3 + Low H 2 SO 4 LOHS: Low O 3 + High H 2 SO 4 HOLS: High O 3 + low H 2 SO 4 HOHS: High O 3 + high H 2 SO 4 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: LO 3 = 0.30 ppm HO 3 = 0.61 ppm LS = 0.48 mg/m 3 HS = 1.00 mg/m 3 LOLS = 0.31 ppm + 0.41 mg/m 3 LOHS = 0.31 ppm +1.04 mg/m 3 HOLS = 0.60 ppm + 0.52 mg/m 3 HOHS = 0.60 ppm + 0.86 mg/m 3 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. HO 3 doubled Type 1 lesions and increased Type 2 lesions 25-fold. Additions of H 2 SO 4 to O 3 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 O 3 exposures but was not affected by coexposure to H 2 SO 4 . Macrophage FcR binding: No effects were observed (no data for LO 3 and HO 3 ). Macrophage Phagocytosis: All levels of exposure (no data for LO 3 and HO 3 ) 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/m 3 2 day study: 144-2758 μg/m 3 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/m 3 CAP-UF 269-556 μg/m 3 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. Increasedpercentages 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/m 3 of CAP-F and increased at 3613 μg/m 3 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/m 3 (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/m 3 Time to Analysis: 6 h/day for 3 day, 5 h post-exposure pulmonary function. 2 day post-exposure for BALF collectionPulmonary 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, SO 2 , NO 2 , nitrate, sulfate
Particle Size: 0.01- 2.5 μm Route: Nose-only Inhalation
Dose/Concentration: 315.6 μg/m 3 (Low) or 684.5 μg/m 3 (High) Time to Analysis: Low: Exposed for 6 h. Sacrificed 36 h post-exposureHigh: Exposed for 4.5 h. Sacrificed 36 h postexposurePulmonary 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/m 3

, CO: 3.5 ± 0.1 ppm, NO 2 : 2.2 ± 0.3 ppm, SO 2 : <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-1 β 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/m 3 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 mononucle ar 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) (NO 2 , CO, SO 2 )
Particle Size: 10 μm (diameter) Route: Open-Top Exposure Chamber
Dose/Concentration: 33.86 ± 2.09 μg/m 3 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 TiO 2 (DuPont)
Particle Size: NR Route: Whole-body Inhalation
Dose/Concentration: 10, 50 or 250 mg/m 3 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 doseinduced 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) (NO 2 , SO 2 , CO)
Particle Size: 2.5, 10 μm (diameter) Route: Open-Top Chamber
Dose/Concentration: PM 2.5 : filtered chamber- 2.9 ± 3.0 μg/m 3 , nonfiltered chamber- 16.9 ± 8.3 μg/m 3 ; Outdoor concentration: PM 10 - 36.3 ± 15.8 μg/m 3 , CO- 1.7 ± 0.7ppm, NO- 89.4 ± 31.9 μg/m 3

, SO 2 - 8.1 ± 4.8 μg/m 3 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. Kill ed 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/m 3 DEE-ER PM: 7 μg/m 3 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. Vagoto my 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/m ouse; 0.005, 0.05, 0.5 mg/kg PSB: 0.1, 1 or 10 μg/m ouse; 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 PM 10 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: PM 2.5 ; FA: 160 nm; UFA: 50-75 nm Route: Nose-only Inhalation
Dose/Concentration: CAPS 80, 66 μg/m 3 ; avg 73 μg/m 3 FA: 299, 280, 119, 203 μg/m 3 ; avg 225 μg/m 3 UFA: 140, 565, 416, 750 μg/m 3 ; avg 468 μg/m 3 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 respiratoryrate 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 particlesPSC: 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 increasesin 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/m 3 P-20: 139 or 112 μg/m 3 P-I: 99 μg/m 3 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/m 3 ; Average Fe concentration: 96 μg/m 3 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; Fe 2 O 3 ; soot: 60% EC, 40% OC) (CO, NO X )
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/m 3 , Soot- 250 μg/m 3 , Fe+Soot- Fe: 45 μg/m 3

, Total PM: 250 μg/m 3 ; Neonates: Fe+Soot- Low: Fe- 30 μg/m 3 , Total PM: 250 μg/m 3 , High: Fe- 100 μg/m 3 , Total PM: 250 μg/m 3 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-1 β , intracellular ferritin, and NFκ B 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-1 β , NF- κ B, 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 ac tivity, and increased IL-1 β 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: PM 2.5 , NO 2 and CB (Sao Paulo, Brazil)
Particle Size: PM 2.5 Route: Whole-body Inhalation
Dose/Concentration: PM 2.5 : 46.49 μg/m 3 Control: 18.62 μg/m 3
NO 2 : 59.52 μg/m 3 Control: 37.08 μg/m 3 CB: 12.52 μg/m 3 Control: 0 μg/m 3 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/m 3 comprised of: NO 2 1.6 ppm NO 4.5 ppm CO 7.5 ppm Hydrocarbons 4.3 ppm Formaldehyde 0.26 mg/m 3 Suspended particulates 4.3×10 6 /cm 3
Particle Size: <10 μm Route: Whole-body Inhalation
Dose/Concentration: DEP 300 μg/m 3 Time to Analysis: Single exposure for 1 h. Parameters measured 6 h post exposure. Transcription Factors: Exposure induced increasedcytoplasmic and nuclear immunoreactivity of phosphorylated p38 MAPK in br onchial 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 positivelyassociated 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 R SPM-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), CO 2 (0.35%), O 2 (20.1%), PM 2.5 , PM 10 )
Particle Size: PM 2.5 , PM 10 Route: Whole-body Inhalation
Dose/Concentration: WS: 60 g, PM 2.5 : 363 ± 23 μg/m 3 , PM 10 : 502 ± 34 μg/m 3 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-1 β , 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- β 1, 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, NO 3 , SO 4 , NH 4 , metals
Particle Size: ~0.25 μm Route: Whole-body Inhalation
Dose/Concentration: Low: 30 μg/m 3 Mid-low: 100 μg/m 3 Mid-high: 300 μg/m 3 High: 1000 μg/m 3 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/m 3 Mid-low: 100 μg/m 3 Mid-high: 300 μg/m 3 High: 1000 μg/m 3 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/m 3 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/m 3

, Medium- 30.3 ± 11.8 μg/m 3 , High- 59.1 ± 28.3 μg/m 3 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 airwayreactivity 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 byexposure. 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/m 3

, Medium- 30.3 ± 11.8 μg/m 3 , High- 59.1 ± 28.3 μg/m 3 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/m 3 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- κ B 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/m 3 ; CB: 20 mg/m 3 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-1 β 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: PM 10-2.5 PMF: PM 2.5 -B: Borken, Germany (rural)-D: Duisburg, Germany (industrialized)
Particle Size: PM 10-2.5 , PM 2.5 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 concentrationvs 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 radicalsincreased with exposure. Relative intensity was: PMCD, PMF-D, PMC-B, PM F-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 PM 2.5 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: PM 2.5 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/m 3 OC = 269 μg/m 3 NO = 45 ppm NO 2 = 4 ppm CO = 30 ppm THV = 2 ppm
HWS: EC = 43 μg/m 3 OC = 908 μg/m 3 NO or NO 2 = 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/m 3 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/m 3 for HWS males exposed to HWS. Cytokines: IL-1 β 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/m 3 . Protein was unaffected by DE. HWS exposure showed male-only effects at 100 and 300 μg/m 3 but not at 1000. AP was unaffected by DE or HWS except for slight decline induced by HWS at 1000 μg/m 3 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/m 3

. BALF GSH to (GSH+GSSG): No effects for DE were observed. HWS significantly decreased the ratio in both males and females at 1000 μg/m 3 . 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, NO 2 , SO 2 , THC) (PM 2.5 compositionEC, OC, SO4, NH4, NO 3 )
Simulated downwind coal emission atmospheres (SDCAs) (fly ash, gas-phase pollutants, sulfate aerosols, NO, NO 2 , SO 2 ) 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/m 3 , SDCAs: 317-1072 μg/m 3 , RD: 306-954 μg/m 3 ; GEE: CO104 ppm, NO- 16.7 ppm, NO 2 - 1.1 ppm, SO 2 -1.0ppm, THC- 12 ppm; SDCAs: CO- <1 ppm, NO0.19-0.62 ppm, NO 2 - 0.10-0.37 ppm, SO 2 - 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 inducedincreases 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/m 3 Fall2 = 260 μg/m 3 Fall3 = 369 μg/m 3 Winter1 = 815 μg/m 3 Winter2 = 190 μg/m 3 Winter3 = 371 μg/m 3 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, follo wed 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/m 3 PM 2.5 including 600 μg/m 3 PM 1 Time to Analysis: 4 h/days for 3 consecutivedays. 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 whilenumber of macrophages increased in bronchiolealveolar duct regions at both time periods. Cytokines: MIP-2 and transferrin increased at 18 h. IL1 β 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/m 3 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 CO 2 : 2170, 6540 ppm NO: 7.0, 22.8 ppm NO X : 8.6, 28.3 ppm SO 2 : 0.83, 3.09 ppm NH 4 : ND Measured Major Components: NO, SO 2 , 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/m 3 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 nottime-dependent, though perhaps some small dosedependence was observed. The followinghistopathology 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 infemales than males and appeared to be dosedependent. The major 3 types of tumors are thefollowing:: 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: SO 2 = 0.11 mg/m 3 NO X = 0.49 mg/m 3 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/m 3 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-1 β 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, NO X , 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/m 3 ; CO- 15-106.4 ppm, NO- 2.2-18.9 ppm, NO X - 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 beingsignificant 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-ladenmacrophages 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, Spl een, 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 bothCB14 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-1 β . 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 PM 2.5 (collected from stacked filter air sampler in Shanghai, China)
Fe: FeSO 4 Zn: ZnSO 4 PMF: PM 2.5 + FeSO 4 PMFZ: PM 2.5 + FeSO 4 + ZnSO 4 Major Measured Components: Fe 26 ppm, Zn 9 ppm, S 61 ppm
Particle Size: PM 2.5 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 m 2 particles/m 3 , Mass specific surface area- 807 m 2 /g Route: Whole-body Inhalation
Dose/Concentration: 172 μg/m 3 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 (ZnSO 4 , aerosolized)
Particle Size: NR Route: Nose-only Inhalation
Dose/Concentration: 9.0 ± 2.1 μg/m 3 , 35 ± 8.1 μg/m 3 , 123.2 ± 29.6 μg/m 3 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 PM 2.5 - 10 Collected from San Joaquin Valley, CA
Particle Size: PM 10-2.5 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/m 3 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/m 3 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 R 2 = 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/m 3

, High- 632.9 ± 47.61 μg/m 3 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. DEPreduced NEP activity. Plasma extraversion dosedependently increased and was greatest in capsaicin animals. Respiratory permeability dose-dependently increased. IL-1 β 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 researchengine at 75% throttle) (EC34.93-601.67 μg/m 3 , OC- 1.90- 11.25 μg/m 3 , Sulfates 0.94-17.96 μg/m 3 , Na- 4.07-4.78 ng/m 3 , Mg0.60-0.86 ng/m 3 , Ca- 5.05-10.66 ng/m 3 , Fe- 3.17-6.44, Cr- 0.68- 1.31 ng/m 3 , Mn- 0.11-0.22 ng/m 3

, Pb- 0.97-1.24 ng/m 3 )
Particle Size: 7.5-294.3 nm Route: Nose-only Inhalation
Dose/Concentration: Low- 35.3 ± 4.9 μg/m 3

, High- 669.3 ± 47.6 μg/m 3 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 Zn 2+
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 TLR 2 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-1 β , MIP-1 α , 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-1 β and MIP-1 α mRNA expression slightly. DEP had no effect. LPS significantly increased IL-1 β and MIP-1 α 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 inflammati on without the alveolar hemorrhages. mRNA Expression of TLRs: DEP-OC, DL, DOL and LPS increased TLR 2 . 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 inducedincreases. 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 peroxyn itrite 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 signi ficantly 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 dec reased 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/m 3 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-1 β observed. No effect on TNF- α or NF- κ B-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 valuedecreased 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)
TiO 2 Particle Size: SRM1648: avg 1.4 μm TiO 2 : 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 byOva/Mch challenge was significantly and dosedependently increased at doses of SRM1648 ≥ 500 μg . TiO 2 /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 TiO 2 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/m 3 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/m 3 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, butlinear 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 thehigh 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/m 3 BC: 3, 12, 25, 43 μg/m 3 OC: 40, 107, 281, 908 μg/m 3 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/m 3 with a dose dependent decrease at 300 and 1000 μg/m 3 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 and15w/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/m 3 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/m 3 level. (p<0.05 at all levels) Splenic B cell proliferation was increased at the 30 μg/m 3 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
O 2 , CO, NO 2 , NO, SO 2 O 2 : 20.9- 20.5% (Lo, Hi) CO: 0.9-5.4 ppm NO 2 : 0.25-1.13 ppm NO: 2.5-10.8 ppm SO 2 : 0.06-0.32 ppm H 3 N 2 : NR
Particle Size: NR Route: Inhalation; Oropharyngeal aspiration (virus)
Dose/Concentration: DE: 529 or 2070 μg/m 3 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/m 3 had significantly greater levels of HA mRNA compared to air-exposed mice. HA levels not significantly altered in mice exposed to 2.0 mg/m 3

. DE Exposure on the Influenza-induced Inflammatory Response: f IL-6 mRNA levels were significantly greater when exposed to 0.5 mg/m 3 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 increasedBAL 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/m 3 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 alonesignificantly increased expression of SP-A in airexposed. Exposure to 0.5 mg/m 3 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/m 3 DE exposed. Decrease seen in expression of SP-A protein in lungs of mice exposed to 0.5 mg/m 3 DE prior to infection. Levels of SP-D mRNA and protein were significantly decreased in lungs of mice exposed to 0.5 mg/m 3 of DE prior to infection compared with mice exposed to air or 2.0 mg/m 3 DE prior to infection. Exposure to 0.5 mg/m 3 of DE prior to infection with influenza decreased levels of SP-D, especially in airways. Mice exposed to 2.0 mg/m 3 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, NO 2 , SO 2 , NH 3 )
Particle Size: NR Route: Whole-body Inhalation
Dose/Concentration: Low(L)- 6.6 ± 3.7 PM/m 3

, Medium(M)- 30.3 ± 11.8 PM/m 3 , High(H)- 59.1 ± 28.3 PM/m 3 , 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-2increased in the HF group and was dosedependent. Eosinophils dose-dependently decreased. OVA-specific IgE increased in the Hgroup, 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+OVAconcentrations 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 mi ce treated with OVA and challenged with PBS, both groups cytokine production was low, only IL-5 levels were significant in the C BP+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)
TiO 2 : fine and ultrafine
OVA: Ovalbumin
Particle Size: F CBP: 260.0 nm UF CBP: 14.0 nm
F TiO 2 : 250.0 nm UF TiO 2 : 29.0 nm Route: Intranasal Droplet
Dose/Concentration: CBP: 200 μg (3.3 mg/mL)
TiO 2 : 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 TiO 2 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 TiO 2 or CBP leads to peribronchial and perivascular inflammatory infiltrates (mostly neutrophils). Exposure to OVA alone, or combined with fine TiO 2 and fine CBP had no effects on lung histology.
Ultrafine Stimulate Local Immune Responses: TiO 2 and CBP particles stimulated the local immune response against co administered OVA antigen. Fine TiO 2 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 TiO 2 Increase OVA-specific IgE and IgG1 Levels: Levels of OVA specific IgE were significantly increased in animals exposed to the UF TiO 2 + OVA compared to F TiO 2 or OVA-only. Average IgE level in mice exposed to ultrafineCBP+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 TiO 2 and CBP. IFN-y levels were significantly increased in ultrafine CBP+OVA treated animals. F TiO 2 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/m 3 OVA 40.5 ± 6.3 mg/m 3 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/m 3 OVA 42.3 ± 5.7 mg/m 3 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 synergisticeffect with OVA on inducing airway hyperresponsiveness (AHR) in rats. DEP alone hadno 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 sensitizedrats. 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 werestimulated 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: PM 10 and PM 2.5 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: PM 10 and PM 2.5 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/m 3 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 instilla tion 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. DEPexposure 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-trkAadministration 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/m 3 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 increasedmacrophages by 161% compared to airexposed, vehicle-sensitized mice, while OVAsensitized mice had 69% increase. Anti-p75administration 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 orNKT 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 wasnot 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 NO X , 1.99 ± 0.02 NO 2 , 10.02 ± 0.12 NO, 0.18 ± 0.002 SO 2 and 1769.2 ± 13.2 CO 2 (all in ppm). DE gas: 11.93 ± 0.13 NO X , 2.93 ± 0.06 NO 2 , 8.91 ± 0.09 NO, 0.11 ± 0.003 SO 2 and 1838.8 ± 15.3 CO 2 (all in ppm)
Particle Size: 0.4 μm (MMAD) Route: Whole-body Inhalation
Dose/Concentration: Exposure to: 0, 1.0 mg/m 3 or 1.0 mg/m 3 DE gas only (0.04 mg/m 3 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 a llergen, 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-1 α in CLN cells from SBP-immuni zed 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/m 3 1796 ppm CO 2 12.09 ppm NO X 0.18 ppm SO 2 Composition of filtered DE Gas: DEP: 0.04 mg/m 3 1839ppm CO 2 11.93ppm NO X 0.11 ppm SO 2 Sugi Basic Protein (SBP)- allergen
Particle Size: 0.4 μm (average diameter) Route: Whole-body Inhalation
Dose/Concentration: 1.0 mg DEP/m 3 or 1.0 mg DEP/m 3 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-1 α 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/m 3 (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. mRNAlevels of inflammatory cytokines IL-2, IL-5, IL12 α , IL-12 Β 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)
NiSO 4 , CuSO 4 , VOSO 4, Na 3 VO 4 Particle Size: NR Route: Cell Culture; seeded into 6-well plates (3- 4.5×10 5 cells/3 mL/well) or 24-well plates (0.6- 1×10 5 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 NiSO 4 alone and NiSO 4 with MALP-2 produced 10 and 50 fold increases, respectively, in IL-6 production. Exposure of HLF to CuSO 4 , VOSO 4 and Na 3 VO 4 , with and without the presence of MALP-2, did not produce as dramatic results as seen with Ni. The action of NiSO 4 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 PM 2.5 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: PM 2.5 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 PM 2.5 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-1 Β , IL6 and MIP-2 in comparison to control and to mice exposed to Zerbst PM. PM 2.5 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 daysafter 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 PM 2.5 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 increasedlevels 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% O 2 , 0.4wt ratio OC/EC; High dose: 20.7% O 2 , 0.4wt ratio OC/EC) (CO, NO X , SO 2 )
Particle Size: Diameter: ~240 nm Route: Inhalation
Dose/Concentration: Low- 514 ± 3 μg/m 3 , High2026 ± 38 μg/m 3 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-13increased 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 m 3 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: Didnot 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/m 3 , 5 day concentration: 313 ± 119 μg/m 3

, July concentration: 16-185 μg/m 3 , September concentration: 81-755 μg/m 3 ; 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 PM 2.5 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:
NO X : 2.0-43.3 ppm CO: 0.94-29.0 ppm
SO 2 : 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/m 3 (low level) or 10027 μg/m 3 (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 andlow 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 & Ai rway 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 lowor 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-infectedmice 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/m 3 PM, MidLow- 100 μg/m 3 PM, Mid-High- 300 μg/m 3 PM, High- 1000 μg/m 3 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, Part icle 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/m 3 ; OC: 244 ± 144 μg/m 3 , EC: 10 ± 4 μg/m 3 , Sulfate: 79 ± 131 μg/m 3 , Nitrate: 39 ± 67 μg/m 3 , Ammonium: 39 ± 59 μg/m 3 ; Urban dust (Fe, Al, Ca, Si): 18 ± 6 μg/m 3 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/m 3 High - 3 mg/m 3 Time to Analysis: 7 h/day, 5 days/wk, 1 or 3 mo Lung Histopathology: DEP-laden macrophages accumulated in the alveoli andperibronchial 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-1 Β 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- κ B 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/m 3 High - 3 mg/m 3 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-1 Β , 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/m 3 3. Air + Der f : 1 mg Der f 4. DE 3.0 mg/m 3 + 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 nonciliatedcells and epithelial cell hypertrophy. Sootcontaining macrophages were found in the alveolar tissue spaces. Accumulated lymphocytes were present in the peribronchiolarlymphoid 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-1 α in all strains. MCP-1 also increased but not significantly. DE+Der f increased IL-5, RANTES, eotaxin, MCP-1 and MIP-1 α 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 IL1 Β , MIP-1 α , 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 m 2 /g 56 nm - 45 m 2 /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-1 Β 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-1 α , KC, IL-1 Β ).
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 m 2 /g 56 nm - 45 m 2 /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-1 α . 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-1 α , 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 m 2 /g 56 nm - 45 m 2 /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/m 3 1.0 mgsoot/m 3 3.0 mg soot/m 3 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: PM 2.5 Route: Cell Culture (Splenocytes resuspended to cell density of 1×10 6 /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: PM 2.5 Route: Cell Culture
Dose/Concentration: 1×10 6 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)
O 3 - (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) O 3 : 2 ppm (avg 1.98 ± 0.08 ppm) OVA sensitization: 10 mg Time to Analysis: OVA sensitization, DEP, O 3 and OVA Challenge on d21- 23 Exposed to O 3 for 3 h and DEP for 1 h AH and BAL measured 1 day after last challenge Airway Responsiveness : OVA + O 3 + DEP exposure group had significantly higher methacholine-induce Penh than sham group or OVA group. Total cells, proportion of eosinophils and neutrophils : The OVA + O 3 + DEP group was significantly higher than OVA group and OVA+ O 3 group. IL-4: OVA + O 3 , OVA + DEP and OVA + O 3 + DEP IL-4 level increased compare to OVA group. IFN- γ : Levels significantly decreased in OVA + DEP and OVA + O 3 + DEP compared to OVA + O 3 . 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: 3×10 5 cells infected with 320 hemagglutination units (HAU)
DEas : For A549 cells: 6.25, 12.5, 25 μg/cm 2 . For bronchial and nasal cells: 22 or 44 μg/cm 2

. 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 30min 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/cm 2 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/cm 2 for nasal cells and approaching significance at 44 μg/cm 2 for bronchial cells. Exposure of both types to 44 μg/cm 2 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 ofboth 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 reversedthe 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 PM 10 - EHC-93 obtained (Environmental Health Canada, Ottawa, ON, Canada) RSV - Human RSV (long strain/lot18D) (American Tissue Culture Collection, Bethesda, MD)
Particle Size: PM 10 (0.35 μm MMAD) Route: Cell Culture
Dose/Concentration: PM 10 : 500 μl/well (100 μg/ml MEM) RSV exposure:: 1 ml/well (6×10 6 pfu/ml MEM) Groups: PM 10 +RSV RSV+PM 10 RSV only PM 10 only negative control Time to Analysis: PM 10 - 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 PM 10 . More than 95% of AM exposed to PM 10 engulfed PM. AM exposed to PM 10 showed significant increase in mean side scatter incomparison to negative control and RSVinfected AM. No significant difference between AM exposed only to PM 10 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: PM 10 exposure inhibits. All RSV-treated groupsshowed significantly greater proportion of RSVimmunopositive cells compared with negative control. PM 10 +RSV showed significantly smaller proportion of RSV-immunopositive cells compared with RSV group. RSV+PM 10 groupsimilar to RSV group. Proportion of RSVimmunopositive AM was influenced by the sequence of exposure to RSV and PM 10 . 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 PM 10 . Negative control and PM 10 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 stimulatedall three cytokines measure (IL-6, IL-8 and TNFα ) compared to negative control. IL-6: PM 10 significantly reduced RSV-induced IL-6 production. IL-6 was affected by the sequence of exposure to PM 10 and RSV (PM 10 +RSV vs. RSV+ PM 10 ). IL-8: PM 10 significantly decreases RSV-induced IL-8 production and baseline. No affect on sequence of exposure. TNF- α : production was increased when exposed to RSV, PM 10 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 ultr afine (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/m 3 -UF at 150 m: 283 μg/m 3 F at 50 m or 150 m: average 400 μg /m 3 OVA sensitization: 50 μg/5 μl OVA challenge: 30 mg/m 3 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: PM 2.5 ; UF: PM0.15 Route: Whole-body Chamber
Dose/Concentration: 50 m - F: 394 ± 94 μg/m 3 50 m - UF: 297 ± 189 μg/m 3 150 m - F: 387 ± 68 μg/m 3 150 m - UF: 213 ± 95 μg/m 3 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
V 2 O 5 , VOSO 4 , SiO 2 TiO 2 , Fe 2 (SO 4 ) 3 , NiSO 4 , LPS
Particle Size: 1.95 μm (MMAD) Route: Cell Culture
Dose/Concentration: ROFA: 0, 2.5, 5, 10, 15, 20 μg/cm 2 LPS: 100 ng/mL
V 2 O 5 : 0, 0.15, 0.3, 0.61, 1.25, 2.5, 5, 10, 20 μg/cm 2
NiSO 4 , Fe 2 (SO 4 ) 3 , TiO 2 , SiO 2 : 0, 1.23, 2.5, 5, 10, 20 μg/cm 2
VOSO 4 : 0, 0.145, 0.29, 0.58, 1.16, 2.32 μg/cm 2 Time to Analysis: LPS: 0, 6, or 18 h ROFA: 0, 2,4,6 h V 2 O 5 : 0, 0.25, 0.5, 1, 2, 4, 6, 8h NiSO 4 , Fe 2 (SO 4 ) 3 , TiO 2 , SiO 2 , VOSO 4 : 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/cm 2 significantly increased inducible TAP expression.
Soluble Metals in BTE: V 2 O 5 inhibition of LPS and IL-1 β induced TAP gene expression increases with exposure time and dose. NiSO 4 exhibits non-significant dose dependent suppression of inducible TAP gene expression. Fe 2 (SO 4 ) 3 , TiO 2 and SiO 2 were found to have no effect.
A549: Results with ROFA and V 2 O 5 in BTE were replicated using the A549 cell line and IL-1 β to induce hBD2 gene expression. Cellular Viability: Was not significantly affected in ROFA doses below 20 μg/cm 2 and V 2 O 5 /VOSO 4 doses below 2.5 μg/cm 2

. 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 (1×10 6 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 bythe 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: PM 0.1 - PM 2.5 Route: Inhalation OVA - Intraperitoneal Injections; Aerosol Exposure
Dose/Concentration: PM - 235-256 μg/m 3 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/m 3

, CO: 3.5 ± 0.1 ppm, NO 2 : 2.2 ± 0.3 ppm, SO 2 : <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-1 Β 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+OVAsignificantly 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-1 α

. 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/m 3 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/m 3

. 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 (0.5×10 6 AM/well)
Dose/Concentration: 20 μg/mL
surface area: 159 ± 4m 2 /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 cm 3 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 NO 2 , <0.01 ppm SO 2 and 103.1 ± 9.2 μg/m 3 DEP.
Particle Size: NR Route: Whole-body Inhalation
Dose/Concentration: 100 μg/m 3 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/m 3; September 313 μg/m 3 Time to Analysis: First rats were sensitized (days 1-3) and challenged (days 14-16) with saline (control) or OVA by intr anasal 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 animalsexposed 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 M HC 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 (5.94×10 10 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 producedthe 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/m 3

, Medium- 30.3 ± 11.8 μg/m 3 , High- 59.1 ± 28.3 μg/m 3 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 throughoutgestation. 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 5×10 4 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-totalgroup 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 timepoints. 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 2.5×10 4 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%) TiO 2 Particle Size: TiO 2 = 0.3 μm average, 1.0 μm max SRM 1648 = 0.4 μm (mean diameter) Route: Cell Culture (625,000 cells/cm 2 in 96 well plate)
Dose/Concentration: 0, 7.8, 15.6, 31.2, and 62.5 μg/cm 2 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 - 1270m 3 ) AERex5d - urban aerosol 5 day sample (total air volume - 6230m 3 ) 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 1in 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 BALcells 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-1 Β 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.)
TiO 2 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 TiO 2 : 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 TiO 2 .) 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 TiO 2 .) 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 didnot 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)
O 3 (positive control) L. mono: Listeria monocytogenes (strain L242/73 type 4B)
Particle Size: DEP, EHC-93: NR Route: DEP/EHC-93: intranasal droplet; O 3 : Whole-body inhalation
Dose/Concentration: DEP/EHC-93: 50 μg (1.0 mg/ml)
O 3 : 2mg/m 3 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)
O 3 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 O 3 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 O 3 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 O 3 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 O 3 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) inducedan 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 samplesproduced 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) pretreatedwith 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 showedonly 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 antibodyresponse 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:
O 2 : 4.3 ± 0.07 ppm NO: 9.2 ± 0.30 ppm
NO 2 : 1.1 ± 0.05 ppm
SO 2 : 0.2 ± 0.10 ppm Low composition:
O 2 , NO, NO 2 , SO 2 below detection limits
Particle Size: NR Route: Whole-body Inhalation OVA immunization and challenge: intranasal
Dose/Concentration: High = 2000 μg/m 3 Low = 500 μg/m 3 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/m 3 CO: 10.6 ppm
NO 2 : 7.3 ppm
SO 2 : 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/m 3 Time to Analysis: Cells were exposed to varying concentrations of suspended DEP for up to 24 h.
NF- κ B: 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 withsuspended 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 after12 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- κ B / STAT6 Activation: (it has been suggested that NF- κ B plays a role in the transcriptional regulation of eotaxin gene expression) Cells exposed to 1-25 μg/mL DEP for 6 h increased NF- κ B. 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- κ B 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. Inaddition pre-treatment with NAC attenuated NFκ B 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- κ B 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 0.5m 3 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/m 3 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 stainedtissue 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 significantincrease in the expression of CD83 and a nonsignificant increased expression of HLA-DR andCD86. 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: Sr 2+

, Ni 2+ , Cd 2+ , Al 3+ , Pb 2+
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 SrCl 2 , NiSO 4 , CdCl 2 or AlCl 3 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 : Al 3+ and Ni 2+ enhanced antigen-mediated release. 10-7 M AlCl 3 released 23% of Β

hexoaminidase compared to antigen alone, which induced 11% release of Β

hexoaminidase. Cd 2+ , Sr 2+ and Pb 2+ enhanced antigen-mediated release to a lesser extent. Ni 2+ , Al 3+ , Sr 2+ and Cd 2+ depicted a dosedependent relationship with antigen-mediated Β

hexoaminidase release. Antigen-Induced Protein Phosphorylation : Addition of the antigen induced the anticipated phosphorylation of multiple proteins in C57 mast cells The presence of Ni 2+ and Pb 2+ mediated an increase in phosphorylation of several of the proteins and Al 3+ 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 = 1×10 6 cell
DG 75 = 3×10 6 cells IgE PMBC 1×10 6 /mL B-cells 0.5×10 6 /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/m 3 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/m 3 , High632.9 ± 47.61 μg/m 3 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-1 β was significantly higher for the low-exposure group. IL-12 was significantly lower in the capsaicin high-exposure group. TNF- α increased in thehigh-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/m 3

, OC- 1.90-11.25 μg/m 3 , Sulfates 0.94- 17.96 μg/m 3 , Na- 4.07-4.78 ng/m 3 , Mg0.60-0.86 ng/m 3 , Ca- 5.05-10.66 ng/m 3

, Fe- 3.17-6.44, Cr- 0.68-1.31 ng/m 3 , Mn0.11-0.22 ng/m 3 , Pb- 0.97-1.24 ng/m 3 )
Particle Size: 7.5-294.3 nm Route: Nose-only Inhalation
Dose/Concentration: Low- 35.3 ± 4.9 μg/m 3 , High669.3 ± 47.6 μg/m 3 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 bycapsaicin. 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-exposuregroup 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 : WashedDEP+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-1 α 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-1 α and Whole DEP+OVA caused an even greater increase in MIP-1 α

. 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/m 3 (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-1 β 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/m 3 (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-1 β , 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 m 2 /g; CB: 0.1- 0.6 μm Route: IT Instillation of Listeria; Cell Culture (2.5×10 5 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-1 Β 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/m 3 (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/m 3 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-2R α in response to ConA stimulation. DEP-treated infected rats had increases in ConA-inducible CD4+/IL-2R α + and CD8+/ IL-2R α +. 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 (concentra ted ambient PM 2.5 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/m 3 ; 60-600 μg/m 3 . ,Study 2- Mean-107 μg/m 3 ; 65-150 μg/m 3 (S.pneumoniae 2-4×10 7 ) 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, lav ageable 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 ef fect 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-1 β 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: PM 2.5 Fe 2+, Mn 2+ , Ni 2+ : 0.4 μm (MMAD) Route: Nose-only Inhalation, IT instillation (S. pneumoniae)
Dose/Concentration: Single metals/NYC PM: 65- 90 μg/m 3 ; 15-20×10 6 (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 ratsexposed to inhaled Fe significantly reduced Blymphocyte proliferation at 48 h, but did not affect T-lymphocyte production. Inhaled Ni, forthe 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)
TiO 2 Carbon Black (CB) (Sigma, St. Louis, MO) Streptococcus pneumoniae: strain ATCC6303
Particle Size: UAP = NR; TiO 2 /CB = NR; CAPs: ≤ PM 2.5 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 kill ed bacteria measured 2 h after bacteria exposure. Binding, Internalizat ion 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: TiO 2 and CB had no effect on J774 binding or internalization of S. pneumo. TiO 2 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 NO 4 2- , LPS, endotoxins)
Particle Size: PM: 2.5, 10 μm Route: Ambient Air Exposure
Dose/Concentration: Mexico City: PM 10 : 78 μg/m 3 , PM 2.5 : 21.6 μg/m 3 , Pb in TSP: <0.4 μg/m 3 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- κ B, iNOS, cyclooxygenase-2, glial fibrillatory acid ic 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 H 2 O soluble gases, O 3 , NO X , SO X 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/m 3 , avg F concentration: 441.7 μg/m 3 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- κ B DNA binding in brain. TNFα increased with F+UF. IL-1 α 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 catalyticconverter 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/m 3 , Low dose: Mass concentration: 30.4 μg/m 3 Time to Analysis: 5 h/day, 3days/wk, 6 wk; 24 h postexposure. Activated AP-1 dose-dependently increased. Activated NF- κ B 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/m 3 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/m 3 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, characterizedby 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, CO 2 , NO, NO 2 , SO 2 )
Particle Size: 26.21 ± 1.50 nm (diameter) Route: Whole-body Inhalation
Dose/Concentration: 148.86 ± 8.44 μg/m 3 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-1 β , 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)
TiO 2 Particle Size: NR Route: Intranasal Instillation
Dose/Concentration: DEP, TiO 2 : 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 TiO 2 . Analyzed 19 or 48 h later. Protocol 1b: Pregnant mice DEP, TiO 2 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 TiO 2 increased IL-1 β , TNF- α , IL-6 and KC compared to nonpregnant controls. Offspring of DEP, CB or TiO 2 exposed mice had increased AHR and airway inflammation. TiO 2 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/m 3 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 m 2 /g (SA) Route: Whole-body Inhalation
Dose/Concentration: 20 mg DEP/m 3 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 mg 2 /g, density 2.1 g/cm 3 Route: Inhalation
Dose/Concentration: 19.1 ± 1.13 mg DEP/m 3 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) (NO 2 , SO 2 , CO)
Particle Size: 2.5, 10 μm (diameter) Route: Ambient Air Exposure
Dose/Concentration: PM 2.5 : filtered chamber- 2.9 ± 3.0 μg/m 3 , nonfiltered chamber- 16.9 ± 8.3 μg/m 3 ; Outdoor concentration: PM 10 - 36.3 ± 15.8 μg/m 3 , CO- 1.7 ± 0.7 ppm, NO- 89.4 ± 31.9 μg/m 3

, SO 2 - 8.1 ± 4.8 μg/m 3 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. Kill ed 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: PM 10 : 35.5 ± 12.8 μg/m 3 ; CO: 2.2 ± 1.0 ppm; NO 2 : 107.8 ± 42.3 μg/m 3 ; SO 2 : 11.2 ± 5.3 μg/m 3 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/m 3 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/m 3 ; Average Fe concentration: 96 μg/m 3 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- November21, 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/m 3 , Urban-Industrial- 38.9 ± 10.5 - 115.3 ± 25.3 μg/m 3 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/m 3 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/m 3 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/m 3 (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/m 3 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/m 3 ). 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% PM 2.5 comprises air pollution)
Particle Size: 2.5 μm (diameter) Route: Open-Top Chamber
Dose/Concentration: PM 2.5 - 27.5 μg/m 3 ; NO 2 - 101 μg/m 3 ; CO- 1.81 μg/m 3 ; SO 2 - 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 ai r 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/m 3 , Filtered- 6.5 μg/m 3 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 fo llicles decreased. Mating time increased and fertility and pre gnancy indices decreased. The mean post-implantation loss rate increased, which was influenced by both pre- and post-gestational exposure. Fetalweight 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, NO 2 )
Particle Size: 90% <0.5 μm Route: Inhalation
Dose/Concentration: High dose total group: PM1.71 μg/m 3 , NO 2 - 0.79 ppm; Low dose total group: PM- 0.17 μg/m 3 , NO 2 - 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, NO 2 filtered, low dose total DE, low dose PM, NO 2 filtered, clean air control. Offspring sacrificed day 96 after birth. All groups had significantly less daily sperm production than the control. PM and NO 2 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/m 3 TSP 9.4+17 μg/m 3
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/m 3 ; CO: 2.67 ppm, NO 2 : 0.23 ppm, SO 2 : <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/m 3 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/m 3 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/m 3

. 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/m 3 ). 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/m 3

. 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/m 3 , Pregnant mice: 0.1, 3.0 mg DEP/m 3 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 andaccessory reproductive glands were significantly greater in DEexposed mice. Blood testosterone concentration was 8X higher than the control at 1.0 mg DEP/m 3 . 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, 20m 3 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/m 3 (summer), 21.8-61 μg/m 3 (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 PM 10 , as well as PAH and BaP. PM 10 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 oilmethyl 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; PM 10 and PM 2.5 from 6 urban locations with different traffic intensities.) (organic extracts)
Particle Size: NR Route: Cell Culture
Dose/Concentration: Mutagenicity assay: 2.5, 9, or 18m 3 sampled air in 100 μL DMSO; DNA adduct assay: 5 μL DMSO containing PM 10 or TSP from equivalent 50m 3 sampled air. PM 2.5 concentration equivalent to 35m 3 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 PM 2.5 extracts were higher compared to PM 10 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 PM 2.5 . Neither transition metal composition nor radical generating capacity of PM correlated with mutagenic potential. Total PAH and carcinogenic PAH levels from PM 10 and PM 2.5 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/m 3 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/m 3 . 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/m 3 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 NO X .

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/m 3 air (CALUX BaP Equivalent (BaPEq)), 21 mg/m 3 air (CALUX TCDD Equivalent (TCDDEq)); Cleaned APE: 7.87 mg/m 3 air (CALUX BaPEq), 0.614 mg/m 3 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) (Plze ň , Prague, Ústí, Z ď ár - 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/cm 3 ): 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 washigher 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 α
napthoflavone 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 sample s, 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 PM 2.5 and PM 10 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. PM 2.5 was more active than PM 10 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 (m 3 air); Luciferase: 0.17, 0.51, 1.26, 5.01 extract equivalents (m 3 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 m 3 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/m 3 ; Non-strike day: 43.01 μg/m 3 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-1 α , IL6, IL-8, TNF- α . The cytokines increased 4-18-fold at the highest dose. Cell viab ility 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 PM 10 (Prague (Czech Republic), Ko ˇ sice (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 φ X174-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 PM 10 ) (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 PM 2.5 and PM 10 (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, 0.1m 3 /ml equivalents PM 2.5 ; 0.82, 1.25, 1.63m 3 /ml equivalents PM 10 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, PM 2.5 extracts had greater genotoxic potential than PM 10 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/cm 2 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: O 2 , Fe (Fe from Fe 3 O 4 ) Street particles: Fe from Fe 2 O 3 Particle Size: 10 μm (diameter) Route: Cell Culture
Dose/Concentration: Comet: 5, 10, 20, 40 μg/cm 2 ; 8-oxodG: 10 μg/cm 2 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/cm 2 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, PM 10 was somewhat more potent than PM 2.5 .



### 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/m 3 ; Mass concentration dust storm day: 221.83 ± 69.89 μg/m 3 Time to Analysis: 24 h; cultures 4 h. OC, NH 4 + , NO 3 - were higher in normal weather PM 2.5 . SO 4 2- , Ca 2+ were higher in dust storm PM 2.5 . Fe, Al, Ca, Mg were 5x higher in dust storm PM 2.5 . 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 andwithout 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/cm 2 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 m 3 /ml equivalents of PM 10 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 withthe 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 PM 2.5 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. PM 10 caused less LDH release, IL-8 stimulation and free radical activity than LU dust particles that contained PM 2.5 . Chelation had little effect on PM 10 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) PM 10 (Prague, Czech Republic; Ko ˇ sice; 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: Č ervenohorské 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 m 3 /ml; Dioxin: TSP+GP- 8, 1.33, 0.22, 0.04 m 3 /ml, PM 2.5 +GP: 4, 0.66, 0.11, 0.02 m 3 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. PM 2.5 +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/cm 3 ): 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-1 α , 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-1 α , FGF-6, FGF-9, VEGF-D, fra-1 and p21. Concentration and Time Responses: Concentration and time-dependent increases occurred for FGF-9, IL-1 α , 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-1 α , 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/m 3 , EOM- 57.7 mg/total UPM; Ibirapuera Park: UPM- 32 μg/m 3 , 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 fromboth 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/cm 2 ; 10, 25, 50, 100 μg/cm 2 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. ΔΨ m 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 ΔΨ m 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; PM 10 : high volume sampling system, Athens; PM 2.5 : 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 H 2 O 2 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 PM 2.5 average than Beijing. It wa s 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 m acrophages. Toxicology, 230: 207-18. 155123 Abou Chakra OR; Joyeux M; Nerriere E; Strub MP; Zmirou-Na vier D (2007). Genotoxicity of organic extracts of urban airborne particulate matter: an assessment with in a personal exposure study. Chemosphere, 66: 1375-81. 098819 Adamson IYR; Vincent R; Bakowska J (2003). Differential pr oduction 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 charge d 2- and 10-"mu"m particles activate vanilloid receptors, increase cAMP, and induce cytokine releas e. 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 vitr o 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 differen t zones of Mexico City. Environ Health Perspect, 110: 715-720. 156204 Amakawa K; Terashima T; Matsuzaki T; Matsumaru A; Saga i M; Yamaguchi K (2003). Suppressive effects of diesel exhaust particles on cytokine release from human and mu rine 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 metalloprot ease-1 in human lung epithelial cells via a NADP(H) oxidase/NOX4 redox-dependent mech anism. Am J Physiol Lung Cell Mol Physiol, 293: L170-181. 156212 Andre E; Stoeger T; Takenaka S; Bahnweg M; Ritter B; Ka rg E; Lentner B; Reinhard C; Schulz H; Wjst M (2006). Inhalation of ultrafine carbon particles tr iggers biphasic pro-inflammatory respon se 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 fl y ash collected from different sites within a power plant. Mol Cell Biochem, 255: 257-265. 097199 Apicella C; Custidiano A; Miranda S; Novoa L; Dokmetjia n J; Gentile T (2006). Differen tial 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 wi th chronic allergic pulmonary inflammation. Toxicol Pathol, 36: 680-686. 187137

Note: Hyperlinks to the reference citations throughout this doc ument 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 af ter 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 ch emokines 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). Arylhydrocarbon 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 airw ay 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--spontane ous hypertensive versus Wistar Kyoto rats. Toxicol In Vitro, 20: 52-62. 097608 Bagate K; Meiring JJ; Gerlofs-Nijland ME; Cassee FR; Wi egand H; Osornio-Vargas A; Borm PJA (2006). Ambient particulate matter affects cardiac recovery in a Lange ndorff ischemia model. I nhal Toxicol, 18: 633-643. 096157Bao 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 Poll ution-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 oxi dative stress. Environ Health Perspect, 113: 1032-1038. 088592 Becker S; Mundandhara S; Devlin RB; Madden M (2005). Re gulation 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; Buch anan KC; Batra SK; Smiley RA (2004). In vivo effect of wood smoke on the expression of two mucin gene s 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 meta bolic 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 proinfla mmatory 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; E mmert B; Westphal G; Muller M; Hallier E; Bruning T (2007). Strong mutagenic effects of diesel engine emissions us ing 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 catal ytic 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). Syst emic 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; Fal zano L; Colone M; De Berardis B; Paoletti L; Arancia G; Fiorentini C (2004). Fine environmental particulate engenders alterations in hum an 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 a ssociated 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 arte ries 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). Fr esh 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 AJ FC; Martins-Junior HA; Bustilho JOWV; Saiki M; Sato IM; Saldiva PHN (2005). Diesel emissions significantly influen ce composition and mutagenicity of ambient particles: a case study in Sao Paulo, Brazi l. 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 infla mmation 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; Ne l 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). Interacti on 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 spont aneously 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 cel l lines. Cell Biol Toxicol, 20: 221-239. 096682 Chauhan V; Breznan D; Thomson E; Karthikeyan S; Vincent R (2005). Effects of ambient ai r 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 am bient particles (CAPs) in mice IV Characterization of acute and chronic effects of ambient air fine particul ate matter exposures on heart-rate variability. Inhal Toxicol, 17: 209-216. 087218 Chen LC; Nadziejko C (2005). Effects of subchronic exposures to concentrated am bient particles (CAPs) in mice V CAPs exacerbate aortic plaque development in hype rlipidemic mice. Inhal Toxicol, 17: 217-224. 087219 Cheng MD; Malone B; Storey JM (2003). Monitoring cellular re sponses of engine-emitted particles by using a direct aircell interface deposition techniqu e. 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 remo deling. 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 e xpression. Inhal Toxicol, 19: 1121-1133. 096557 Corey LM; Baker C; Luchtel Daniel L (2006). Heart-rate vari ability 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 inhalati on 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 Nanoparticle s. 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-reperfusi on 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). Ro le 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 engi ne 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 H ealth Perspect, 112: 814-819. 066329 de Haar C; Hassing I; Bol M; Bleumink R; Pieters R (2005). Ultrafine carbon black pa rticles cause early airway inflammation and have adjuvant activity in a mouse alle rgic 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-adm inistered antigen in mice. Clin Exp Allergy, 36: 1469- 1479. 144746 de Haar C; Kool M; Hassing I; Bol M; Lambrecht BN; Pi eters 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 traf fic-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; Robe rts JR; Zhang X-D; Antonini JM; Ma JKH (2005). Exposure of Brown Norway Rats to diesel exhaust particles Prio r 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; Laur ent 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 associat ions 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 ag ed, compromised rats. Inhal Toxicol, 16 Suppl 1: 41-53. 087354 Elder A; Gelein R; Finkelstein JN; Driscoll KE; Harkema J; Oberdorster G (2005). Effect s of subchronically inhaled carbon black in three species I Retention kinetics, lung in flammation, 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 mu tagenic 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). In flammation, edema, and peripheral blood changes in lungcompromised rats after instillation with combustion-de rived and manufactured nanoparticles. Exp Lung Res, 32: 363-378. 097066 Farraj AK; Haykal-Coates N; Ledbetter AD; Evansky PA; Gave tt SH (2006). Inhibition of pan neurotrophin receptor p75 attenuates diesel particulate-induced enhancement of allerg ic 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 particle s 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 IF N-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 lipopolys accharide 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 ambi ent air particulate matter exposure induces molecular alterations associated with vascular disease progression wi thin 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). Distin ct 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 differ ent endpoints for in vitro measurement of genotoxicity of extractable organic matter associated with ambien t 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). Microb ial 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; Abouka is 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 co mposition of ambient PM2.5 influences severity of allergic airways diseas e 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 alve olar macrophages. Toxicol Lett, 157: 129-137. 096689 Geng H; Meng Z; Zhang Q (2006). In vitro responses of rat alve olar 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 . Envir on Sci Technol, 43: 4729-4736. 190353 Gerlofs-Nijland ME; Boere AJ; Leseman DL; Dormans JA; Sa ndstrom 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 c ontrasting 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 concen trated 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-re lated 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 part icles (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; Koda vanti 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 particle s. 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 pulm onary 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 synerg istic 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 e xhaust inhalation produces hypertensive gene expression pattern in health y 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 pulmona ry cells. Inhal Toxicol, 19 Suppl 1: 23-28. 156488 Greenwell LL; Moreno T; Richards RJ (2003). Pulmonary antioxi dants 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). To xicity of tire debris extract s on human lung cell line A549. Toxicol In Vitro, 19: 1001-1008. 097841 Gunnison A; Chen LC (2005). Effects of s ubchronic exposures to concentrated ambient particles (CAPs) in mice VI Gene expression in heart and lung tissu e. Inhal Toxicol, 17: 225-233. 087956 Gurgueira SA; Lawrence J; Coull B; Murthy GGK; Gonzal ez-Flecha B (2002). Rapid incr eases 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-Ce deno 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 offs pring. J Toxicol Environ H ealth A Curr Iss, 70: 688-695. 091235 Hamoir J; Nemmar A; Halloy D; Wirth D; Vincke G; Va nderplasschen 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 le vels 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). Di esel exhaust particles exert acute effects on airway inflammation and function in murine allergen provoca tion 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 partic les from six European cities. Inhal Toxicol, 19: 227-246. 096630 Harder V; Gilmour P; Lentner B; Karg E; Takenaka S; Zi esenis A; Stampfl A; Kodavanti U; Heyder J; Schulz H (2005). Cardiovascular responses in unrestrained WKY rats to i nhaled ultrafine carbon particles. Inhal Toxicol, 17: 29-42. 087371 Harkema JR; Keeler G; Wagner J; Morishita M; Timm E; Ho tchkiss 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 Pseudom onas 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 dies el engine emissions. Am J Respir Cell Mol Biol, 28: 451- 463. 097046 Heidenfelder BL; Reif DM; Harkema JR; Cohen Hubal EA; H udgens EE; Bramble LA; Wagner JG; Morishita M; Keeler GJ; Edwards SW; Gallagher JE (2009). Comparative microarr ay 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; Schwar ze PE (2005). Cytokine release from alveolar macrophages exposed to ambient particulate matter: heterogeneity in relati on 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 cel ls 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; S ugawara 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 mycobacteria l infection. Exp Lung Res, 31: 405-415. 088285 Hirano S; Furuyama A; Koike E; Kobayashi T (2003). Oxidativ e-stress potency of organic extracts of diesel exhaust and urban fine particles in rat heart microvesse l 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, genotox icity 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 interleuki n-6 in male rats. Toxicol Sci, 103: 137-148. 156574 Huang SL; Hsu MK; Chan CC (2003). Effects of submicromete r 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 meta l 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). Tr affic related air pollution as a determinant of asthma among Taiwanese school child ren. 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. 156582Ichinose 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; Sh imada 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; Yosh ino S; Yamaki K; Yoshikawa T (2007). Effects of components derived from diesel exhaust particles on lung physiolo gy 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; Sh imada 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 an tigen. 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 mi ce. Basic Clin Pharmacol Toxicol, 99: 346-352. 190142 Ishihara Y; Kagawa J (2003). Chronic di esel exhaust exposures of rats demons trate 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 atmos pheric 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 exposur e 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 particle s. 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-se gregated 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 in flammatory 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 respirator y 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 proinf lammatory 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). Po sitron 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). Quantitati ve measurements of the ge neration 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 s yncytial 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 indu ce 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). Compar ison of genotoxic and inflammatory effects of particles generated by wood combustion, a road simulator and collect ed 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). Ge notoxicity of airborne particulate matter: the role of cell-particle interaction and of substances with adduct-forming a nd 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 la vage 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 th e respiratory system after inha lation of ozone and sulfuric acid aerosol mixtures. Inha l Toxicol, 18: 295-303. 088596 Kleinman M; Sioutas C; Stram D; Froines J; Cho A; Chak rabarti B; Hamade A; Meacher D; Oldham M (2005). Inhalation of concentrated ambient particulate matter near a heavily trafficked road stimulat es 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 traf ficked road stimulates antigen-induced airway responses in mice. Inhal Toxicol, 19 Suppl 1: 117-126. 097082 Knuckles TL; Dreher KL (2007). Fine oi l combustion particle bioavailable constituents induce molecular profiles of oxidative stress, altered function, and cellular injury in cardiomyocytes. J Toxicol Envir on 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 pot ential 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 inju ry 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 exhaus t particles stimulates expressi on of Ia and costimulatory molecules associated with antigen presentation in rat pe ripheral blood monocytes but not in alveolar macrophages. Toxicol Appl Pharmacol, 209: 277-285. 088303 Kooter IM; Boere AJ; Fokkens PH; Leseman DL; Dorm ans JA; Cassee FR (2006). Re sponse of spontaneously hypertensive rats to inhalation of fine and ultrafine partic les from traffic: experimental controlled study. Part Fibre Toxicol, 15: 3-7. 097547 Kristovich R; Knight DA; Long JF; Williams MV; Dutt a 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 us ing 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; Laga dic-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 pa rticles 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). Ef fects of Asian dust event particles on inflammation markers in peripheral blood and bronchoalveolar lavage in pul monary 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 pulmona ry 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 pulm onary 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 mi ce. Environ Toxicol Ph armacol, 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). Comparis on 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 m ouse 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; Gudmundss on A; Swietlicki E; Ljungman AG (2007). Wear particles generated from studded tires and pavement induces infla mmatory reactions in mouse macrophage cells. Chem Res Toxicol, 20: 937-946. 155934 Lippmann M; Hwang J; Maclejczyk P; Chen L (2005). PM s ource 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). Ca rdiovascular 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 He lper 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). Characterizati on 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 fi ne 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 cycl e. 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 m onocyte-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 wors ens 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 exha ust particles in mouse epidermal cells through phosphatidylinositol 3-kinase/Akt sign aling 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). Res ponses 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; Ta sat 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; Taki zawa 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 stat e 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; Terashim a 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 leve ls 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 sulf ur 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). Pa rticulate 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 Pu erto 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 um bilical vein endothelial cells. Inhal Toxicol, 19 Suppl 1: 91-98. 156778 Mori T; Watanuki T; Kashiwagura T (2007). Diesel exhaust pa rticles disturb gene expressi on in mouse testis. Environ Toxicol, 22: 58-63. 096564 Morishita M; Keeler G; Wagner J; Marsik F; Timm E; Dvonc h 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 ci ty 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). Am bient 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). Ai rborne 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 hyperten sive 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 throm bosis. Toxicol Appl Pharmacol, 186: 38-45. 087931 Nemmar A; Inuwa IM (2008). Diesel exha ust 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; Hoylaer ts MF (2003). Pulmonary inflammation and thrombogenicity caused by diesel particles in hamsters: role of hi stamine. Am J Respir Cr it 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 expo sure to carbon black induces in flammatory response in rats. Circ J, 72: 144-149. 156812 Nozaki JI; Yamamoto R; Ma L; Shima M (2007). Trial to ev aluate effects of ambient particulate matter on health: A preliminary study using two-dimensional gel electr ophoresis. Environ Health Prev Med, 12: 138-142. 097862 Nurkiewicz TR; Porter DW; Barger M; Castranova V; Boegehol d MA (2004). Particulate matter exposure impairs systemic microvascular endothelium-dependent dilati on. 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 inflamma tion after pulmonary partic ulate 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 microvasc ular 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; Pi eters R; Lovik M (2005). Ambient air particles from four European cities increase the primary ce llular response to allergen in the draining lymph node. Toxicology, 207: 241- 254. 088655 Nygaard UC; Ormstad H; Aase A; Lovik M (2005). The IgE adj uvant 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 cap acity 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 al veolar 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 meta ls on metabolism. Chemosphere, 51: 1121-1128. 042292 Okeson CD; Riley MR; Riley-Saxton E (2004). In vitro alveolar cy totoxicity of soluble components of airborne particulate matter: effects of serum on toxicity of tran sition 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 spermatogene sis. 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). Combusti on-derived ultrafine particles transport organic toxicants to target respiratory cells. Envir on Health Perspect, 113: 956-963. 088257 Pereira CEL; Heck TG; Saldiva PHN; Rhoden CR (2007). Ambien t 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-te rm 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 a nd 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). Hypersensiti vity of prediabetic JCR: LA-cp rats to fine airborne combustion particle-induced direct and noradrenergic-med iated 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 m acrophage phosphorylated ERK1 and -2 and iNOS expression during ongoing alteration in iron homeostasis. J Toxicol Environ Health A Curr Iss, 69: 935-951. 156888Radomski A; Jurasz P; Alonso-Escalano D; Drews M; Morandi M; Malinski T; Radomski MW (2005). Nanoparticleinduced platelet aggregation and vascular thrombosis. Br J Ph armacol, 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 guine a 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; Gonzal ez-Flecha B (2005). PM-induced cardiac oxidative stress and dysfunction are mediated by autonomic stim ulation. 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; Ha milton A (2005). Comparison of the sensitivity of three lung derived cell lines to metals from combustion derive d 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 extr act. 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 anal ysis of airway injury following pollution particle exposure. J Toxicol Environ Hea lth 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 infecti on 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 h ealthy 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-We gman 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 character ization of wintertime and springtime urban-air particles with an aerodynamic diameter below 10 μ m 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. Toxico l Appl Pharmacol, 191: 86-93. 113782 Santini MT; Rainaldi G; Ferrante A; Romano R; Clemente S; Mo tta A; De Berardis B; Balduzzi M; Paoletti L; Indovina PL (2004). Environmental fine particulat e 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). Identifi cation 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 res ponse. 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 biologi cal 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 spectrometr ic analysis. Environ Sci Technol, 39: 6153-6161. 088368 Schober W; Belloni B; Lubitz S; Eberlein-Konig B; Bohn P; Sa ritas Y; Lintelmann J; Matuschek G; Behrendt H; Buters J (2006). Organic extracts of urban aeros ol (< 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 (2 008). 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 exhaus t 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 neutr ophil 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 emissi ons. 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 bronchoal veolar 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 par ticles 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; Sc hins RPF (2003). Temporal va riation of hydroxyl radical generation and 8-hydroxy-2'-deoxyguanosine fo rmation by coarse and fine particulate matter. Occup Environ Med, 60: 315-321. 088248 Shwe T-T-W; Yamamoto S; Kakeyama M; Kobayashi T; Fujimak i H (2005). Effect of intratrach eal 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; Warre n B; Cocker DR 3rd (2008). Tr imethylamine 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 cardi otoxicity 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 fo r 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-Ryche tska 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 heal thy 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 act ivities 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 ci ties: 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-A cetylcysteine-Treated, iNOS- and IL-4-Deficient Mice. Inhal Toxicol, 16: 835-843. 087981 Stevens JP; Zahardis J; MacPherson M; Mossman BT; Petru cci 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). Mate rnal 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 a nd 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 di et-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 f actor 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; K udoh 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; Sa met JM (2006). Inhibition of protein tyrosine phosphatase activity mediates epidermal growth factor receptor signa ling 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 endotheli al 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 recepto r-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; Bedj a 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 interact ions 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 infl ammatory 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; Vin cent 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 pr eproET-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 ex haust 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 microrad iography 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 so luble mediators released by particle-exposed lung cells. Toxicol Sci, 106: 233-241. 157056 Tozuka Y; Watanabe N; Ohsawa M; Toriba A; Kizu R; Ha yakawa K (2004). Transfer of pol ycyclic 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 affect s 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 e xhaust 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 moto rcycle exhaust particulate in MCF-7 human breast cancer cells and immature female ra ts. 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). Expos ure 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). Compar ative study of the formation of oxidative damage marker 8- hydroxy-2'-deoxyguanosine (8-OHdG) adduct from the nucle oside 2'-deoxyguanosine by transition metals and suspensions of particulate matter in relation to meta l content and redox reactivity. Free Radic Res, 39: 1071-1081. 096432 Veranth J; Kaser E; Veranth M; Koch M; Yost G (2007). Cyt okine responses of human lung cells (BEAS-2B) treated with micron-sized and nanoparticles of metal oxides compar ed 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-d erived 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; Sc oriza 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 surf ace charge of visible particulate matter predicts biological activation in human bronchial epithelial cel ls. Toxicol Appl Pharmacol, 178: 144-154. 024599 Veronesi B; Makwana O; Pooler M; Chen LC (2005). Effects of subchronic exposures to con centrated 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; Verh eyen G; Schoeters G (2005). Flow cytometric characterisation of antigen presenting dendritic cells afte r in vitro exposure to diesel exhaust particles. Toxicol In Vitro, 19: 903- 907. 096872 Vogel CFA; Sciullo E; Wong P; Kuzmicky P; Kado N; Mats umura F (2005). Induction of proinflammatory cytokines and C-reactive protein in human macrophage cell line U937 expos ed to air pollution particulates. Environ Health Perspect, 113: 1536-1541. 087891 Walczak-Drzewiecka A; Wyczolkowska J; Dastych J (2003). E nvironmentally relevant metal and transition metal ions enhance Fc epsilon RI-mediated mast cell activ ation. 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 intratrach eal instillation in rats. J Toxicol Sci, 98: 231-239. 156144 Wan ECH; Yu JZ (2006). Determination of sugar compounds in atmospheric aeros ols 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; Va n Houten B; Bonner JC (2003). Vanadium-induced STAT-1 activation in lung myofibroblasts requires H2O2 a nd 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 arrhythmia s in a rat model of myocardial infa rction. 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 cons cious 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 aer osolized diesel exhaust particles in a murine model for ovalbumin sensitization. J Immi gr 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 spontan eously hypertensive rats Part II: pulmonary responses. Inhal Toxicol, 16: 407-419. 055636 Wichers LB; Rowan WH, 3rd; Nolan JP; Ledbetter AD; McG ee JK; Costa DL; Watkinson WP (2006). Particle deposition in spontaneously hypertensive rats exposed via whole-body inhalation: measured and es timated 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 instillati on 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 nanopa rticle-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 exha ust. 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 neur ogenic inflammation in rats. Arch Toxicol, 77: 638-650. 097707 Wottrich R; Diabate S; Krug HF (2004). Biological effects of ultrafine model particle s 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 phosphor ylation. 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 Ce ll 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 Envi ron 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; Siout as C; Borok Z; Kim KJ; Cra ndall ED (2007). Nanoparticle effects on rat alveolar epithelial cell monolayer ba rrier 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 re lated 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 En viron 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 r ecruitment of monocytes into atherosclerotic plaques. Am J Physiol Heart Circ Physiol, 294: H944-H953. 157162 Yauk C; Polyzos A; Rowan-Carroll A; Somers CM; Godschal k 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 di esel 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 repeat ed 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-med iated 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 ne utrophils after intratracheal instillation with diesel exhaust particle s 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 pulm onary 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. Envir on 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 street s 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). Pulm onary responses of acute exposure to ultrafine iron particles in healthy adult rats. Environ Toxicol, 18: 227-235. 087940