A - A* * * -

Response of Mouse Lung to Carbon Deposition

During Injury and Repair

Ian Y.R. Adamson and Heather L. Prieditis

Department of Pathology, University of Manitoba, Winnipeg R3E OW3 Canada

Increased respiratory disease and. d'iy AWor-':

ras associated wi i

of fine p e a po a W j

ined t.he possibility that de i o eenz

inert particles to previously injured lun

may accente pumonar d by investigating how the lung handles sMall caron:

particles delivered during acute injury or

during fibrotic repair. Mice received 2 .g

carbon by intratracheal instillation into,"

lungs already showing acute injury, 3 d

after bleomycin (BL), or into lungs ith

fibrosis, 4 weeb after IL..At3 s ater

BL, injury to the tyep` I voola .p......

resulted in high p els in lavae

'fluid. Intlingr carbon Itthstu idcd

*a,a.tl}arge¢ a 9.,wincrease in inflam..mat*ry cdts 'though m i r t inser?.,-

tiumn ada a rtion wa .,,,,w.up

to 16 wees le H al..e:

mice wasequal tothat fnd aer l faoe

In the mice that received ca n4w

after bleomycin, fibrotic repar.

occurred, and the epitheli sprface as.,

restard beorte pDart caro,the> susqun * ,

tio: cdmost paie

interstitiun, and carbon reti .t 16

weeks was not differe frm th : the

carbon-only group. t.0.l pa,ticles.# 0 nt

fibrotic luing diad not indufce a4fion#It

:fibroblast growth or colagenip

The results indiate instili oparticulates to the alveoli at a tim f t

al injury results inin

to the interiim. H , d of

pure carbo t injured lupsp aloes n i.-

ther stimulate an. on fibtoi pr cs,

although it alters the patens.' of p.aricle

deposition and retention in the :lu.i Ky

words: air pollution, cabon pa lare,0

bleomycin, lung.injury, phaC`YPOtosiA.

Xnvron Healch Perpectl3:7

Human exposure to various inhaled particulates may result in lung disease, which has

been attributed to the nature and toxicity

of specific particle types, for example silicainduced pulmonary fibrosis (1-3). One of

the most common inhaled air pollutants is

the carbon particle, and deposits are frequently seen upon autopsy of human

lungs, particularly lungs of city dwellers

(1,4). It is generally assumed that carbon

itself is inert, and any associated lung disease arises from the effects of co-inhaled

toxic substances, such as mineral dusts,

metal ions, or acidic gases. Recent epidemiologic evidence, however, points to the

need for more investigation into the health

effects of inhaled particulate matter in general. Studies now link exposure to fine particles with increased mortality, and there is

evidence that diminished lung function in

persons with lung disease accompanies

inhalation of particulate air pollutants

(5-8). In a recent study, the daily mortality

rate in six U.S. cities was associated with

the level of air pollution, after correcting

for other factors such as smoking (8). The

strongest correlation with mortality was

found in levels of fine respirable particles

rather than with aerosol acids or gases such

as nitrogen dioxide (8).

Many experimental studies have been

published to describe the deposition, clearance, and pathologic effects, if any, of

various particles introduced into normal

lungs (9,10), but not much is known

about possible synergistic effects of

depositing particles, whether toxic or inert,

into abnormal lungs. Fine particulates

(< 2.5 pm diameter) such as soot can reach

the most distal regions of the lung, and

although such particles are usually considered inert, there is little information on

their effects on previously injured lungs. If

fine particles are deposited into alveoli during an acute injury phase, damage at the

air-blood barrier may increase particle

translocation to the interstitium (11). On

the other hand, it is also possible that the

increased number of inflammatory cells

recruited to the alveoli could enhance

phagocytosis and subsequent clearance.

There may also be differences in deposition

and clearance if particles are introduced

into a fibrotic lung in which function as

well as structure is altered (12). In many

examples of fibrosis, activation of macrophages is believed to be a key event in

cytokine production (13,14). How these

activated pulmonary macrophages then

respond, even to an inert particle, is not

known. During phagocytosis and attempted clearance, there may be synergistic

effects within the macrophage to increase

cytokine secretion so that phagocytosis of

an inert particle may become an extra

fibrogenic stimulus.

We studied these questions on how the

injured lung responds to deposition of fine

particulates using intratracheal (IT) instillation of colloidal carbon as an inert

particle. Carbon was given to mice a few

days after IT bleomycin, which induces

acute injury to the alveolar wall, and to

mice 4 weeks after bleomycin when the

epithelial surface is restored but fibrosis has

developed. Lung responses such as injury,

inflammatory cell numbers, particle

retention, and the production of fibrosis

were compared over a subsequent 16-week

period.

Materials and Methods

Swiss mice (25-g males) were divided into

5 groups of 40 which received intratracheal injections while under mild nembutal anesthesia. Group 1 received 2 mg of a

carbon suspension (carbon black in

hydrolyzed gelatin, 40-nm diameter;

Faber-Castell Corp., Newark, New Jersey)

in 0.1 ml water; group 2 received 0.15

units of bleomycin (Bristol-Myers Squibb

Co., Evansville, Indiana) suspended in 0.1

ml water; group 3 received the same dose

of bleomycin, then 3 days later received 2

mg carbon; group 4 was instilled with

bleomycin, then the same dose of carbon 4

weeks later; and group 5 received 0.1 ml

distilled water only. Animals were killed in

groups of four by barbiturate overdose at

the following times after bleomycin or

water (groups 2 and 5) or after carbon

(groups 1, 3, 4): days 0, 1 and 3, weeks 1,

2, 4, 8, 12, and 16; mice received 2 pCi/g

tritiated thymidine 1 hr before death.

A tracheotomy was performed on each

animal, and the lungs were washed four

times with 1 ml saline. We pooled the

bronchoalveolar lavage (BAL) fluid and

determined the total cell count by hemocytometer. The cell suspension was centrifuged; a cytospin preparation was made

and stained by the Giemsa method; differential counts of polymorphonuclear leukocytes (PMN) and alveolar macrophages

(AM) were made on 500 cells per slide.

We determined the total number of cells

of each type for each time studied, and the

mean ± SE for four mice per group was

calculated. We used the supernatant of the

lavage fluid from each mouse to determine

total protein content as an index of lung

injury (15,16).

After lavage, the bronchus leading to

the right lung was clamped; this lung was

removed, weighed, and frozen for biochemical analysis. The left lung was inflated with 0.5 ml of 2% buffered glutaraldehyde and removed; most of the tissue was

processed for embedding in glycol

methacrylate. Sections (0.75 jim thick)

from three random blocks per animal were

prepared for histology and for autoradiography with the use of Kodak NTB2 emulsion. We determined the percentages of

3H-thymidine-labeled cells at each time by

Address correspondence to I.Y.R. Adamson,

Department of Pathology, University of Manitoba,

236-770 Bannatyne Avenue, Winnipeg R3E 0W3

Canada.

This research project was supported by a grant

from the Medical Research Council of Canada.

Received 11 July 1994; accepted 26 October

1994.

72 Environmental Health Perspectives
- eem Ie wan*9m 1--

counting 3000 alveolar lung cells per animal. The means and SEs were calculated

for each group and plotted as time after

carbon instillation. Significant differences

were determined using ANOVA or

Student's t-test as appropriate.

The right lung of each mouse was

homogenized in water, and biochemical

assays were performed on duplicate samples. We determined total protein, and as

an index of collagen content, we determined hydroxyproline (HYP) levels after

hydrolysis with hydrochloric acid (16,1X).

In addition, six extra mice per group were

killed at the end of the experiment (16

weeks) to assess the amount of carbon

retained in the whole lung. The lungs were

removed, immediately chopped, and incubated in 40% potassium hydroxide

overnight in a 80°C waterbath. When the

tissue was completely digested, the solution

was centrifuged at 1500 rpm for 15 min,

and a residue was obtained. This was

washed twice in distilled water, resuspended in water, and transferred to a weighed

tube. The residue was dried to constant

weight. Means ± SEs were calculated for

each group of six, and the significance of

any difference from control and from carbon only groups was determined.

Results

Lung Morphology

Mice that received only water showed normal lungs by light microscopy. The lungs

of animals instilled with only carbon had

increased cellularity, particularly in the first

2 weeks when most carbon was phagocytized in alveoli by AM and PMN, as previously described (18,19). A few large AM

with carbon persisted for several weeks,

and some carbon was seen in interstitial

macrophages, particularly in peribronchiolar locations.

Lungs examined 3 days after bleomycin

showed diffuse injury to the alveolar wall

and an inflammatory response of AM and

PMN (Fig. IA). After these mice received

carbon, additional inflammatory cells were

seen in the alveoli and, as the experiment

progressed, it appeared that more carbon

was present in the pulmonary interstitium

and remained there as fibrosis developed

(Fig. 1B). Lungs examined 4 weeks after

bleomycin only showed areas of extensive

fibrosis (Fig. 2A). When these animals

received carbon, an inflammatory response

with increased AM and PMN in the alveoli

was seen a few days later, particularly in

less fibrotic areas of lung. Most of the carbon appeared to be cleared by 4-8 weeks

when the inflammatory response was

reduced, and little carbon was found in

fibrotic areas of interstitium (Fig. 2B).

There were no apparent differences in the

severity of fibrosis seen morphologically in

any of the bleomycin groups at the end of

the experimental period.

Bronchoalveolar Lavage

Values obtained for the numbers of AM

and PMN and for the protein level in

water-injected controls were not different

from noninjected (time 0) mice and so are

not shown at each time to simplify the figures. The number of AM recovered after

bleomycin alone increased fourfold in the

first week, and, though it fell somewhat, it

was still greater than baseline over the 16-

week period (Fig. 3). After carbon only,

AM numbers peaked at 8-10 times normal, then fell slowly. A similar response in

AM numbers was seen in groups that

received bleomycin 3 days or 4 weeks before

carbon instillation (Fig. 3). A similar pattern was seen when the PMN response was

quantitated. A small increase in lavaged

cells was found soon after bleomycin alone,

whereas the increase was much greater after

carbon injection (Fig. 4). A comparable

PMN response in intensity and duration

was found after instilling carbon 3 days

after bleomycin or 4 weeks later in fibrotic

lungs.

To quantitate lung injury, particularly

any permeability change at the air-blood

barrier, protein levels were measured in

BAL fluid. Normal values were low, at

around 90 jig/ml, and carbon instillation

caused a sharp but transient increase (Fig.

5). A similar pattern was seen in mice with

fibrotic lungs due to bleomycin administration 4 weeks before receiving carbon.

Figure 1. (A) Mouse lung, 3 days after bleomycin, shows diffuse alveolar injury (arrows) with cell debris,

red blood cells, and inflammatory cells in alveoli. (B) Lung section from mouse to which carbon was

instilled 3 days after bleomycin, killed 8 weeks later. Many carbon-laden macrophages (arrows) are seen

in fibrous interstitium. x655.

Figure 2. (A) Mouse lung, 4 weeks after bleomycin only, shows extensive interstitial fibrosis while alveoli

(A) are mostly clear of cell debris and inflammatory cells. (B) Lung section from mouse to which carbon

was instilled 4 weeks after bleomycin and killed 8 weeks later. Little carbon (arrows) is seen in the fibrotic interstitium, while a few carbon-laden alveolar macrophages are seen in the airspace (A). x655.

Volume 103, Number 1, January 1995 73
A . e -

0 2 4 6 10 12 14 16

Weeks after Carbon

Figure 3. The number of alveolar macrophages (AM) recovered by bronchoalveolar lavage (BAL) from the

four treatment groups up to 16 weeks after receiving carbon. There were no statistical differences

among the three groups that received carbon.

4 M arboIn| - | | -

Figure 4. The number of plvolyrmocrponuchargeukcye recovered by bronchoalveolar (A)fo lavage h

~~~~~~leomycin-u

fonly t t groups r we(e no0s.01a)

fh~~~~~~~~~~~~~~A Blomci th days i Carbon

3500 _ _ _ _ _ _ 1 s

||S____~~~~~~~~~~~~~~~~~~~~~~~~~------ -emcnWeBeomcis days}e+Carbon_

Figur 4_$enme fplmrhncla ekcts(M )rcvre ybocolelrlvg BA)Th 3da blo yipls-crbn valu wa infcnl ihrta hti te rusa a

onl( <0.1)

The highest levels of alveolar protein were

found in mice in the acute injury phase up

to 1 week after bleomycin instillation,

when levels were 16-20 times normal.

These values were obtained in BAL after

bleomycin only and in mice that received

carbon 3 days after bleomycin. In these

groups, protein levels did not return to

normal for several weeks (Fig. 5).

Lung Fibrosis

We examined autoradiographs of lung to

determine if there was evidence of extended cell proliferation, particularly in the

fibroblast population. In the first week

after injecting carbon, the percentage of

thymidine-labeled cells increased in all

experimental groups (Fig. 6). This early

increase appeared mainly due to interstitial

monocyte/macrophage labeling, though

mice that received bleomycin also showed

some epithelial and endothelial cell labeling in the first 2 weeks. Labeling in the

carbon-only group dropped sharply to control values by 2 weeks, while DNA synthesis was higher in all bleomycin groups (Fig.

6). After 1 week, labeling in the various

bleomycin groups was similar, and in each

case, the predominant labeled cell type was

the fibroblast.

The similar fibrotic response in the

three bleomycin groups was confirmed by

the HYP measurements. Mice that

received carbon only showed a small

increase in HYP, not different from water

controls, and consistent with aging (Fig. 7).

Animals in groups that received bleomycin,

with or without carbon at different stages

of the post-bleomycin response, showed a

rapid rise in tissue HYP beginning at 2

weeks. Thereafter, all bleomycin groups

were equivalent, and HYP levels were all

significantly greater than the control or

carbon-only groups (Fig. 7).

Lung Residue

An amorphous, nonparticulate residue

weighing 0.2 + 0.05 mg was obtained

from control lungs, and a similar weight

of residue was obtained from fibrotic

lungs after bleomycin alone (Fig. 8).

Particle retention in mouse lung 16 weeks

after instilling the large dose of carbon

could be quantitated; the residue was

black and weighed significantly more than

that from controls. A similar weight of

residue was measured from lungs that

received carbon only and carbon 4 weeks

after bleomycin. The highest weight of

residue was recovered from lungs that

received carbon during the acute injury

phase, 3 days after bleomycin. The residue

from this group weighed significantly

more than that obtained from all other

groups (Fig. 8).

Discussion

Baseline studies on deposition, clearance,

and pathologic effects that determine

whether a particle is toxic or inert," are

usually carried out on normal animal lungs

after inhalation or intratracheal instillation.

However, in human terms, exposure to

particulate air pollutants often occurs when

the lung is compromised. It is possible that

exposure to a particle such as carbon,

which causes no effects in normal lung,

may induce a different reaction when

introduced to lungs at different stages of

an injury-repair cycle. In general, particles

deposited in the lung are mostly cleared by

the mucociliary system, which can eliminate free particles as well as those engulfed

by AM. As the dose increases, an adaptive

increase in AM occurs, and at higher levels

PMN also migrate to the alveoli to augment phagocytosis and clearance (18,19).

These initial responses occur whether the

particle is considered inert or toxic (3) and

were seen here in all carbon-injected

groups. There were differences, however,

in the subsequent fate of particles, which

can be related to the integrity of the alveolar epithelium.

Little is known about particle handling

by the lung when there is alveolar epithelial cell injury. The normal epithelium is a

barrier to particle translocation, and it is

only crossed in overload situations when

particles are transported across type I cells

Environmental Health Perspectives

250

20o

x

150

100

74
A - ** - -e aa - - I

to reach the interstitium, where they are

phagocytized by interstitial macrophages

(18-20). Particles may remain in this location for long periods, and in the case of

carbon, no fibrotic reaction ensues. In the

present study we created a diffuse alveolar

injury by instilling bleomycin 3 days

before giving a large dose of carbon.

Because transepithelial passage increases

with particle number (18,20), we used a

level near the maximum deliverable in a

single dose to a mouse to help detect any

possibile changes in the fibrotic response

due to particle exposure of injured lung.

Intratracheal bleomycin induces type 1

epithelial necrosis (21) and results in a

high level of leaked protein that is measured in BAL fluid. Although carbon does

not increase this level of damage, the

epithelial injury present for about 2 weeks

allows many free particles to reach the

interstitium for phagocytosis by interstitial

macrophages. This was seen morphologically, and later the increased translocation

and retention of carbon was confirmed by

the increased weight of a black lung

residue recovered at 16 weeks. The

increase in particle translocation to the

interstitium occurred despite a brisk

inflammatory cell response to carbon that

increased the number of AM and PMN in

the alveoli. Most of these cells would have

recently arrived in the alveoli and so were

likely immature and poor phagocytes. The

observation that acute inflammatory lung

injury retards particle clearance has also

been reported in calves with viral pneumonitis, in which clearance of cobalt oxide

2000

I80

1600

lam

Ej 12W

-M

600n

UO

02

m

400

200

U la

Weeks after Carbon

Figure 5. Protein content of bronchoalveolar lavage (BAL) fluid. Values in the first week after bleomycin

alone or bleomycin 3 days plus carbon were significantly higher than the other groups (p < 0.01).

4,.0 3 .5||fi|lEEEEEEaa|~- Carbon

111 __ _ _ -O~~~~~~~0- Bleomycin_

1111 - _____ ",~~~~~~~~Beomycin (4 wookss+ Carbon_

M 3.0 1 ___Bleomycin (3 days) + Carbon_

r .5

0.5

20s~~~~~~~~~ek aferCabo

.X

s

6ln_ dfr c

0q2- 4 B 10 12 14 1

aR~~~~~~~~ekafter_ Carbon

Fiue6ecetgso llln el ta noprae rtaedtyiie(3T tvrou ie.N

sigifiandifrne nlbln atr fe ekwrese ntetrebemcnijce rus

was reduced and particles were sequestered

in the interstitium (S).

The fact that particles deposited in the

alveoli during epithelial injury subsequently results in an increased interstitial burden constitutes a potential hazard. In several studies, a clear link has been established between interstitial macrophage

activation by particles such as silica or

asbestos and the subsequent development

of pulmonary fibrosis (22,23). This also

occurs in other examples of fibrosis, such

as the pulmonary response to bleomycin

alone, in which activated macrophages,

particularly those in the interstitium, have

been identified as the prime source of

cytokines that stimulate fibroblast proliferation and collagen deposition (24). It was

thought possible that an extra burden on

these macrophages, even by an inert particle, might further stimulate an activated

cell in a synergistic reaction. However that

did not happen in these experiments using

pure carbon; despite the extra particulate

load retained up to 16 weeks, no additional fibrotic response was seen or measured

biochemically. Although we used particle

instillation, our results are in agreement

with an earlier publication in which

adding titanium dioxide as an "inert" particle to inhaled asbestos did not increase

fibrosis over that induced by asbestos

alone (25).

A different pulmonary response was

seen when carbon was instilled to the lung

4 weeks after bleomycin. At this time,

epithelial repair was complete, and alveolar

protein levels had returned to near normal,

even though interstitial fibrosis had been

produced. Instilling carbon induces the

usual inflammatory response, though particles were mostly deposited in the less

fibrotic areas of lung. This may be the

result of relative variations in ventilation

due to the fibrotic process. In one previous

study of inhaled particle retention in

fibrotic lungs of hamsters, it was noted

that diseased areas of lung received fewer

particles (12). Greater deposition of carbon

in the more functionally normal regions of

lung, together with the restoration of the

epithelial barrier before at the time of

instillation, allows a more regular pattern

of phagocytosis and clearance, which would

explain why the retained carbon at 16

weeks was equal to that of the carbon only

group (Fig. 8). Despite the likely presence

of activated macrophages in these

bleomycin-injected lungs (24), few instilled

particles reached the interstitium, and there

was no additional fibroblast stimulation as

judged by the equal levels of HYP after

bleomycin, with or without carbon.

The results demonstrate that deposition of a large number of inert particles in

a single dose to the lung during a phase of

Volume 103, Number 1, January 1995 75
A - A*I -

300

=211

threbleomycin 20 grup wer found . _

0Boryi -

10 -* Bloyi (4 weks Cabo

300

>~~~~~~~~ek afte_-l-.rc

Mean-OSEsonfrcroBnyadbemcn rus rm2wesowralbeomycin grus{ r

thebl_ _ _ __i groupsc wer found.Cabo _

- 2.0

,1.0

wes afercarbo (C instBIlainp < 0.1 cm

._ _

Is

X~ 5

p 0.0

C BL BL{3 dasys) BL({4 weeks)

TIC .IC

Figure 8. Weight of lung residues (mean + SE) 16

weeks after carbon (C) instillation. tp < 0.01 corn

pared to all other groups; *p < 0.01 compared to

bleomycin (BL) alone or water control.

epithelial injury or of fibrotic repair does

not exacerbate the fibrotic response.

However, it is possible that repeated exposure of injured lung to inert or to toxic

articulates found in urban environments

may produce a different outcome. In particular, exposure of the lung at a time of

epithelial injury is potentially more serious

due to the likelihood of increased particle

translocation and retention in the pulmonary interstitium.

REFERENCES

1. Parkes &JR. Occupational lung disorders.

London:Butterworths, 1974.

2. Heppleston AG. Pulmonary toxicology of silica, coal and asbestos. Environ Health Perspect

55:111-127 (1984).

3. Bowden DH. Macrophages, dust and pulmonary disease. Exp Lung Res 12:89-107 (1987).

4. Spencer H. Pathology of the lung, 4th ed.

Oxford:Pergamon Press, 1985;413-510.

5. Utell MJ, Samet JM. Particulate air pollution

and health: new evidence on an old problem.

Am Rev Respir Dis 147:1334-1335 (1993).

6. Schwartz J. Total suspended particulate matter

and daily mortality in Cincinnati, Ohio.

Environ Health Perspect 102:186-189 (1994).

7. Xu X, Wang L. Association of indoor and outdoor particulate level with chronic respiratory illness. Am Rev Respir Dis 148:1516-1522 (1993).

8. Dockery DW, Pope CA, Xu X, Spengler JD,

Ware JH, Fay ME, Ferris BG, Speizer FE. An

association between air pollution and mortality

in six U.S. cities. N EnglJ Med 329:1753-1759

(1993).

9. Brain JD, Valberg PA. Deposition of aerosol in

the respiratory tract. Am Rev Respir Dis

120:1325-1373 (1979).

10. Oberdorster G. Lung clearance of inhaled

insoluble and soluble particles. J Aerosol Med

4:289-330 (1988).

11. Slauson DO, Lay JC, Castleman WL, Neilsen

NR. Acute inflammatory lung injury retards

pulmonary particle clearance. Inflammation

13:185-199 (1989).

12. Sweeney TD, Brain JD, Tryka AF, Godleski JJ.

Retention of inhaled particles in hamsters with

pulmonary fibrosis. Am Rev Respir Dis

128:138-143 (1983).

13. Elias JA, Freundlich B, Kern JA, Rosenbloom

J. Cytokine networks in the regulation of

inflammation and fibrosis in the lung. Chest

97:1439-1445 (1990).

14. Schapira RM, Osornio-Vargas AR, Brody AR.

Inorganic particles induce secretion of a

macrophage homoglogue of platelet-derived

growth factor in a density and time dependent

manner in vitro. Exp Lung Res 117:

1011-1024 (1991).

15. Lowry OH, Rosebrough NJ, Farr AL, Randall

RJ. Protein measurement with the Folin phenol

reagent. J Biol Chem 193:265-269 (1951).

16. Young L, Adamson IYR. Epithelial-fibroblast

interactions in bleomycin-induced lung injury

and repair. Environ Health Perspect

101:56-61 (1993).

17. Woessner JF. Determination of hydroxyproline

in connective tissue. In: The methodology of

connective tissue research. Oxford:Joynson and

Bruvvers, 1976;227-233.

18. Adamson IYR, Bowden DH. Dose response of

the pulmonary macrophagic system to various

particulates and its relationship to transepithelial passage of free particles. Exp Lung Res

2:165-175 (1981).

19. Bowden DH, Adamson IYR. Pathways of cellular eflux and particulate clearance after carbon instillation to the lung. J Pathol

143:117-125 (1984).

20. Morrow PE. Dust overloading of the lungs:

update and appraisal. Toxicol Appi Pharmacol

113:7-12 (1992).

21. Adamson IYR. Drug-induced pulmonary

fibrosis. Environ Health Perspect 55:25-36

(1984).

22. Adamson IYR, Letourneau HL, Bowden DH.

Comparison of alveolar and interstitial

macrophages in fibroblast stimulation after silica and long or short asbestos. Lab Invest 64:

339-344 (1991).

23. Brody AR, Bonner JC, Overby LH, Badgett A,

Kalter V, Kumar RK, Bennett RA. Interstitial

pulmonary macrophages produce plateletderived growth factor that stimulates rat lung

fibroblast proliferation in vitro. J Leuk Biol

51:640-648 (1992).

24. Phan SH, Kunkel SL. Lung cytokine production in bleomycin-induced pulmonary fibrosis.

Exp Lung Res 18:29-43 (1992).

25. Davis JMG, Jones AD, Miller BG. Experimental studies in rats on the effects of asbestos

inhalation coupled with the inhalation of titanium dioxide or quartz. Int J Exp Pathol 72:

501-525 (1991).

76 Environmental Health Perspectives