The comparison of a fibrogenic and two nonfibrogenic dusts by bronchoalveolar lavage

TOXICOLOGY AND APPLIED PHARMACOLOGY 102,26828

l(1990)

The Comparison of a Fibrogenic and Two Nonfibrogenic Dusts by Bronchoalveolar Lavage ROBERTC.LINDENSCHMIDT, KEVIN E.DRISCOLL,MARY A.PERKINS,JANET JAMES K. MAURER,ANDKATHLEENA.BELFIORE Thr Procter

& Gamble

Conzpat7~~.

Miami

I’allq,

Laboratories.

P.O. Bo.x 3Yl7.5*

Cincinnati,

M.HIGGINS, Ohio

45247

The Comparison of a Fibrogenic and Two Nonhbrogenic Dusts by Bronchoalveolar Lavage. LINDENSCHMIDT. R. C.. DRISCOLL.K. E.. PERKINS. M. A.. HIGGINS. J. M., MAURER, J. K., AND BELFIORE, K. A. (1990). Toxicol. .4ppl. Pharmacol. 102, 268-281. Analysis of bronchoalveolar lavage fluid (BALF) appears to be a sensitive approach to characterizing an acute inflammatory response within the lung. More work, however, is needed to determine if analyses of BALF endpoints can predict chronic responses(i.e., fibrosis). The objective of the present study was to compare the dose and temporal pulmonary response of a known fibrogenic agent, silica. and two known nonfibrogenic agents, aluminum oxide and titanium dioxide. Animals were instilled with silica (0. 0.2, I .O,or 5.0 mg/lOO g body wt), titanium dioxide (I .Oor 5 mg/lOO g body wt). aluminum oxide (1.0 or 5.0 mg/lOO g body wt) or saline. Animals (n = S/group) were terminated I, 7, 14. 28. and 63 days following instillation, and the BALF was characterized by biochemical and cellular assays. Histopathological changes were determined at 60 daysafter exposure. The biochemical results demonstrated BALF levels of lactate dehydrogenase (LDH). fi-glucuronidase (BG), N-acetylglucosaminidase (NAG), and total protein (TP) increased in a dose-related fashion at the earlier time points for all test materials, with the magnitude of change being greatest for silica. The temporal response for these parameters was significantly different for the two classesof materials. With time, the response for the librogenic dust steadily increased, while the levels for the nonfibrogenic dusts decreased toward normal values during the 2-month study period. Of the cellular changes, total cell numbers, neutrophils, and lymphocyte numbers were the most sensitive markers of the pulmonary response. As shown with the biochemical parameters. the cellular response to silica increased with time while that of the nuisance dusts did not. It was also found that, similar to inhalation studies. high doses of a nuisance dust may result in toxicity/inflammation. This toxicity at high dose levels emphasizes the importance of choosing relevant doses when comparing potentially fibrogenic and nonfibrogenic dusts. In conclusion, the persistent and progressive changes seen in the biochemical (LDH, TP, BG, NAG) and cellular parameters (total cells. neutrophils and lymphocytes) following silica administration correlated with the fibrotic response which occurred after exposure to this material. The lessdramatic and transient changes seen with aluminum oxide and titanium dioxide correlated with the inert nature of these nuisance dusts. The results of this study indicate evaluation of BALF may provide a means to predict the chronic pulmonary response to a material. rig1990 Academic Press. Inc.

Inhalation is a major route of exposure in occupational settings. In order to determine appropriate industrial hygiene measures, the potential toxicity of respirable particles must be defined. Evaluating respiratory effects of a material via a chronic inhalation study is a costly and time-consuming process. A number of investigators are trying to develop 004 1-008X/90 $3.00 Copyright Q 1990 by Academic Press. Inc. All rights of reproduction in any form reserved.

268

shorter-term in viva assays to assessthe potential fibrogenic response of inhaled materials (Beck et al., 1982; Benson et al.. 1986; Henderson ez al., 1978, 1985; Henderson, 1984; Sykes et al., 1983). These new approaches are based on analysis of cellular and biochemical endpoints in bronchoalveolar lavage fluid (BALF). Lavage of the bron-

BAL AFTER

EXPOSURE

choalveolar region samples epithelial lining fluid of conducting airways and alveoli, sites of pulmonary contact for inhaled materials. Previous studies have indicated analysis of BALF is a sensitive means of characterizing the acute inflammatory response within the lung (Brain and Beck, 1985; Henderson, 1984: Henderson d ul., 1985: Hunninghake et al.. 1979; Reynolds, 1987). However, early changes in these cellular and biochemical constituents may also be predictive of more chronic lung responses (e.g., fibrosis). More work is needed to determine if early changes in BALF endpoints can predict chronic responses (i.e., fibrosis). To identify and validate a battery of shortterm predictors for long-term effects, it is important to use appropriate positive and negative controls. In the present study, we assessed a number of potentially useful biochemical and cellular changes observed in BALF from F344 rats following instillation of various doses of silica (fibrogenic agent) and titanium dioxide and aluminum oxide (nonhbrogenic nuisance dusts). The rat was used since a major inhalation database exists for this species. A number of bronchoalveolar lavage parameters have been previously studied and correlated with various histologic responses (Beck et al., 1982; Henderson et al., 1985; Sykes et al., 1983). However, the present studies were designed to further characterize (1) magnitude of the responses to both fibrogenic and nuisance dusts; (2) time course for these responses for up to two months after exposure: (3) dose-response relationship for these materials: (4) intratracheal dose levels at which these,comparisons can best be made; and (5) histopathologic changes at the final time point. METHODS Animals. Male Fischer-344 rats (COBS CDF/crlBr) weighing approximately 225 g were obtained from Charles River Breeding Laboratories. Rats were given a conventional laboratory diet (Purina Chow pellets) and

TO INSTILLED

DUSTS

269

tap water ad libiturn during a 1-week acclimation period and throughout the 63-day study period. On Day 0 rats were randomized into study groups based on body weights. Ekperitnental freatmmt. Crystalline silica, aluminum oxide, and titanium dioxide from Fischer Scientific (with a mean particle size f SD of 2.2 + 1.1. 5.3 f 2.3, and 2.2 _+ 1.5 pm, respectively) were prepared in 0.9% sterile saline at aconcentration such that each rat would receive a constant dose volume (0.1 ml/l00 g body wt). Rats were anesthetized by intraperitoneal injection of sodium pentobarbital and placed on a vertical tilt restraint board. Dose suspensions were sonicated 15 min prior to filling the syringe. Each dosed aliquot was pipetted up and down in the dose syringe immediately prior to dosing to insure uniformity. Using a modified pediatric laryngoscope to illuminate the larynx each rat was intratracheally instilled with either saline (vehicle control). 0.2. 1.0, and 5.0 mg silica/l00 g of body wt. or titanium dioxide or aluminum oxide at 1.0 and 5.0 mg/lOO g of body wt. Bronchoalveolar lavage. On Days I, 7, 14, 28, and 63 postexposure. bronchoalveolar lavage was performed on five animals/study group according to a technique previously described (Brain and Beck. 1985). Briefly. animals were anesthetized with sodium pentobarbital and exsanguinated. Lungs were excised and weighed. Lavage was performed on excised cannulated lungs using sterile. room temperature Dulbecco’s calcium and magnesiumfree phosphate-buffered saline (PBS-GIBCO). Lungs were first lavaged with two separate 6-ml washings which were each left in the lung 30 set, aspirated. and reinstilled for an additional 30 sec. These two lavage samples were pooled and centrifuged at 300gat 4°C for 15 min and the resultant cell-free supernatant was immediately analyzed for the various biochemical parameters. Additionally. lungs were lavaged three times with 8 ml of PBS. This was shown in pilot experiments to increase cell yield by an average of 60%. Cell pellets from all washes were combined for cell counting and differentiation. Acellrrlar assays. Parameters measured in cell-free supernatant were selected for their potential to be good indicators of lung injury. Lactate dehydrogenase (LDH). alkaline phosphatase. acid phosphatase, and N-acetylglucosaminidase (NAG) were assayed on a Hitachi 705 autoanalyzer using Boehringer-Mannheim Diagnostics for LDH and alkaline phosphatase, Boehringer-Mannheim Biochemicals for NAG. and Sigma for acid phosphatase. @-Glucuronidase (BG) was assayedaccording to a modified Sigma method on a Beckman DU 50 spectrophotometer at 550 nm (Fishman et a/., 1967). Total protein (TP) was determined using a Bio-Rad method (Bradford. 1976). Elastolytic activity was measured by radial diffusion in agar gels containing bovine ligament elastin (Werb and Gordon, 1975).

270

LINDENSCHMIDT

Cellular evulztatiur2. Cell pellets from all five lavages per animal were resuspended in PBS and pooled for evaluation. Total cell numbers were determined using a hemocytometer and cell viability was assessed by trypan blue exclusion. Cells (loo/rat) were differentiated on cytocentrifuge-prepared slides after staining with eosin and methylene blue. Hisrological evahutior~ offi.cs~rc~. On Day +60 of study. three rats from each group were humanely killed and the lungs perfused with and saved in 10% neutral-buffered formalin (Buckingham and Wyder, I98 I ). Subsequently. they were routinely trimmed. processed. sectioned. and stained with hematoxylin and eosin. Masson’s trichrome-stained sections were used to evaluate for increased collagen (i.e., fibrosis). Stafistic.s. Statistical evaluation was made by analysis of variance techniques. Provided that Bartlett’s test of homogeneity of variance was not significant. treated groups were compared to the control group or to one another using the least significant difference (LSD) criterion. When Bartlett’s test was significant. comparisons were made using Wilcoxon’s rank sum test, a f-test technique which makes allowance for unequal variances. All statistical tests were conducted at a 5%‘. two-sided risk level.

RESULTS

No differences in animal body weights were observed between control or treated groups. Significant increasesin lung weights, however, were seen in the silica-treated rats (Fig. 1). These lung weights increased with time and dose. This effect was seen at each dose level of silica with the exception of the 0.3 mg/ 100 g body wt group on Day 14. Lung weights for the rats treated with the nuisance dusts were not significantly different from controls.

ET AL.

creased steadily to Day 63. BG was significantly elevated on Day +l and steadily increasedto Day 63. A dose-responserelationship occurred with NAG, TP, LDH, and BG. Acid and alkaline phosphatase levels were not significantly affected by silica exposure. Additionally, elastase activity was not detected after silica exposure (data not shown).

B. =Iluminw~~ o.t;ide and titanium

dio.vide.

Mean levels of the biochemical constituents following nuisance dust exposure are shown in Figs. 2 to 5. As with silica. LDH, NAG. and BG were significantly (p < 0.05) elevated over control levels. However, these changes were significantly less than those induced with silica at comparable doses and times. With both nuisance dusts, significantly elevated LDH and BG occurred on Day 1 at both doses, while NAG was significantly increased on Day 1 at only the high dose. The general trend was for these enzymes to gradually decreaseover time. Only LDH and BG were significantly elevated at Day 63 (high dose groups). No consistent alterations occurred in alkaline or acid phosphatase levels and elastaseactivity was not detected (data not shown).

Silica. Mean cellular counts for silica-exposed rats are presented in Table I. Dose-related increasesin total cells were observed at all time points. with the greatest responseoccurring on Day 63. Neutrophils were significantly increased (11 < 0.05) at all time points versus controls and accounted, to a large extent. for the increases in total cells. Changes in macrophage numbers did not demonstrate a consistent doseil. Silica. Mean levels of the biochemical response pattern following silica exposure constituents of BALF following silica admin- until Day 63 at which point significant inistration are shown in Figs. 3 to 5. Levels of creases were observed at the two highest NAG, TP, and LDH were significantly (p doses.Lymphocytes were slightly but signih< 0.05) increased on Day 1, were decreased cantly elevated on Days 1 to 38, with a on Day 7 relative to Day I, and then in- marked increase on Day 63. Treatment-re-

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Days atter

Day +14 lntratr8cheal

271

DUSTS

1.6 $

1.4

: _a

1.2

s P 3

1 0.6 0.6

Day +1

Day +7

Day +26

Day +63

lnstlllatlon

FIG. I. Lung weight changes over 63 days following exposure to silica (0.2, 1.0. and 5.0 mg/lOO wt). Values are expressed as treatment over control 4sterisk indicates significant difference from atPc0.05(*)orp
lated decreases in cell viability served.

.-iliminwn

were not ob-

0.x-i& and titmium

dimi&.

The effect of aluminum oxide and titanium dioxide on free lung cell populations is shown in Table 2. Total cells on Days 7 through 63 were significantly elevated at the high dose for aluminum oxide and titianium dioxide, with the magnitude of change decreasing with time. Increases in total cells at the high dose of each nuisance dust were primarily due to an increase in neutrophils and lymphocytes. At the low dose of each nuisance dust, the only significant change was an increase in neutrophils on Day 1, which returned to a control level by Day 7. No treatment-related decreases in cell viability were seen.

‘1. Silica (Fig. oh). The changes seen were most often associated with alveolar duct re-

g body control

gions and at times were perivascular. At each dose, there were accumulations of macrophages and neutrophils in the alveoli, interstitial inflammation, type II cell hyperplasia. and necrosis principally of alveolar macrophages. Lymphoid hyperplasia and macrophage accumulations in the lymphoid tissue became more readily apparent as the dose increased. Birefiingent particulates were visible and associated with alveolar macrophages. Granulomas were observed at the mid and high dose. With the Masson’s trichrome stain, increased prominence of collagen, interpreted as reflecting minimal fibrosis, increased in incidence but not in severity as the dose increased.

B. .-llwrinum o.ride amI titunium dit).x-i& (Fi,gs. 6~’ ar& 6~0. The changes were again associated with the alveolar ducts. Both dusts were visible and often were present in alveolar macrophages. At the high dose of each, there was minimal interstitial inflammation.

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LINDENSCHMIDT

ET AL.

Increased alveolar macrophages and minimal type II cell hyperplasia were more readily observed with aluminum oxide than with titanium dioxide. DISCUSSION The present study was conducted to assess the potential of using short-term exposures to predict long-term effects in the lung. Selected cellular and acellular BALF parameters following exposure to silica, a fibrogenic material, and aluminum oxide and titanium dioxide. nuisance-type materials, were evaluated. Major differences in the magnitude and temporal nature of the responses occurred, clearly distinguishing the two classes of materials. Exposure to fibrogenic dust resulted in a steady increase in many of the BALF parameters measured, even at the low dose. In contrast, exposure to nuisance dusts caused an early peak in response followed by a steady return toward control values. Analyses of BALF provided a sensitive means of assessing acute pulmonary toxicity to silica. Statistically significant changes in the cellular and biochemical endpoints occurred at even the lowest concentration of silica where the microscopic changes were less dramatic. Valuable biochemical parameters included LDH, a cytoplasmic enzyme released following damage to the cell membrane; NAGS and BG, indicators of increased phagocyte activity and/or toxicity to macrophages and neutrophils within the lung (Bentwood and Henson. 1980: Keeling and Henson, 1982); and TP, an indicator of vascular damage. With silica, these parameters, in general, increased with dose and time. A

(3/l)

~SUp!UlluW~Oonl6l~~0oV-N

FIG. 2. Effect of silica (Si), aluminum oxide (Al), titanium dioxide (Ti). or saline (0.0) on N-acetylglucosaminidase levels in BALF on Days I. 7. 14,28. and 63 following intratracheal instillation. Dose levels (in parentheses) were as mg/lOO g body wt. Asterisk indicates significant difference from control at p c 0.05 (*) or p < 0.01 (**).

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similar response to silica has been shown by others (Moores et al., 198 1: Sykes et al.. 1982). The time course of these changes corresponds to the known progressive nature of silica toxicity following a single intratracheal dose (Gross et al., 1984). Nuisance dusts caused significant increases in LDH, NAG, BG, and TP. The magnitude of these elevations was significantly less than that seen with silica at comparable doses and times. Unlike with silica, the changes were less dramatic and transient. These enzymes tended to gradually decrease over time, with only the high dose groups being significantly elevated at Day 63 (LDH, NAG, and TP). BALF cell numbers after a single silica exposure similarly demonstrated a progressive response. Total cells were increased at all doses, a response due largely to increased neutrophils as previously observed (Moores et al., 1981; Sykes et al.. 1982, 1983). Lymphocytes were also significantly increased at all silica doses, especially on Day 63. Interestingly, alveolar macrophage numbers did not appear to correlate with the silica exposure except at the final time point where the increases were dose-related and significantly different from controls. This difference in the macrophage response, compared to the other cell types, may reflect granuloma formation seen histopathologically. These cells can become more adherent upon activation (Van Oss et al., 1984) a response which could change the efficiency with which they are retrieved by bronchoalveolar lavage. Mechanisms underlying the inflammatory cell recruitment were not addressed in the present study. However, it has been demonstrated previously that macrophages after ingesting silica or other particles can release chemotactic

(3/l)

u!WoJd

WK’l

FIG. 3. Effect of silica (Si), aluminum oxide (Al), titanium dioxide (Ti). or saline (0.0) on total protein levels in BALF on Days 1. 7, 14.28, and 63 following intratracheal instillation. Dose levels (in parentheses) were as mg/lOO g body wt. Asterisk indicates significant difference from control at p < 0.05 (*) orp < 0.01 (**).

274

LINDENSCHMIDT

ET

AL

factors that attract PMNs and/or monocytes (Bowden, 1987: Hunninghake et al.. 1978). suggesting a role for the macrophage in particleinduced inflammatory cell recruitment. As with silica, nuisance dust-induced elevation in total BALF cells was primarily due to an increase in neutrophils. Unlike silica, however, the initial increase was transient and was significantly greater than controls only for the high dose groups at the later time periods. Histologically. at 2 months after silica instillation, fibrosis was detectable at all dose levels and granulomas occurred at the mid and high doses. These silica-induced changes appeared to be dose-related. Previously. Gross and coworkers ( 1984) reported occasional fibrotic nodules in rats 24 weeks after a similar intratracheal dose of silica. Since the selected BALF changes were significant at all doses and all the earlier time points, the BALF constituents appear to be sensitive indicators of the subsequent pulmonary tissue damage. Significant differences in the biochemical or cellular endpoints did not exist between the two nuisance dusts. The minimal and generally transient increase of these parameters is consistent with their established lack of significant lung toxicity in both man and animals (TLV. 1986- 1987). Histologically. however. there did appear to be a slight difference between the two nuisance dusts with the response to aluminum oxide being slightly more severe. This minimal diflerence could be due to different chemical properties. or to the larger particle size ofaluminum oxide compared to titanium dioxide used in the present study. Lung weights steadily increased following silica administration, likely reflecting the accumulation of cells and fluid. The nuisance dusts produced only a small and transient increase in

FK;. 4. Effect of silica (Si). aluminum oxide (Al). titanium dioxide (Ti). or saline (0.0) on lactate dehydrogenase levels in BALF on Days I. 7. 14. 2X. and 63 following intratracheal instillation. Dose levels (in parentheses) were as mg/lOO g body wt. .Asterisk indicates significant difference from control at II< 0.05 (*) or 17 i 0.0 I (**).

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AFTER

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c l

l

l*hW

*

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INSTILLED

DUSTS

275

lung weight at the high dose of titanium dioxide. This is consistent with the biochemical and cellular data which indicated a more limited response with the nonfibrogenic dusts. Although intratracheal instillation is obviously not the normal route of exposure. previous studies have shown that alterations in BALF to instilled or inhaled particles were qualitatively similar (Henderson et al.. 1985). It is important, however, to be able to compare the doses between the two routes of exposure. Doses used for the present study were chosen to cover those feasible to attain during a short-term inhalation study (e.g., 50 mg/m3 for 6 hr), up to doses clearly exaggerating a feasible inhalation dose. Although cellular and biochemical responses of the nuisance dusts decreased over time, there was a persistent effect on the biochemical and cellular endpoints at the top dose of aluminum oxide and titanium dioxide. This could be explained by an overload phenomenon that has been described to occur in a number of inhalation studies (Lee et al., 1985, 1986a,b: Morrow. 1986; Vostal. 1986). In these studies. even nuisance dusts. at a high enough level, caused significant pathological responses. A number of investigators (see Morrow, 1986. for list of references) have shown that deposition of large amounts of inert dust in the lungs (> l-2 mg/ g lung tissue) leads to inhibited phagocytic removal of dust, resulting in a prolongation of dust clearance from the lung. This overload results in a persistent inflammatory response. The high dose in the present study (-9.1 mg/ g lung for a 200-g rat with a lung weight of 1.1 g) clearly exceeds this level and the high dose of the instilled nuisance dusts resulted in an “overload” response. Until clearance mecha-

oxide (Al). titaFIG. 5. Effect of silica (Si). aluminum nium dioxide (Ti). or saline (0.0) on &glucuronidase levels in BALF on Days 1,7. 14. 28. and 63 following intratracheal instillation. Dose levels (in parentheses) were as mg/lOO g body wt. Asterisk indicates significant difference from control at 17 < 0.05 (*) or p < 0.0 1 (**).

276

LINDENSCHMIDT

ET AL.

TABLE I BRONCHOALVEOLAR

LAVAGE

FI.UID CELL NUMBERS AFTER INTRA~RACHEAL INSTILLATION OF SILKA (.I'?

SE)"

Termination day Treatment

Total cells

Macrophages

Neutrophils

Lymphocytes

Day 1 Saline Silica 0.2 mg Silica 1.O mg Silica 10.0 mg

I .2 1 F 0.41 6.27 zk0.56* 5.43 + 1.10* 10.32 + I .65*

1.l2iO.41 1.05 kO.14 I .66 k 0.11 -.’ 39 k 0.49

0.05 4.8 I 3.56 7.33

i 0.02 AIo.so* k I .26* + I .52*

0.01 i-O.01 0.28 I 0.06* 0. I3 +- 0.03* 0.40~0.1

Day 1 Saline Silica 0.2 mg Silica 1.Omg Silica IO.0 mg

3.80 i 0.70 5.41 + 1.20 8.46 i- 1.85 1 I .Y5 +- 7.97*

3.70 2.18 4.00 3.24

0.0’ 2.96 4. II 7.75

+ 0.0 I f 0.74* * 2.33*

0.07 0.27 0.79 0.95

Day 14 Saline Silica 0.2 mg Silica I .O mg Silica 10.0 mg

2.99 4.84 9.65 12.26

t 0.70 + 0.99 !I 1.03* + I .38*

2.89 + I .YOi 2.87 * 3.85 +

0.03 2.48 5.77 7.40

* t f t

0.01 0.42* 0.72* I .05*

0.07 1 0.04 0.46 + 0.13* 1.15 -eo.33* I .06 I 0.23*

Day 28 Saline Silica 0.2 mg Silica 1.Omg Silica 10.0 mg

I.19 3.43 6.00 9.06

20.19 + 0.44* t 0.79* f 2.68*

1.07 kO.19 1.17t0.16 I .34 f 0.13 2.62 k 0.74*

0.01 2.09 4. I7 7.31

+ 0.00 + 0.30* f 0.66* i 1.60*

0.04 t 0.03 0.17 -t 0.02* 0.49 t o.os* I. 13 k 0.09*

1.82 -to.14 9.15 + 1.16* 22.40 f 1.29* 44.18 t 3.50*

1.76 kO.14 2.34 t- 0.30 4.16 f 0.76* 8.48 f I .02*

0.03 6.45 14.53 28.12

* 0.00 + 0.95* t- 0.81* f 1.76*

0.03 0.43 2.97 7.76

Day 63 Saline Silica 0.2 mg Silica 1.O mg Silica 10.0 mg

? 0.67 I!! 0.45 -t 1.26 IL 0.44 0.66 0.46 0.48 0.54

f 0.66*

k k f k

0.03 0.08* 0. I3 0.74*

-+ 0.0 I * o.os* t 0.6 1* t 1.08*

’ N = 5; all values are X 106. * Significantly different from saline control group mean: p < 0.05.

nisms can handle the lung burden, an inflammatory response will persist. This response to a nonrealistic dose impairs the ability to distinguish a fibrogenic dust from a nuisance dust in a short-term assay. In this respect, the most appropriate dose in the present study was 1 mg/lOO g body wt. This dose easily differentiated the fibrogenic from the nonfibrogenic materials, while still being high enough so that a potential adverse effect would not be missed. There appear to be both an early phase and a late phase in the rat lung response to these instilled materials. The acute response to sil-

ica resulted in larger increases in biochemical and cellular parameters than equal doses of nuisance-type dusts. The most useful parameters characterizing the early response were LDH, NAG, TP, and BG. A significant and persistent influx of PMNs was also a prominent factor. The initial response, however, does not appear to predict the potential for chronic injury since the early responses (Days + 1 and +7) of the low dose silica and the high dose nuisance dust were similar, but only the low dose silica resulted in fibrosis. Unlike the nonfibrogenic dusts, silica exposure resulted in a later phase of biochemical and cellular

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TABLE BRONCHOALVEOLAR Termination Treatment

LAVAGE

FLUID

INSTILLED

277

DUSTS

2

CELL NUMBERS

AFTER Alz03 OR TiOz EXPOS~JRE (.Y + SE)”

day Total

cells

Macrophages

Neutrophils

Lymphocytes

Day I Saline Al2O3 1 .O mg AlzO, 10.0 mg TiOz 1.0 mg TiOz 10.0 mg

2.26 6.56 5.90 6.66 7.82

0.2 I 1.34 0.52 1.34 0.52

2.06 2.42 1.79 2.36 3.74

kO.17 + 0.98 f 0.37 f 0.41 * 0.48

0.16 + 0.08 3.55+0.31h 3.85 ?I 0.41 h 4.18 + l.03h 3.99 i: 0.49h

0.05 XL0.02 0.58 AI 0.2 I 0.26 k 0.08 0.15 * 0.10 O.l6~0.(14

Day 7 Saline A&O, 1 .O mg AlzOx 10.0 mg Ti02 I .O mg Ti02 10.0 mg

1.84+0.19 2.14kO.24 9.24 ? I .06h 2.74 It 0.34 8.54 f 1.44h

1.82 1.88 3.84 2.62 4.36

kO.17 t 0.33 f 0.64h t 0.34 k 0.60”

0.01 0.19 5.01 0.03 3.79

37 0.00 +- 0.07 t 0.83’ t 0.01 i I .06”

0.03 0.02 0.37 0.09 0.36

f 0.0 1 + 0.01 ~fr 0.18 f 0.0 I f 0.1 I ’

Day 14 Saline A&O3 I .O mg AljO 10.0 mg TiOz 1 .O mg TiO? 10.0 mg

3.86 2.54 8.72 2.62 6.78

k 0.38 f 0.24 f 1.67h to.18 k 0.96”

2.84 f 0.36 2.28 k0.18 5.04 f 1.60 2.52iO.18 3.66 + 0.39

0.01 0.21 3.45 0.07 2.96

k k k t +-

0.03 0.02 0.22 0.02 1.56

* * zk it

Day 28 Saline A1203 I .O mg A&O3 10.0 mg Ti02 1 .O mg TiOz 10.0 mg

1.76 + 0.19 1.72 + 0.25 4.48 zk 0.32h 1.4OkO.14 3.20 f 0.50h

1.74 * 0.21 1.50 * 0.19 2.48 kO.17 1.3OkO.13 I .98 f 0.32

0.00 t 0.00 0.16rtO.04 1.93It0.15* 0.10 +- 0.03 1.12 k 0.26’

0.03 f 0.02 0.04 + 0.02 0.13f0.03h 0.01 f 0.01 0.09 f 0.0 I h

Day 63 Saline Ai20, 1 .O mg A1203 10.0 mg TiOz l.Omg TiOz 10.0 mg

1.90 f 0.13 1.68 kO.18 3.64 t 0.82 1.62kO.21 3.92k 0.75 h

1.82kO.18 1.56kO.16 2.14i0.35 1.46 k 0.20 2.12k0.24

0.06 r 0.04 0.06 + 0.02 1.43 r0.51h 0.12~0.02 I .67 +- 0.50h

0.00 0.03 0.05 0.02 0.12

f f Iff i

” N = 5; all values are X 106. ’ Significantly different from saline control

group

0.01 0.07 0.87” 0.03 0.63”

* + f + k

0.03 0.0 I 0.05 h 0.0 1 0.03’

0.00 0.02 0.03 0.0 1 0.06

mean; y < 0.05.

changes which not only persisted, but also increased in magnitude with time after instillation. This progression was most dramatic between Days +28 and +63. This is consistent with the known progressive nature of silicainduced lung pathology. This late phase was dose dependent and seemed to correlate well with the pathological changes.

In conclusion, the present study indicates that when trying to predict the toxicity and fibrogenic potential of a new material, it is important to (1) use appropriate parameters to monitor changes; (2) use positive and negative controls to position the results of an unknown material; (3) follow parameters over a sufficient time period to distinguish progressive versus

278

LINDENSCHMIDT

FIG. 6. Lungs from (b) silica;(c) aluminum

rats 60 days following exposure oxide: and (d) titanium dioxide

ET

AL.

to 5 mg/lOO g body (H&E. .z: 100).

wt: (a) sterile saline (control):

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279

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nonprogressive changes; and (4) use appropriate dose levels for making comparisons. ACKNOWLEDGMENTS Thanks go to C. Hudson and J. Englehart for their help with clinical chemistry.

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