Document wgvV9aj1GJ5g9vBd9aYRzYYM3

LUNG REACTION TO CHRONIC INHALATION OF THREE CHRYSOTILES IN THE FISCHER 344 RAT. I. MORPHOMETRY Kent E. Pinkerton1 , Philip C. Pratt 1, Fred 2Miller and James D . Crapo Departments of Medicine and Pathology Duke University and Durham V.A. Medical Center Durham, North Carolina 27710 2 Environmental Protection Agency Research Triangle Park, North Carolina 27711 Running Title: Lung Reaction to Three Chrysotiles Send Correspondence and Reprint Requests to: Kent E. Pinkerton, Ph.d. Box 3177 Duke University Medical Center Durham, North Carolina 27710 '.i I ; 7 8 0., UCC 007680 ABSTRACT LUNG REACTION TO CHRONI^ INHALATION OF THREE TYPES OF CHRYSOT'lLEl ASBESTOS DURING THE LIFESPAN iyi/ i U : F ,THE FISCHER 344 RAT 1/ ' The quantitation of lung reaction to aerosolized chrysotile asbestos was determined by using morphometric techniques at the light and electron micro scopic levels of magnification. Three different preparations of chrysotile were used to expose male and female Fischer 344 rats 7 hours per day, S^days per week for 12 months. Randomly selected animals were examined following 3 t and 12 months of exposure. Some animals exposed for 12 months were kept in filtered air for an additional 12 months before evaluation. Fiber characterization of each aerosolized chrysotile preparation was done using light and scanning electron microscopy. Fiber length and fiber diameter measurements demonstrated unique size distribution characteristics for each aerosolized chrysotile preparation. Exposure to each type of chrysotile caused different patterns of lung reaction. Lung reaction was most severe in the epithelial and interstitial compartments. Partial resolution of the epithelial reaction occurred during the postexposure period, while the interstitial reaction persisted. The dif ferences in lung reaction to these three types of chrysotile are likely to be a result of differences in fiber length and in deposition of fibers within the lung parenchyma. Both short and long fibers elicit lung reaction. How ever, long fibers {> 50 pm) cause a more intense reaction than do short fib- . A ; 780? iv UCC 007681 ers (< 30 im). Fiber localization within the lung tissue compartments and ultrastructural changes which occurred following chrysotile inhalation were examined by light and transmission electron microscopy at each of the three time periods studied. . ^" Bedause^of the extensive period of time covered by this study, the ef fects o(f aging on morphological changes in the lung were examined oven'the l ! 1/ lifespan/of the Fischer 344 rat. Ji * ' ' ) .[ t UCC 007682 A : 780o CHAPTER 4 LUNG REACTION TO CHRONIC INHALATION OF THREE /CHRYSOTILES IN THE FISCHER 344 RAT I. -MORPHOMETRY / INTRODUCTION Lung injury caused by the inhalation of asbestos was first in 1907 in the autopsy findings of a cardroom worker in an asbestos factory. Extensive areas of this worker's lungs had become fibrotic and numerous "spicules of asbestos" were found both free and within the lung parenchyma (Murray, 1907). The term "asbestosis" was first used to identify this type of lung injury in 1927 (Cooke and McDonald). The induction of pulmonary fibrosis by inhalation of either the amphibole or serpentine class of asbestos dust in humans and animals is well documented (Vorwald et al., 1951; Wagner et al_., 1974; Seal, 1980; Davis et al_., 1980). Chrysotile, a member of the serpentine group, makes up 90% of the total asbestos produc tion today. The use of chrysotile in insulation, ceiling tiles, brake linings, cement products, hairdryers and numerous other materials greatly enhances the number of people coming into contact with this type of asbestos. The biological effects of chrysotile have been studied using both natural and unnatural means of fiber exposure in laboratory animals. The fibrogenic and carcinogenic effects of chrysotile have been confirmed by inhalation (Vorwald et al_. , 1951; Wagner et al_., 1974) and intratracheal and intrapleural injection of fibers (Suzuki, 1974; Wagner et al., 1973, 1980). 89 UCC 007683 A ; 78 0 3 90 In vitro studies have also implicated chrysotile to be both cytotoxic (Sakabe et^'aT., 1971; Allison, 1971; Beck et al., 1971; Richards et al.. 1977) and hemolytic (Harington et al_., 1975; Desai et al_., 1975). The purpose of this study is to quantitate the degree and pattern of lung injury caused by the inhalation of three different chrysotile prepara- Quantitative measurements of alveolar tissue structure in the lungs of rats following increasing periods of fiber inhalation and after a post exposure period of time allows us to examine the acute and chronic effects of fiber inhalation for three types of chrysotile. Employment of morpho metric techniques provides an objective means of comparing the relative fibrogenicity and degree of lung injury caused by each type of aerosolized chrysotile. In addition, we can identify those tissue compartments in the alveolar regions of the lung which are most affected by fiber exposure and the changes which occur in each alveolar tissue compartment following different periods of fiber inhalation. UCC 007684 r\ O 91 Animals t- - MATERIALS AND METHODS Three hundred thirty (330) age-matched specific pathogen-free male and female Fischer 344 rats (weighing 125-150 grams) were acquired from Charles River Laboratory, Inc. Ten rats were randomly selected and killed. Total body necropsies found these animals to be free of (1) external and internal parasites, (2) salmonella in the feces, (3) respiratory pathogens, e.g.. Mycoplasma pulmonis. Streptococcus pneumoniae and Corynebacterium kutscheri, (4) murine viruses as determined by serology and (5) disease of all major organs. Forty animals of each sex from the remaining 320 animals were assigned by random selection to either a control group or one of the i|iree exposure groups. f Crysotile Preparations The three asbestos samples used in the inhalation studies were UICC B chrysotile, Jeffrey Mine chrysotile and Coalinga Mine chrysotile. A description of the origin and physical characteristics of each chrysotile sample has been reported (Pinkerton et al_., 1982). UICC B Chrysotile Reference Sample - This preparation, a grade 4 chry sotile (Timbrell et ^1_., 1968A) was obtained from eight different Canadian chrysotile mines and mixed according to the proportional production of each mine during the year of 1950. The approval of this preparation as a reference standard was made in 1966 by the Union Internationale Contre Cancer (UICC) as an attempt to standardize asbestos materials used in exper imental research. After aerosolization in our chambers, this preparation had fibers longer than 100 um in length, but 93% of all fibers were less than 10ym in length, and no fiber, regardless of length, exceeded 2 ym in diameter (Pinkerton et al_. , 1982). ^ <781 1 UCC 007685 92 Jeffrey Mine Chrysotile - Identified as Plastibest 20 by Johns Manville, Iftis form of chrysotile is a grade 4 asbestos used by the plastics industry. The mine is located in Asbestos, Quebec, Canada. This sample was originally purchased by the National Institute of Environmental Health Sciences (NIEHS) for oral ingestion studies. The material was prepared by roller milling and air classification using standard industrial techniques, followed by passing the material through a Hurricane Pulverizer three times to open fiber bundles. In the aerosolized state, fibers longer than 100 pm were present, 92% of all fibers were less than 10 pm in length and no fibers exceeded 2 pm in diameter, regardless of length (Pinkerton, et al_., 1982). Coalinga Mine Chrysotile - Identified as C0F-25 by Union Carbide,jthis unique form of chrysotile was mined from the New Idria serpentinite'`mass located in the Diablo range in California. The deposit is unusual in*that the fibers are randomly oriented as a mat, rather than as parallel fibers running in veins and the mining process is done with bulldozers. The deposit is almost pure chrysotile. The fibers are short in length and are not of spinning grade. The processing of this material was done by roller milling and water classification. In the aerosolized state, fibers greater than 100 pm were present, 66% of all fibers were less than 10 pm in length and many fibers exceeded 2 pm in diameter. In general, as the length of the fiber increased, the diameter of the fiber also increased (Pinkerton et al., 1982). For fibers longer than 5 pm in length and under 3 pm in diameter, it was found by light microscopy that the Jeffrey Mine chrysotile contained a significantly greater fraction of fibers in the 40-50 pm, 50-100 pm and >100 pm length intervals than that found in the UICC B or Coalinga chrysotile samples in the aerosolized state. Fiber analysis by scanning electron microscopy also revealed that of all aerosolized fibers longer than 10 pm in UCC 007686 A : 78 1 2 93 length, the percent with a diameter less than 0.6 ym was 92.9% for Jeffrey chrysotile^1"85.3% for UICC B chrysotile and 12.0% for Coalinga chrysotile {Pinkerton et aK , 1982). Exposure Conditions . 3 Four 5.0 m stainless steel chambers were used to house in each 40 male and 40 female rats in hanging 17 x 33 x 15 cm wire cages, two animals per cage. The animals were continuously maintained in the chambers. Three chambers were used to expose animals to one of the three chrysotile prepara tions by fiber aerosolization using a modified Timbrell generator (Timbrell e_t _aj_., 1968) which was operated 7 hours/day, 5 days/week for 12 months. The controls breathed filtered air and were not exposed to chrysotile. -f The total chamber dust mass concentration was monitored daily to main tain a constant dust concentration throughout the duration of the exposure periods. These measurements were made by sampling 100 liters of air from the chamber at a flowrate of 10 liters/minute and collecting the airborne fibers and particles on a 25 mm glass fiber filter held in a Gelman filter holder. After the samples were collected, the filters were removed and weighed and the dust concentration in each exposure chamber was expressed in mg/m3 . S.ize selective dust samples were also taken daily using a Casella type 113A Gravimetric Dust Sampler to determine the "respirable" dust concentration in each chamber. With the Casella Sampler placed inside the exposure chamber, samples were collected for 6 hours at a flowrate of 2.5 liters/minute. A multihorizontal elutriator system within the instrument was calibrated to prevent particles whose equivalent aerodynamic diameter exceeded 7.1 ym from collecting on the filter. The filter was weighed at the end of the sampling period to determine the "respirable" mass A; 7H: 3 UCC 007687 94 concentration in the exposure chamber. A summary of the daily means for the total and Respirable dust concentrations are given in Table 1. Animal Sacrifice and Tissue Preparation Four animals of each sex were randomly selected from each exposure chamber and the control chamber after 3 months of fiber inhalation and again after 12 months of fiber inhalation. Full body necropsies were performed on these animals and will be reported elsewhere. The remaining animals were removed from the exposure chambers following 12 months of fiber inhalation and maintained behind a limited access barrier. Twelve months following the end of dusting, four animals of each sex were randomly selected from the surviving animals in each exposure group and the control group. The percent of survival was the following for each group: 61% in animals exposed to Coalinga Mine chrysotile, 53% in animals exposed to UICC B chrysotile, 52% in animals exposed to Jeffrey Mine chrysotile and 64% in animals which served as controls. The necrospy findings and lifespan characteristics of these randomly selected rats and the surviving animals will be reported el sewhere. The rats used in this study were anesthetized by an intraperitoneal injection of pentobarbital (50 mg/kg) and the trachea cannulated. The abdomen was opened, both hemidiaphragms punctured to collapse the lungs and fixative was instilled intratracheally via the tracheal cannula at a pres sure of 20 cm of water. The size of the cannula and the tubing connecting it to the fixative were adjusted to give a flowrate equal to 115 ml/minute when the cannula is not inserted into the trachea. The fixative was 2% glutaraldehyde buffered to 7.4 using Na cacodylate and having a total osmol ality of 350. After a minimum of 30 minutes fixation the lungs were removed from the chest cavity and the lung volume was measured by water displacement UCC 007688 A 1 78 l 4 95 (Scherle, 1970). This technique of lung fixation has been shown to fix the lungs at Jpproxinately 75% of their physiologic TLC {Hayatdavoudi et al., 1981). 1 Light Microscopy The right cardiac lobe from each animal was embedded in paraffin. Serial sections 10 urn thick were made through the entire lobe. Every fortieth section was placed on a glass slide and stained with hematoxylin and eosin. These were used to determine the parenchymal fraction of the lung as described in the morphometric analysis section of this paper. Electron Microscopy ^ Four sites from the peripheral region of the left lunq in each animal were randomly selected. These sites were chosen from the ventral and dorsal portions of the cephalic and caudal regions of the lung. The tissue samples from each site were washed in cacodyl ate buffer, dehydrated in a graded series of alcohol, transferred to increasing concentrations of propylene oxide, and embedded in Epon. Sections 0.5 ym thick were cut and stained with toluidine blue to verify the presence of alveolar tissue. Parenchymal tissue was considered to include alveoli and alveolar ducts. Airways with ciliated epithelium and blood vessels with a diameter greater than 25 ym were considered to be nonparenchymal tissue. Sections containing paren chymal tissue were cut using a diamond knife, placed on copper 200 mesh grids and poststained with uranyl acetate and lead citrate. Morphometric Analysis The morphometric procedures followed in this study have been well described by Weibel (1979), Crapo and Greeley (1978), Crapo, et aK (1978) and Woody, <rt a_L (1980). The fraction of the total lung volume which A ;7B b UCC 007689 96 constitutes the parenchymal region was determined by point counting each of the step sections taken through the right middle lobe of each animal. These paraffin step sections were directly placed in a Durst photographic enlarger and projected, at a constant magnification, onto 11 x 14" photographic paper. The resulting print was a negative image of the section. These prints were placed under a test lattice containing 224 points and the distribution of these points falling on either parenchyma or non-parenchyma were determined. Parenchymal areas included alveoli and alveolar ducts. Nonparenchymal areas included blood vessels greater than 25 um in diameter, interlobular connective tissue, bronchioles and bronchi. The above tech nique was found to be faster than doing the same analysis using an autoi|ptic sampling stage light microscope. Fifteen random electron micrographs were taken of each of four sections prepared from peripheral lung sites for each animal and printed on 11 x 14" paper at a final magnification of 8500. Morphometric analysis of these point were done as described previously (Crapo, et aK, 1978). Numerical density of lung cells (N ) was determined by using the formula Ny = N^/D, where is the number of nuclear profiles per unit area on a thin section and D is the mean caliper diameter of the nuclei (Greeley et al., 1978; Woody et aK , 1980). The nucloar profiles fcund in 100 consecu tive grid squares (25 grids per section for 4 sections per animal) totally covered by the tissue section were counted. Grid squares containing non parenchyma 1 portions of the lung were emitted. Each nuclear profile was classified according to coll type using the unbiased counting rule described by Gunderson (1977). It was found that the area of the grid square on any single grid varied less than 2%, but that variation did occur between grids. Therefore, one of the grid spaces for each section was photographed at 500 X and its area determined by digitization. The total area counted for nuclear ft i76'.o UCC 007690 97 profiles for each grid was calculated as the number of grid squares counted times the area of a single grid space for that grid. The mean caliper diameter (Du) of each cell type was determined by taking light micrographs of serial nuclear profiles 0.5 ym thick through the entire nuclei of randomly selected cells from each of the major alveolar cell types. Each micrograph was enlarged to 4000 times actual size and the outline of all profiles making up one nucleus was traced using a Hewlett Packard 9874A Digitizer and serially reconstructed in three dimensions with a Hewlett Packard 9845A Computer. Computer analysis of' this data, as described by Woody et aj_. (1980), was used to determine the mean caliper diameter for each recon structed nucleus. ^ Lung Ashing and Silica Determination The cranial, caudal and accessory lobes of the right lung from animals exposed to the three chrysotile preparations for 3 and 12 months were dried to a constant weight at 105C and then ashed at a temperature of 460C in a platinum crucible. The ash was mixed with anhydrous sodium carbonate and heated to 1Q00C. Distilled water was added to the fused mass and the solu tion was decanted several hours later* in H^O^. HC1 and H^SO^ were used to remove any remaining material from the crucible. This mixture was allowed to evaporate and then distilled water was added to bring the total volume to 50 ml. This solution was then mixed with the following compounds: ammonium molybdate, oxalic acid-sulfuric acid, and ammonium ferrous sulfate. The mixing of these compounds resulted in reduction of silicomolybdic acid to a blue colored solution which was colorimetrically analyzed with a Technicon Autoanalyzer. Benusil, a silica compound, was used, as the standard to determine the amount of silica present in solution (King et aj_., 1955). h ;78 1 7 UCC 007691 98 Statistical Analysis All statistical comparisons were made by using a one-way analysis of variance followed by Duncan's multiple comparison test to determine the significant changes occurring between and within the various control and treatment groups. ,, i UCC 007692 A i7a 13 99 RESULTS % Body Weight and Fixed Lung Volumes The mean body weight and fixed lung volumes are given in Table 2. The mean body weight for female rats exposed to each type of chrysotile was decreased following 3 months exposure compared to control. With continued exposure to chrysotile, the mean body weight was not significantly different from the age-matched control values. The mean body weight of the male rats was relatively unchanged from that of the controls with the exception of a decreased body weight in males exposed for 12 months to Jeffery chrysotile and an increased body weight in males one year following the end of exposure to Coalinga chrysotile. The mean fixed lung volumes were significantly increased compar|d to control in male rats following 3 months exposure to all fibers. The fixed lung volume remained significantly elevated in the male rats exposed to Jeffrey chrysotile following 12 months exposure to 21% greater than control and one year after the end of exposure to 36% greater than the age-matched control values. Female rats exposed to Jeffrey chrysotile for 12 months and UICC B exposed females kept an additional year in filtered air also had a mean lung volume significantly greater than the age-matched controls. Only females exposed for 12 months to either Coalinga or IJICC B chrysotile had a significantly smaller mean lung volume than the control value. Light Microscopy The mean parenchymal fraction found for the lungs of each group of animals ranged from 0.79 to 0.82. This range in values was not statisti cally significant, but was a result of neoplastic changes occurring in the parenchymal portions of the lung. Most lung tumors were found peripherially one year following the end of fiber exposure. Th<e me^i^Rjer^ent of the lung UCC 007693 100 with tumor was 0.1% in the animals exposed to Coalinga chrysotile, 0.7% in the animalf-exposed to UICC B chrysotile and 1.4% in the animals exposed to Jeffrey chrysotile. These tumors consisted of adenomas, squamous cell carcinomas and bronchiolar metaplasia. The exclusion of these neoplastically altered regions of the parenchyma from the parenchymal fraction of the lung did not significantly affect the morphometric results of this study. Electron Microscopy The volumes and surface areas of the three major components of the parenchymal portion of the lungs, air, capillary blood and tissue are given in Table 3. Hale rats exposed to Jeffrey chrysotile were the only group to have a total alveolar airspace volume significantly greater than controls at all three time periods, with 13%, 17% and 29% increases respectively jOver the age-matched controls. The pulmonary capillary blood volume was not significantly changed from controls in any of the exposed groups. Total alveolar tissue volume increased in male rats following 3 and 12 months exposure to each chrysotile preparation. One year following the end of fiber exposure, the alveolar tissue volume in male rats exposed to UICC B showed a progressive increase which1 was significantly greater than that occurring in controls. Female rats showed similar trends toward greater parenchymal tissue volume after each chrysotile exocsure but these changes did not reach a statistical level of significance except for those exposed to the Jeffrey chrysotile which had a 154% increase in tissue volume after 12 months exposure and an 84% increase one year following 12 months expo sure. The absolute surface area of the alveolar epithelium and the capillary endothelial lumen demonstrated no substantial changes between the controls and treatment groups for both males and females. The capillary endothelial A i 782C UCC 007694 101 surface area in females one year following the end of exposure to UICC B chrysotile^ was significantly decreased from control. However, a similar trend was riot seen in the corresponding males exposed to UICC B chrysotile. Therefore, we have not attributed any biologic significance to these differ ences. The alveolar tissue volume of the lung is subdivided in figure 1 into its three basic components; epithelium, interstitium and endothelium. The volume of endothelium was unchanged from control values for each exposure group except for a 49% and 57% increase respectively in the male and female animals exposed to Jeffrey chrysotile for 12 months and then to filtered air for 12 months. Exposure to all three types of chrysotile for 3 ninths resulted in an increased alveolar epithelial volume in male and female^irats. Following 12 months exposure, the epithelial volume was also significantly increased for all three treatment groups compared to the age-matched con trols. The increase in epithelial volume was greatest in the animals exposed to Jeffrey chrysotile for 12 months, being 175% and 2C1% in males and females respectively. Following 12 months exposure to UICC B chryso tile, the alveolar epithelial volume in the male and female rats was 88% and 75% greater than control respectively. Only the animals exposed to Ccalinga chrysotile showed a decrease in the epithelial volume from 3 months of expo sure to 12 months of exposure with a 20% decrease in the males and a 19% decrease in the females, but the absolute epithelial volume was still significantly greater than control. One year following the end of 12 months fiber exposure, the alveolar epithelial volume in all exposure groups decreased for both male and female animals. The alveolar interstitial volume also increased in the lungs of male and female rats following 3 months exposure for all three chrysotile samples. Following 12 months exposure, the interstitial volume in males and A 1 782 1 UCC 007695 102 females dusted with Jeffrey chrysotile increased 146% and 157% of the re , fr spective control values. The alveolar interstitial volume in the lungs of male rats exposed to Coalinga and UICC B chrysotile for 12 months had also significantly increased by 53% and 50% of the age-matched control value respectively. One year following the end of 12 months exposure, the inter stitial volume in the lungs of male and female rats exposed to Coalinga and Jeffrey chrysotile had increased by an average of 12% from the interstitial volume recorded at the end of fiber exposure. In contrast, the alveolar interstitial volume in the lungs of the age-matched male and female controls increased by an average of 24% during the same time period. Only the alveo lar interstitial volume in male rats exposed to UICC B chrysotile ft^r 12 .1 months increased significantly during the postexposure year, by 83% from the value seen immediately following the end of 12 months exposure. * The changes occurring in total volume of alveolar type I and alveolar type II epithelium are given in Table 4. After 3 months exposure, total alveolar type II cell volume had more than doubled in the lungs of male and female rats from all three treatment groups. At the end of 12 months expo sure, the type II epithelial volume continued to increase in males and females exposed to UICC B chrysotile (233% and 250% of control) and Jeffrey chrysotile (420% and 395% of control). The alveolar type I cell volume was also significantly increased in males following 12 months of exposure to each of the fiber preparations. One year following the end of fiber expo sure, the total volume of alveolar type II cells had decreased for both male and female rats in all three treatment groups, although male rats exposed to UICC B chrysotile and female rats exposed to Jeffrey chrysotile had a significantly greater type II epithelial volume than that of the respective controls. During the postexposure year, the volume of the type I epithelium remained relatively unchanaed. " A ; 7.82 2 UCC 007696 103 Table 5 divides the alveolar interstitium into two parts; the cellular *- component fhd the noncellular or matrix component. After 3 months exposure, the total volume of cells in the interstitium increased in the lungs of all treatment groups for both sexes. Following 12 months exposure, the only groups to maintain a significantly increased interstitial cell volume com pared to the age-matched controls were the male and female rats exposed to Jeffrey chrysotile. One year after the end of exposure, males and females exposed to Jeffrey chrysotile and males exposed to UICC B chrysotile had significantly greater interstitial cell volumes compared to controls. The interstitial matrix volume had begun to increase after 3 months exposure in both males and females from all three exposure groups. Aft*r 12 months exposure, a marked increase in the interstitial matrix volume had occurred in the male animals from each of the treatment groups. Compared to the control value, the interstitial matrix volume was 60% greater in male rats exposed to Coalinga chrysotile, 54% greater in males exposed to UICC B chrysotile and 143% greater in male rats exposed to Jeffrey chrysotile. The total volume of the interstitial matrix continued to increase during the postexposure period in control and treatment groups of both sexes. A unique change in the interstitial matrix volume during this postexposure period was seen in the lungs of rats which had been exposed to UICC B chrysotile. While the controls and groups exposed to Coalinga and Jeffrey chrysotile showed a gradual increase in the total interstitial matrix volume of the lungs, male rats and female rats to a lesser degree had a significant increase in the volume of the interstitial matrix volume during the post exposure year. The increase in matrix volume in the males was a 4 to 5 fold greater increase than seen in the corresponding controls or the animals which had been exposed to Coalinga or Jeffrey chrysotile. UCC 007697 A ! 7823 104 The computerized serial reconstruction of nuclei to determine the mean nuclear diameter of the major alveolar cell types in the rat lung was done in four rats from the controls age-matched to the animals exposed to chrysotile for 12 months. Two controls of each sex were used and three rats which had been exposed for 12 months to Jeffrey chrysotile (2 males and 1 female). The animals exposed to Jeffrey chrysotile for 12 months were selected because they demonstrated the most dramatic lung reaction of all exposure groups. If reaction of the lung cells to asbestos caused a change in the mean nuclear diameter of each cell type, animals exposed for 12 months to Jeffrey chrysotile would be most likely to demonstrate these changes most clearly. The results of this study are given in Table |. A jackknifed weighted mean for each cell type was determined (Greeley, et; a1., 1978) and values were compared between males and females, and between exposed animals and controls. It was found that there was no statistically significant difference between sexes or between controls and animals exposed to Jeffrey chrysotile for alveolar type I, alveolar type II, interstitial or macrophage nuclei. A difference was noted for the endothelial mean nuclear diameter between the exposed group and controls. It was decided to pool the data for each alveolar cell type to obtain an overall mean nuclear diameter for each class of cells. These values were used to calculate the cell density of each alveolar cell type. The mean nuclear diameter found for rats exposed to Jeffrey chrysotile for 12 months was used to calculate the endothelial cell number for that specific exposure group and time period. The endothelial cell number calculated for all other exposure groups and time periods used a pooled endothelial mean nuclear diameter. It should be noted that the use of either endothelial mean nuclear diameter does not cause significance changes in endothelial ceil number that would have been created or lost by the use of one value over twe other. A ; 7824 UCC 007698 105 Changes in alveolar cell numbers are given in Table 7. The total number of^ells which make up the alveolar region of the lungs was most dramatically increased in male and female rats exposed to Jeffrey chrysotile for 12 months. In males the total number of cells had more than doubled, while in females the total cell number had increased to 166% of the corre sponding control value. The three alveolar cell types showing the greatest changes in numbers and responsible for most of the increases in total cell number during and following exposure to the three types of chrysotile were alveolar type II cells, interstitial cells and alveolar macrophages. A significant increase in alveolar type I cell number compared to controls occurred only in the lungs of animals exposed to Jeffrey ^Mine .L chrysotile (Table 7). Alveolar type II cell number was significantly i increased in male rats after 3 months of exposure, 12 months of exposure and one year following the end of exposure for all three treatment groups (Table 7). The greatest type II cell proliferation was seen in the lungs of male rats exposed to Jeffrey chrysotile for 12 months (a 279% increase in compar ison to the age-matched controls). One year following the end of exposure to Jeffrey fibers, the type II cell number in males decreased by 44%, but was still more than double the type II cell number seen in controls. In general, the pattern of alveolar type II cell proliferation in female rats exposed to the three chrysotiles was similar to that seen in the males. In animals exposed to Jeffrey chrysotile from 3 months to 12 months, females had an 84% increase in the number of alveolar type II cells while males had a 73% increase in type II cell number. Type II cell number was the highest in males and females exposed for 12 months to Jeffrey fibers compared to other treatment groups of the same sex and time periods. Figure 2 illu strates the percent change in the number of alveolar type I and alveolar type II cells in pooled male and female rats compared to the age-matched UCC 007699 A ; 782b 106 controls. Figure 2 graphically illustrates the more pronounced changes which occurred in alveolar type II cell number compared to alveolar type I cell number-in all exposure groups. The number of interstitial cells in the alveolar region of the lung for both male and female rats was increased after 3 months exposure to all three chrysotile preparations from 22% to 70% above that of the controls. For animals exposed to Jeffrey chrysotile, the number of interstitial cells continued to increase after 12 months exposure, while animals exposed to Coalinga and UICC 3 chrysotile showed a decrease in the total number of alveolar interstitial cells during this time period in both sexes. One year after the end of exposure, male and female rats which had been expos^i to UICC B and Jeffrey chrysotile demonstrated the highest numbers of inter stitial cells compared to the controls ranging from 46% to 144% of control. This represented a significant increase over control with the exception of the male rats exposed to UICC B fibers. Figure 3 illustrates percent changes in the major components of the interstitium in pooled male and female rats. These are changes in cell number, changes in cell volume and changes in the matrix volume. The total number of alveolar macrophages in the airspaces cf the lung was increased following 3 months of exposure to the three different types of chrysotile (Table 7, Figure 4). After 12 months exposure, the number of macrophages was still at least doubled compared to control in all treatment groups for both sexes. Alveolar macrophage number was greatest in males and females exposed for 12 months to Jeffrey fibers with a 371% and 471% increase over the number seen in the corresponding controls. One year following the end of fiber exposure, only the males which had been exposed to Jeffrey chrysotile had a significantly increased number of alveolar macrophages compared to the controls. Figure illustrates the percent A ; 7823 UCC 007700 107 changes in the numbers of alveolar macrophages for pooled male and female rats. Capillary endothelial cells in control animals comprised approximately one-half of the total number of cells in the alveolar region of the lungs. The total number of endothelial cells in animals exposed to Coalinga and UICC 8 remained relatively unchanged from that of the age-matched controls during the entire period of study (Table 7). In contrast, male and female rats exposed to Jeffrey chrysotile had a significantly increased capillary endothelial cell number following 12 months exposure which remained signifi cantly increased one year after the end of fiber inhalation. The harmonic mean tissue thickness of the air-blood harrier is givyi in Table 9. With the exception of male rats exposed for 3 months to Coalinga and UICC B chrysotile, all exposure groups for both sexes demonstralfed a significantly increased harmonic mean thickness of the air-to-blood tissue barrier during and following fiber inhalation. Animals exposed to Jeffrey chrysotile demonstrated the greatest increase in the harmonic mean tissue thickness during the period between 3 to 12 months fiber inhalation with a 40% and 56% increase for males and females respectively. The arithmetic mean thickness of the air-to-blood tissue barrier is illustrated in Figure 5. This thickness has been subdivided into the arith metic mean thickness of the epithelium, interstitium and endothelium. The thickness of the alveolar epithelium was significantly increased at every time point studied for all three types of chrysotile. However, the thick ness of the epithelium decreased by 23% to 78% in all groups during the postexposure period. The thickness of the interstitium was also signifi cantly increased at almost every time point during and following exposure to each type of chrysotile. The arithmetic mean thickness of the interstitium increased by 60% in animals exposed to Jeffrey c rysotile for 12 months UCC 007701 A; 7827 108 compared to those exposed for 3 months. The interstitial thickness in animals ed|fosed to Coalinga and UICC B chrysotile increased by 3% and 23% respectively during the same time period. During the postexposure period, the interstitial thickness in animals which had been exposed to UICC B chrysotile increased by an additional 25%, while no increase was seen in animals dusted with the other two chrysotiles. The arithmetic mean thick ness of the endothelium was significantly increased in animals exposed to Jeffrey chrysotile at each of the three time periods. Lung Ashing and Silica Determination The results of the lung ashing are given in Table 10. All treatment groups following 3 and 12 months of fiber inhalation had signifidntly 1 greater amounts of silica in the lungs compared to the controls. The silica content of the lungs following 3 months fiber exposure was similar for the three treatment groups. Only the animals exposed to Jeffrey Mine chrysotile had a statistically significant increase in silica content of the lungs between 3 months and 12 months of exposure. UCC 007702 A : 7823 109 DISCUSSION t The degree of pulmonary damage brought about by the inhalation of asbestos has been postulated to be due to either the chemical composition of the fibers, the physical state of the fibers, the amount of fibers deposited in the lungs or some combination of these factors (Wagner, 1965; Seaton, 1975). Although animal experiments can closely control asbestos exposure conditions, the measurement of the relative fibrogenicity of different types of asbestos has been largely subjective and inconclusive, while the relative carcinogenicity of different types of asbestos has been more definitive in that tumors were or were not produced (Wagner et al_., 1974). _Other factors which are likely to contribute to the amount of lung fibrosis produced by different types of asbestos are the degree of fiber penetration int$ the lungs and the pattern of fiber deposition in the lung. Both fiber penetra tion and deposition are related to the size and aerodynamic properties of the fiber (Timbrell, 1965, 1970, 1973). Mew insights into the pathogenicity and fibrogenicity of inhaled chrysotile can be obtained by correlating the physical characteristics of different aerosolized chrysotile preparations and the degree of fiber depo sition achieved with quantitative measurements of lung structure. Inhala tion of all three chrysotiles included in this study resulted in both an epithelial and interstitial reaction in the lung parenchyma. An increase in the volume and in the number of alveolar type II cells and interstitial cells occurred following 3 months of fiber inhalation. With progressive exposure, the epithelial and interstitial reaction was most pronounced in those animals inhaling Jeffrey chrysotile. Following three months exposure to aerosolized fibers, the amount of lung injury in each of the three treatment groups was similar. These A ; 782 3 UCC 007703 no results suggest that inhalation of both short and long fibers can acutely lead to epithelial and interstitial injury and to cell proliferation in these alveolar compartments of the lunq. The shorter and thinner fibers may be able to penetrate more efficiently than longer fibers. Shorter fibers may also be more efficiently cleared from the lungs since alveolar and interstitial macrophages can engulf the entire fiber and bring about its removal through either digestion or clearance of the cell from the lung via the lymphatic and or mucociliary escalator. Differences in the patterns of lung reaction in rats with exposure to each type of chrysotile may be due to differences in the physical character istics of each chrysotile preparation in the aerosolized state. Cca^inga Mine chrysotile, which contained fibers up to 100 ym in length in the aero solized state, had few fibers greater than 30 ym in length that were^also less than 0.6 ym in diameter. Previous studies have suggested that fibers with a diameter greater than 0.5 ym are unlikely to reach the alveolar air spaces of the lung. Therefore, only the shorter fibers (30 ym in length) in the aerosolized Coalinga Mine chrysotile are likely to be respirable. This makes this chrysotile preparation a "relatively short" fiber preparation in comparison to the other two chrysotiles studied. Correlation of the physi cal characteristics of each aerosolized fiber preparation to the patterns of lung injury following exposure suggest that short fibers or fibers less than 30 yin in length are less toxic than fibers of greater length. However, even short fibers less than 30 ym in length can cause lung reaction, particularly in the alveolar epithelium and interstitium. Animals exposed to Jeffrey chrysotile respired a greater portion of fibers which had both a long length (40 to >100 y m) and a small diameter compared to animals exposed to the other two chrvsotile preparations. Animals exposed to Jeffrey fibers demonstrrm d the greatest degree of lung UCC 007704 A .7830 Ill injury during fiber inhalation compared to animals exposed to the other two chrysotilef: These results suggest that fibers in the 40 to > 100 pm length range are less in number, but are the most toxic fiber in the lung paren chyma . Information concerning the amount of chrysotile reaching and being retained in the lungs may be estimated from of the amount of silica remaining in the lungs of dusted rats. Animals exposed to Coalinga chryso tile had a mean of 610 ugm of silica in the lungs following 3 months of fiber inhalation which was unchanged following an additional 9 months of fiber exposure. Animals exposed to UICC B chrysotile shewed a small, but not statistically significant increase in the amount of silica in the ^ungs from 3 to 12 months of exposure. Animals exposed to Jeffrey chrysotiTfe had a 52% increase in silica content in the lungs from 3 to 12 months of expo sure but this was a proportionately, less accummulation of silica than occurred during the first 3 months of exposure (Table 9). The patterns of lung reaction with inhalation of the three chrysotiles correspond with the silica content found in the lungs. Animals exposed to Coalinga and UICC B chrysotile showed relatively no changes in the epithe lial or interstitial tissue compartments from 3 to 12 months of exposure, while animals exposed to Jeffrey chrysotile showed dramatic reactions in both the epithelium and the interstitium. Based on the physical characteristics of the aerosolized fibers and the amount of silica retained in the lungs, it appears that animals exposed to Coalinga chrysotile have reached a state of equilibrium in the fiber load of the lungs in which fibers were cleared at approximately the same rate in which they were deposited. On the other hand, animals exposed to Jeffrey chrysotile had not yet reached equilibrium following 3 months of fiber inha lation. It is possible that the gradual deposition of long fibers in the UCC 007705 A i783l 112 lungs of animals exposed to UICC B or Jeffrey chrysotile is responsible for the increasing silica load being retained in the lungs of animals given these exposures for a total of 12 months. Long fibers which cannot be taken up by one cell and removed from the lungs may eventually become trapped within the interstitial matrix of the alveolar septa and never be removed. Although the means of fiber accumulation cannot be determined by the measurement of silica content in the lungs, the gradual accumulation of a relatively small number of long fibers in the lung parenchyma could lead to significant increases in the silica content of lungs of animals exposed to aerosolized Jeffrey chrysotile for 12 months. One year following the end of fiber exposure, the epithelial reaction in the lungs had, in part, resolved with decreases in the volume and in the i number of alveolar type II cells. The resolution of the epithelial reac tions was most notable in those animals exposed to Jeffrey chrysotile. One year following the end of fiber exposure, the interstitial reaction had not significantly resolved. However, increases in the volume of the intersti tial matrix were seen in each exposure group. The interstitial cell number in animals exposed to each chrysotile preparation did decrease during the postexposure year. A surprising finding was the dramatic increase in the volume of the interstitial matrix in the lungs of animals which had been exposed to UICC B chrysotile (Figure 3). The pattern of these changes occurring during the postexposure year in each exposure group add support to the importance of fiber length and fiber load in causing lung reaction. Animals which had been exposed to Coalinga chrysotile had relatively no changes in lung structure from that seen after 3 months of fiber inhalation with the exception of a significant decrease in the volume of the alveolar type II epithelium and a gradual increase in the volume of the interstitial matrix in the a UCC 007706 "iolar regions of the lung. A;7832 In 113 animals which had been exposed to Jeffrey chrysotile, the lungs also demon strated a Increase in the volume of the alveolar type II epithelium and a gradual increase in the volume of the interstitial matrix. Only in animals which had been exposed to UICC 3 chrysotile did the volume of the inter stitial matrix continue to increase significantly during the postexposure year. This increase in the interstitial matrix volume in the lungs of rats exposed to UICC B chrysotile may reflect the presence of long fibers in the lungs which were not cleared and continued to be a stimulus for the continued deposition of collagen within the interstitium. The analysis of fiber size distribution for aerosolized UICC B chrysotile demonstrated the presence of long fibers with a diameter sufficiently small to allow pen^tra- l tion to the alveolar airspaces of the lung, but with sufficient length to create immense problems in clearance. This same type of fiber was also present in the aerosolized Jeffrey chrysotile, only in proportionately greater numbers. It is feasible that the presence of fewer long fibers in the lungs of animals exposed to UICC B chrysotile resulted in a slower rate of lung fibrosis which continued to progress during the postexposure period. The greater exposure to very long fibers (> 50 um) in the animals exposed to Jeffrey Mine chrysotile stimulated a much more intense initial inflammatory reaction which encased many of these fibers in layers of noncellular, colla genous interstitial matrix thereby reducing their inflammation inducing potential. This would have allowed the inflammatory reaction in these animals to have decreased in intensity during the postexposure year in air while the reaction in the UICC B chrysotile exposed animals progressed at an initially slower but steady pace. The presence of fewer long fibers in the lungs of rats exposed to UICC B chrysotile after 12 months compared to the corresponding rats exposed to Jeffrey chrysotile is indirectly supported by UCC 007707 A ; 7833 114 the significantly smaller amount of silica in the lungs of rats exposed to UICC B fibeife compared to the animals exposed to Jeffrey fibers. In summary, these different patterns of lung reaction are likely to be a result of differences in fiber length and in deposition of fibers within the distal portions of the lung parenchyma. Both short {< 30 pm) and long (40 ym to > 100 pm) fibers are capable of eliciting a tissue response in the lungs which is most pronounced in the epithelial and interstitial compart ments. Exposure to proportionately greater numbers of long fibers (> 50 pm) probably leads to the accumulation of a greater fiber load in the lungs than would occur with exposure to fibers less than 30 pm in length. Fibers less than 30 urn appear to reach an equilibrium in the lungs in terms of dep^ition aqd clearance. Fibers greater than 40 pm may be less efficiently clear ed from the lungs and are likely to cause epithelial and interstitial fac tions until they can be dissolved, cleared or isolated. The proportion of long fibers in the lungs correlates with the intensity of the tissue reac tion. Exposure to large numbers of long fibers {> 50 pm) cause an intense tissue response within the epithelial and interstitial compartments. An exposure to smaller numbers of long fibers appears to cause a less intense, but more prolonged tissue reaction. The progressive tissue reaction seen in this situation may be due to the fact that the initial inflammatory response is not severe enough to totally encase the fibers and thereby remove them as a major source of irritation to the lung parenchyma. -Chapter 5 will illus trates number of these findings.- UCC 007708 A i'7834 115 REFERENCES I, ' Allison AC. (1971) Inhaled particles III (WH Walton, Ed), P. 437, Unwin Bros, Old Woking, Surrey. Beck EG, PF Holt, ET Nasrallah. (1971) Effects of chrysotile and acid-treated chrysotile on macrophage cultures. Br J Med 28:179. Cooke WE. (1927) Pulmonary asbestosis. Brit J Med 2:1024. Crapo JD, DA Greeley. (1978) Estimation of the mean caliper diameter of cell nuclei. II. Various cell types in rat lung. J Microscopy 114:41. Crapo JD, J Peters-Golden, J Marsh-Salin, JS Shelburne. (1978) Pathologic changes in the lungs of oxygen-adapted rats. A morphometric analysis. Lab Invest 39:640. Davis JMB (1972) The fibrogenic effects of mineral dusts injected into the pleural cavity of mice. Brit J Exp Pathol 53:190. . Davis JMB, ST Beckett, RE Bolton, K Donaldson. (1980) A comparison of t^e pathological effects in rats of the UICC reference sample of amositdrand chrysotile with those of amosite and chrysotile collected from the fjctory environment. In: Biological Effects of Mineral Fibres, (JC Wagner, Ed.) p. 285, IARC Scientific Publication, Lyon. Desai R, P Hext, RJ Richards. (1975) The prevention of asbestos-induced hemolysis. Life Sci 16:1931. Gunderson HJG (1977) Notes on the estimation of the numerical density of arbitrary profiles: the edge effect. J Microsc 111:219. Harrington JS, AC Allison, DV Badami. (1975) Mineral fibers: chemical, physi cochemical, and biological properties. Adv Pharmacol Chemotherapy 12:291. Hayatdavoudi G, JD Crapo, FJ Miller, JJ O'Neil. (1980) Factors determining degree of inflation in intratracheally fixed rat lungs. J Appl Physiol 48:389. Hilscher W. (1972) Zbl Allg Path 116:413. King EJ, BD Stacy, PF Holt, DM Yates, D Pickle. (1955) The colorimetric deter mination of silicon in the microanalysis of biological material and mineral dust. The Analyst 80:441. McDonald S. (1927) Pulmonary asbestosis. Brit J Med 2:1025. Morgan A. (1979) Fiber dimensions: their significance in the deposition and clearance of inhaled fibrous dust. In Dusts and Disease, (R Lemen and JM Dement, Eds), p. 87, Pathotox Publishers, Park Forest South. Morgan A, JC Evans, RJ Evans, RF Hounam, A Holmes, SG Doyle. (1975) Studies on the deposition of inhaled fibrous material in the respiratory tract of the UCC 007709 78 3 116 rat and its subsequent clearance using radioactive tracer techniques. Environ^Res 10:196. Morgan A, RJ -Talbot, A Holmes. (1978) Significance of fibre length in the clearance of asbestos fibres from the lung. Br J Cancer 35:146. Murray M. (1907) Department committee for compensation for industrial diseases, Cmd. 3495 and 3496, HM Stationery Office, London. Pinkerton KE, AR Brody, DA McLaurin, B Adkins, RW O'Connor, PC Pratt, JD Crapo. Characterization of three types of chrysotile asbestos after aerosoliza- tion. Submitted to Environ Res. , ,, . , . -v Pinltftr-hjM K.6, Vra* fcrei, - -V . ^ ' !- Richards RJ, PM Hext, R Desai, T Tetley, J Hurif, R Presley, KS Dodason. (1977) Chrysotile asbstos: biological reaction potential. In: Inhaled teV Particles IV, (WH Walton, Ed), p. 477, Pergamon Press, Oxford. Sakabe H, K Koshi, H Hayashi. (1971) Inhaled Particles III, (WH Walton, Ed), p. 423, Unwin Bros., Old Woking, Surrey. Scherle W. (1970) A simple method for volumetry of organs in quantitative stereology. Mikroscopie 26:57. Seal RME. (1980) Current views on pathological aspects of asbestosis (tjpe unresolved questions and problems). In: Biological Effects of Mineral Fibres, (JC Wagner, Ed), p. 217, IARC Scientific Publication, Lyon. Seaton A. (1975) Asbestosis. In: Occupational Lung Disease, (KC Morgan, A Seaton, Eds), p. 124, W.B. Saunders, Philadelphia. Suzuki Y. (1974) Interaction of asbestos with alveolar cells. Environ Hlth Prespectives 9:241. Timbrell V. (1965) The inhalation of fibrous dusts. Annals NY Acad Sci 132:255. Timbrel! V. (1970) The inhalation of fibers. In: Pneumoconiosis Proceedings of the International Conference Johannesburg, (HA Shapiro, Ed), p. 3, Oxford Uhiv. Press, London. ' Timbrell V. (1973) Physical factors as etiological mechanisms. In: Biological Effects of Asbestos, (JC Wagner, Ed.), P. 295, IARC Scientific Publication, Lyon. Timbrell V, JW Skidmore. (1968) Int Conf Biol Effects Asbestos, 2nd Dresden, pp. 52-56. Timbrell V, JC Gibson, I Webster. (1968A) Characteristics of the International Union Against Cancer standard reference samples of asbestos. In: Pneumoconiosis Proceedings of the International Conference, Johannesburg 1969, (HA Shapiro, Ed), p. 28, Oxford University Press, London. Timbrell V, AW Hyett, JW Skidmore. (1968) A simple dispenser for generating dust clouds from standard reference sample of asbestos. Ann Occup Hvq 11:273. A ; 7830 UCC 007710 117 Vorwald AJ, RM Durkan, PC Pratt. (1951) Experimental studies of asbestosis. Arch Incest Hyg Occup Med 3:1. Wagner JC. (1965) The sequelae of exposure to asbestos dust. Annals NY Acad Sci 132:691. Wagner JC, G Berry, V Timbrel!. (1973) Mesotheiomata in rats after inocula tion with asbestos and other materials. Br J Cancer 28:173. Wagner JC, G Berry, JW Skidmore, V Timbrell. (1974) The effects of the inha lation of asbestos in rats. Br J Cancer 29:252. Wagner JC, G Berry, JW Skidmore, FD Pooley. (1980) The comparative effects of three chrysotiles by injection and inhalation in rats. In: Biological Effects of Mineral Fibers, (JC Wagner, Ed), p. 363, IARC Scientific Publication,' Lyon. Weibel ER. (1979) Stereological Methods, Vol. 1, Academic Press, London. Woody D, E Woody, JD Crapo. (1980) Determination of the mean caliper diameter of lung nuclei by a method which is independent of shape assumptions* J Microscopy 118:421. -f J A ; 7837 UCC 007711 22 MEAN DAILY AND CUMULATIVE RESPIRABLE AMD TOTAL CHAMBER DUST CONCENTRATION OF EACH CHRYSOTILE BY MASS DETERMINATION % to in <Si LU U L. > JC jc _C 4--H I i 1 J--' cn CD cn < . g cn r-** O U") CVJ G CO CD in gj CO * lo c 2; 1--. 1--. _1 H: ' <O 1-- cx ^ . g h-- h- ro UJ >- gj t-- -J U1 O cx (X g LU Hco CX O cz Li- CO CJ CM +1 LD CO CD cc CM +1 in *--1 1--t *--* CO CO CM +1 in CM O (/> (/> on LU S- c X > XI jc -C -- J 1 1 p-- CD CD Or =c ts a --I ZJ --4 cc m r LD CM CD : 3" 03 CD CJ --1 CQ CO X !T-- LO LU c - _J I-- 1-- zz CM r- co <c < G cx 0: T7 l-M f--' CL. T"" co CD * r-* i-f CO r-H GO LU -H -H -M LU CJ t-- CC zr LO CTl CO LO 0 CC CM ID O >- CJ --* * * __1 Lu CO CO <D 1--^ h-- < LO CX 0: C LU LU --. ro t-- CD >1 G C CQ CJ LO X >- A-- GJ 4- (XJ 03 4-- 0 t--* CJ CJ CJ G =tfc LU 1--1 1--< __ I G v-~ Z3 CJ O *-- * -J -- LO <3T c r- CX J-- O 1-- I-- G LU >- GJ __1 bZ CM O 4--4 < GJ a C h~ G CO LU O LU > -- 1-- <c ; lu G CJ LU G X CD 1-- LO << CX CX I-- --. j-- c_ rr O LO LU LU O cx zz CM 0 f--4 >- CJ CX 4--' 1--k G C LO LU G Z3 G LU *---1 hO LO >a: D c. 1 CD rx CO CM CO r--1 +1 ID r^ un X xr 1 CD CM ^r in CO 00 0 +1 CO CM CO CD c r^ro C GJ un U 1 CD 10 CO CO 00 4--* (/) 5JZ 1 OY r^. 00 LO CD r--4 *--t C3 r~-4 1--4 CM CM +1 +* CD CD CO O pH r-H r-H SO 4- CD CO CM 1 in co CM 11 c A \A -C -tu c 0 CO X 0 4- LO in t 10 LO II l/i 1 CD CD --4 CD CO r-- in L-C 1 CD LO co CM ID r"*4 LO CO 10 f-H +1 +1 CM 0 CO CD * CO CD A G uo +1 c 0 0 s0 l/> c 0 r** -M m S^ro CJ -1= 0^ uw *. 1_/1 tu-j -3 " ^ V) , 1/) 5 -Q Q_ EX 2 flu flu aj i_ 1 ^ V) QJ G^J 3^ ro > U, ^ CL m *n GJ ^ i HU >> CQ CJ &CJ 4- G 4- >> O r 4t5 CM t 7 i"7 8^r C <u <v > -u* O CJ CL tfl CJ s~ CJ XT 4-> M C* GJ CD in c <u -rU u- Cl CJ st- a cl in 0 c C in *-- ra rMo S s- X +- 0 cV GJ YO U ic CJ 0c O CJ CJ) u> m r-- 3 4-- -c CJ SV JO CJ _o H* n5 X CJ 0 x: -i--> -a c in ctt S- 3 ci 0 .-- JC _Q fO 4s- O r~ a. s- m cu CJ JO S- 3 4- C O CJ in jC -M 4J C 4) in CJ CJ t- -r3 4- in its m OJ c 0 GJ 4U > ^0 1- L. u hj <T3 C p-- aj 3 GJ C 3O GJ O 4fc UCC 007712 *T r~ ?i i r-1 5 n "I A n 4* i t ii 23 TABLE 2 I MEAN BODY WEIGHT AND FIXED LUNG VOLUMES _ ' Body weight (grams) Exposure times 3 months 12 months 12 mo. + 12 mo. air CONTROL 2S913 3773 39111 MALES COALINGA UICC B 3065 36712 44611* 301+10 375+5 4056 JEFFREY 2816 337+14*# 3799 Fixed lung volumes (cm3) Exposure times 3 months 12 months 12 mo. + 12 mo. air 8.60+.31 10.58i.09 12.67.74 9.69.30* 9.44.38 ll.70i.27 9.91,32* 10.21i.12' 12.39.17 9.881.25* 12.76.33* 17.24|1.35*# Body weight (grams) Exposure times 3 months 12 months 12 mo. + 12 mo. air CONTROL FEMALES COALINGA UICC B 182 5 218+8 2987 167+5* 205+7 286+13 1643* 20516 28412 J JEFFREY 163i5* 1986 271+10 Fixed lung volumes Exposure times 3 months 12 months 12 mo. + 12 mo. air 7.481.10 7.80+.27 9.39+.40 6.781.24 6.96+.06* 9.10+.13 7.061.17 6.891.11* 9.031.23 7.101.33 9.35i.06*= 11.25i.48*= All data are mean i SEM, n = 4 *p < 0.05 for comparisons to the age-matched controls. #p < 0.05 for comparisons to the group exposed to Jeffrey chrysotile to both of the other age-matched fiber exposure groups. UCC 007713 A : 783 J 24 A R S O LU TE VO LU M ES O F A LV E O LA R A IR S P A C E , C A P IL L A R Y LU M EN AND A LV E O LA R T IS S U E D U R IN G AM D M IL L tiW U K '. A S B E S T O S IN H A L A T IO N * on n Lbi rn r-K O vo cm ce ffT/LC v c <0 ir <-- m> cm <- r vo rs. d VO CM CM O') Ifl <r m^| d c GO vO oo ^o m ^ CO vn r --' CM CJ on U3 VQ d nnn on to* to- cr> t- c o mnc ft> ojn^IOOV o oo o oi ftj *T Rv CC CM lO Ci lO un VO lO d to o mm acc n a, **- vO VO VO --' vn d -- ID n r-l t m rs i/i n vo rs. is. to O vO O >--i CM nn<M cc d on aO vo co Oon Cojaj- o vn vo CM 1/1 O O O CM ' rvi *-- + +. *i do --< CTY TO" -- o> --i c GO -- o-> rs, r\j d ^ VO CM cm rs, d vo vc TO to o m to* o An l/HO ts. vc o vO vO CO c rsCOrs f=f Lft CM d i/nr is eft TO- vn Cl U7i CM vn rs. d CM O CM TO r to i-- C .c '--. ^ *4 .*- > W -- 4J -- c = g m e c: g - - C ft ft TO C ft C Co = =: -- e: = s C i: a j= uncsjcM^ncMM virnN TO ~~ *f-I LO r- CC Cm vo vo -- on cm + 1 * *! ohk in o d Oi rv co c/nn cm cm co -- CO vo -- CM CO 4-1 *! + 1/1 cc o oino rs -- rs moo ten * d rs to CO -- TO CM Cm -- Cm U v( o m cc<-) (fl is Oi <M fs* CV to toWON mm vo d n cm n ro Cm cm O O CM *3- VO CM <M -*-* on 91 01 CO -- TO Oi C ncstPi rs. on to oDOr cc rs in. cm cm on t O < H; 0-0 0 c -- o u 1/5 co ^ h-- VO UJ s co ft? to 4- < ^ OlU9 il cl co co hcvjh to to CO vo CO rs V CM O vo cm co cm O vo ^rt-H ^ 00 d 00 O vo n on m on VD VO ^Oin cm ro vO CO CM TO d *T -- --* LO VC . Cl +- f ' & Cw JO lit O TO A/> 1/1 M 0> 0; 4 f ^ C*m */ O o. evt 33C a OC3 1f-f w94|- *J t 2^ e o &' & -o CM *- C 4-1 c e & _ L. ftj *j CVl tl ^ f-- tn C C -t) c -* *- * JO VJ oi < L*_ O O CO LlJ ZZ Z* C <LJ < --_J <-J u- < OOO vn tnTO O ^ to cm on m OI Lfl Ps CO CC d ^fvjfl WOC vo Lfl N cm O cn CM ~ in ^ VO -- CO CM CO rs cC VO 4VO CO co cm d Office <*> m vo cm o nmo --< -- VO r ^ ^IHUI --- TO m TO +1 -I o LO vD TO TO TO --c Q i ^1 Ui Vi -- -ft -- y* v* ^ -- ftf^OSSE-- = TO = ft -- +- ft >mCMCM Q- m CNJ CM JZ W G IB W d Otn fUt' ft; >, w >, ns <j ft *J U -- 1- ft 3 <9 ft 4- C Art k. U C TO ft)X I-- CM ft re o i/i &p c Cl c ft ft^^ ft CL^. - UX 3 *- dO ft C ddto 5- C*ft -- c C ft J= ft- ft 3 *4 3 -- p-- ft C C ^ u JZ OW 6 Ft! +J c ft m *> ft fCt vTO. E- -ff-tt ffTOtt tA V> cc cc .X s: ft w u *** ' U n Q (A ft4l i/i cv e. G- c -j ft Gu^>s TO = E3Os ft o c u l. ft- 1 ft ft M c ff CM O C *C ft ft ft -- eft O ft c O TO TO *- I VV V ft ft i 3 3: OL Qt C. vO V/ UCC 007714 A '; 7 8 4- C [ [ .? ~l r i r L. 4- r 4 u. r1 t. r1 TABLE 4 I ABSOLUTE VOLUMES OF THE ALVEOLAR EPITHELIUM r DURING AND FOLLOWING ASBESTOS INHALATION* MALES CONTROL COALINGA UICC B Total volume {mm3/both lungs) Type I epithelium 3 months 12 months 12 mo + 12 mo air 81 4- 6 83 + 3 84 + 8 112 3* 113 + 0* L. 114 -1- 15 93 8 113 12* 116 7 Type II epithelium 3 months 12 months 12 mo + 12 mo air 36 + 9 30 z. 4 28 9 132 4- 14* 82 + 4 62 -i- 15 79 4~ 12 100 - 14* 96 21* JEFFREY 95 + 2 155 4- 7*# 121 + 22 123 + 27* 156 + 37* 73 f 26$ Total volume (mm3/both lungs) Type I epithelium 3 months 12 months 12 mo + 12 mo air Type II epithelium 3 months 12 months 12 mo + 12 mo air CONTROL FEMALES COALINGA UICC B t JEFFREY 78 + 7 57 4- 7 66 -r 6 25 4- 1 20 + 3 18 4- 5 80 t 6 64 4- 8 66 + 8 68 + 6* 56 4- 11* 29 4- 9 63 M. 5 67 8 79 4- 5 79 5 133 9*r 87 + 8 66 4- 7* 70 z 9* 41 11 67 + 5* 99 Jm. 17* 70 4. 6*f t All data mean SEM, n = 4 for each qrouo and time period. * p < 0.05 when comparing the age-matched control values using Duncan's multiple comparison test after first testing for significance using a one-way ANOVA. # p < 0.05 when comparing groups exposed to Jeffrey chrysotile with all other asbestos-exposed groups of the same time period. p <0.05 when comparing a treatment group to its corresponding treatment group at the preceeding time point. UCC 007715 A, ,7841 26 TABLE 5 ^ABSOLUTE VOLUMES OF THE ALVEOLAR INTERSTITIUM DURING AMD FOLLOWING ASBESTOS INHALATIONt CONTROL Total volume (mm3/both lungs) Cellular interstitium 3 months 12 months 12 mo + 12 mo air 67 5 5/ x 8 57 - 7 Noncellular interstitium 3 months 12 months 12 mo + 12 mo air 126 X 16 149 X 13 178 20 CONTROL Total volume (mm3/both lungs) Cellular interstitium 3 months 12 months 12 mo + 12 mo air 59 x 7 49 X 4 56 X 6 Noncellular interstitium 3 months 12 months 12 mo + 12 mo air 80 X 10 109 X 11 153 X 8 MALES COALINGA UICC B JEFFREY 124 + 2* 77 3 81 21 104 5* 80 * 16 127 X 2* 131 + 15* 145 X 16* 168 X 54* 195 X 22* 239 X 15* 287 4- 38 177 X 3* 230 X 25* 439 + 25* 163 + 13 362 X 27*# 422 + 80* f FEMALES COAL INGA UICC B i JEFFREY 97 X 9 47 X 6 53 X 9 82 X 5 138 + 9 168 X 15 72 + 10 62 X 10 46 + 4 140 + 24* 138 X 11*# 107 16*# 134 X 11* 146 X 10 211 X 28* 98 X 5 265 X 21*# 282 X 22*# t All data mean SEM, n = 4 for each group and time period. * p < 0.05 when comparing the age-matched control values using Duncan's multiple comparison test after first testing for significance usina a one-wav ANOVA. # p < 0.05 when comparing groups exposed to Jeffrey chrysotile with all other asbestos-exposed groups of the same time period. 5 p < 0.05 when comparing one treatment group to its corresponding treatment group of the preceeding time point. UCC 007716 A ; 7 8 4 2 27 J J J LU (T> O 00 o r^ CO >j CD c a; E cnzn. o_ +i L >r" *- 4-> S- j occ 03 o to <SI <LOo +TM qj CO a; Ul .O LO 3 -c 5 o +-> c in 4-> J +tO- "O p03 r--" u <u at m in u J LO * CM 00 * * * CT *d"m "O +(3O-/>>J aaXQJ s0LaO,>.t*rO-r-" "O tn QJ M LU <D J= J LU CJ rD-*-- =: E 3. w h . o o LU +i o r^. +1 LO CNJ to r^ +1 CM CO CO l-i r^ "O to > *M c to 5- 'r* O +-> TJ ~a tD C 1-- c CD QJ o fC CL i- cn QJ __1 LU LO cn CD +> c r-- i- to J CJ O a: on C -J oo CD s- L- 3 r-- 1.D E c QJ i-- _i LO <3- o to XC to O h-- CO CO O L. Cj -tU CD LU c K--* av > QC J w x: c -a: co t-- LO ro OZ to CM CO 1-H to i--i t- O m CD +-> 4- 4-> CJ -C -o S- QJ o LU LO *3 Q * J CO X LU E 31 LU O u LU CO t--t t*-* CO 3: c H Li_ h- J U. O O LU HCSi LO CS cC __) t--1 LO il * r-. r-^ to l-- a + 1 Ol J <LsU: qs > a. CM __i >- CO c h- O a *0- E 'E cj / to J3 3 f-- CM S o i-- 3 C o Stc Cr--D r_ 2 /--CJ 4-> CD L. QJ LD S- CD 4-5 CM 3C QJ CO 2 ct- -- ro to co: ~CoJ i "O CD CJ C CJ _ tO E -- t- *r- cm O -L rt) +J r-- O QJ O -Q"C r-- S- _J *- O C U OJ 0 O r- QJ 3 _C LU X CJ to C 4-> j Q- J-- >--* 2 --I O rO *D 0J x: S- cj o CM 4-J CDXZ C a C VD fijf- < <_J o <~ </> r-> O, c a oj p-- r*"l c ai = to Cl CJ 4-^ J O LU LO LU Q. CM co CM X QJ r r--H cc CM * ff to CO CM * *p ro -- 4- CJ "O r- O o -C 3 u U r- en to -- CJ 4U J co r > cc co oc/1 Vo- . <D cd *1 o" 3c to -C O "C "3 C. OJ in Qj 4- c in /i a; O S- rO CJ 3 ---. ul CM o O J t~o L. o 4- QJ O 4- +--> 0J CM "D *r- J CJ 4-J -f- jC a o co l/l 4-> =o _1 OJ to L. o xo oc to 1 u J JT--^_ c E CJ cn o Q LU h-- LO _C t-- rC--J C 4*J CO to Lt*> r= CJ CM L- 4- r--1 _C ID r-- L. O >-, + 4* CJ s- un "O 4- CD 0J 4,--- oO CJ iO O *3 E Cl XO < h* < C3 Q LU --1 C o CJ to CJ i/) O "O C. 3 -- O T3 "O C -- cj C c Q U O S- G- O 4-> p" 4J x: i- r-" 2 CJ tn ai o CJ -C r-- r-- 4- CJ h-- C CJ 1_ 2 LO O c XJ i-tU 4-J .C CJ O "O O o CJ i/i o u r" >>'- V OJ S- L- L. 7T -3 a c Q, 1-- ID CL 4- tD o CM to +- CM CJ t-> o O * -k J CJ --- G 'A ; 78?: UCC 007717 TABLE 7 TOTAL CELL NUMBER OF THE MAJOR PARENCHYMAL LUNG CELLS IN RATS f o r s ig n ific a n c e u s in g a one-w ay AUOVA, cm ^3 c* so c** r- rn CM pfMH rTr CSJ --Cr> of'"'' cc' tncsin CO I**-. O' r-. ^ vo no -- un *i -r-> +i n^trs. --. Os r -- Or-.ro vfi vo {\jp-cg CM 'O c (sj iT- r> c in ^ O fNJ -- K =Mi <\| CO CO CCM N-- C-M & C. CJ 3U u CJ Q. E &~ C r-~ CO CM LT> M Ps tj CuOO ^ U"i LD Os ^ Y--i o CM CM CM CM lOinn +i +: fi O' l--. vC r* &3 **- o. +-* <U U p-- i O' 3 +- EO C c/i m iu 3 CO Lu <C 3< O o o o; vo ro |\jCX--o --I CO -- +1 *1 *! p^. co CM ITT f- to r-K * k \o n co ^ so +< *1 -r Vfi CO O' CM O O CO iO SO VOVO H+< w)m+i to co <o il) C (Vi VO LO iO CO CM O' ininin r-* o CM CM CM mMi O' CO fM r. kC> id ** * O' - lOr-lvO +1 +1 +1 CMC* r-- --* MHO U CM CO (fi 1--4 --< +h *>) *1 r-- co O O"0 *-- -- CM CM Csj ^ tfs >i +i *i *+ w in ^ si cm m r-s CM Cm ^ vo +i .* *i Csj iO fO CM PM ^ n m si CM CM CM VO CM fO CM CO X !** --I ^ SO CO CM CM n^ hCC* ^LD ncsicv h. ^03 CO CO CO 2 O' 4p C ^ *o ** cC <* a. 5^ *n W. d> C w M fi. tt1 ClOM OCM' TC"M** P+-1 C*M1 O-fl X ^ f* CO - & &l a3 J= o W -M O' 2 U &) O O' 07 IQ *r U, |Q c E o E S C7 ^ IA (A w -Q JC JZ .= 3 c** e oc c -- s c- fe W pr> <M CM Qj c. + *j t/i */r t/s ,L -^= -S 4.-> (Q C C C -- o o c EE Qj > e.o.C.M CM /C c c -gig CJ > (O CM CM W CO CM CM O' C C C c o Q x: c. t, (/i w i/* t. o*j J*=. <Q C C C -:$ss c e e = > 0*1 CM CM l/l B2 flS o o mm oo do c C. . -7 I/ UCC 007718 29 TABLE 8 t HARMONIC MEAN TISSUE THICKNESS OF THE AIR-BLOOD BARRIER DURING AND FOLLOWING ASBESTOS INHALATION Air to plasma 3 months 12 months 12 months + 12 mo air MALES .379.030 .397+.016 ,367.004 .478.02Q*# .336.009 .440.016* ,485.017* ,668.044*# .368.013 .499+.027* .580.023* ,707.044*# 3 months 12 months 12 months + 12 mo air .339+.006 318.005 ,373.019 FEMALES ,408.005* .402.017* .402,016* ,442.014* .474.008* .629^.030*# .448.023* .537.G25* .599*.018* * p < 0.05 when comparing to the age-matched control values using Duncan's multiple comparison test after first testing for significance using a one-way ANOVA. # p < 0.05 when comparing groups exposed to Jeffrey chrysotile with all other exposure groups. p < 0.05 when comparing one treatment group to its corresponding treatment group of the preceeding time point. A i 784b UCC 007719 30 TABLE 9 SILICA CONTENT IN THE LUNGS OF RATS EXPOSED TO CHRYSOTILE ASBESTOS GROUP Controls 3+12 months N 17 UGM SILICA/BOTH LUNGS 156 32 - Coalinga chrysotile 3 months 12 months UICC B chrysotile 3 months 12 months Jeffrey chrysotile 3 months 12 months 6 8 6 8 8 8 610 54* 57* * 48* 569 79* 701 60* - 741 46* 1127 66*# All data are mean SEM. 1 * p < 0.05 when compared to the controls. # p < 0.05 when compared to all other exposure groups. -* r' l f r* J* ** K r* A ' "" fart " | 'L 1 UCC 007720 ri 1 t T MALES FEMALES 3 MONTHS EXPOSURE Epithet turn Interstitium HI Endothelium 31 CONT COAL UICC JEFF 12 MONTHS EXPOSURE TISSUE VOLUME (m m 3) CONT COAL UICC JEFF CONT COAL UICC JEFF lOOOr 500 12 MONTHS EXPOSURE* 12 MO. AIR % m m CONT COAL UICC JEFF CONT COAL UICC JEFF Figure 1: ; 7847 Changes in the volume of parenchymal lung tissue follow ing inhalation of three types of chrysotile asbestos {CONT - controls, COAL = Coalinga, UICC = UICC B, JEFF = Jeffrey). Each bar represents the mean of 4 animals and is subdivided into t^e epithelial, inter stitial and endothelial compart nts. *p < 0.05 for comparison to the age-matched "rols. UCC 007721 32 PERCENT CHANGE FROM AGE MATCHED CONTROLS Figure 2: Percent change compared to age-matched controls in the number of alveolar type I (A) and alveolar type II (B) cells in the lungs of rats exposed to Coalinga* UICC B and Jeffrey chrysotile. Each point represents the pooled mean of 4 males and 4 females. UCC 007722 A t ! i ! i B } -y i i C A ; 7 ii 4 J Figure 3: Percent change compared to age-matc! ?d controls in the number (A) and volume 'B) of inters tial cells and volume of interstitial matrix :) in the 1 gs of rats exposed to Coalinga, UICC B and Je rey chryso e. Each point represents thL pooled ' of 4 mal UCC 007723 nd 4 fen,ales I Figure 4: Percent change compared to age-matched controls in the number of alveolar macrophages in the lungs of rats exposed to Coalinga, UICC B and Jeffrey chrysotile. Each point represents the pooled mean of 4 males and 4 females. A "7 ,( ? ^ / u 0 'j UCC 007724 I 3 MONTHS EXPOSURE ^ Epithelium 2- O Interstitium H Endothelium lJ r 35 =1 UICC JEFF ARITHMETIC MEAN TISSUE THICKNESS 12 MONTHS EXPOSURE I ** 1 J 3 ] CONT COAL UICC JEFF ] ] 1 J 1 1 Figure 5: Arithmetic mean thickness of alveolar tissue in control and 1 chrysotile dusted rat lungs. {CONT = control, COAL = A i 7 3 5 1 Coalinga, UICC = UICC B, JEFF = Jeffrey). Each bar represents the pooled mean of 4 male and 4 female rats. 1 *p < 0.05 fe*' comparison to the age-matched controls. UCC 007725