Sites of particle retention and lung tissue responses to chronically inhaled diesel exhaust and coal dust in rats and cynomolgus monkeys.

The usefulness of pulmonary carcinogenicity data from rats exposed to high concentrations of particles for quantitatively predicting lung cancer risk in humans exposed to much lower environmental or occupational concentrations has been questioned. The results of several chronic inhalation bioassays of poorly soluble, nonfibrous particles have suggested that rats may be more prone than other rodent species to develop persistent pulmonary epithelial hyperplasia, metaplasia, and tumors in response to the accumulation of inhaled particles. In addition, rats and primates differ in their pulmonary anatomy and rate of particle clearance from the lung. This paper reviews results of recent Lovelace Respiratory Research Institute (Albuquerque, NM) investigations that directly compared the anatomical patterns of particle retention and the lung tissue responses of rats and monkeys exposed chronically to high occupational concentrations of poorly soluble particles. Lung sections from male cynomolgus monkeys and F344 rats exposed 7 hr/day, 5 days/week for 24 months to filtered ambient air, diesel exhaust (2 mg soot/m3), coal dust (2 mg respirable particulate material/m3), or diesel exhaust and coal dust combined (1 mg soot and 1 mg respirable coal dust/m3) were obtained from a study conducted at the U.S. National Institute for Occupational Safety and Health and examined histopathologically and morphometrically. Within each species, the sites of particle retention and lung tissue responses were the same for diesel soot, coal dust, and combined material. Rats retained a significantly greater portion of the particulate material in the lumens of alveolar ducts and alveoli than monkeys. Conversely, monkeys retained a significantly greater portion of the particulate material in the interstitium than rats. Rats, but not monkeys, had significant alveolar epithelial hyperplastic, inflammatory, and septal fibrotic responses to the retained particles. These results suggest that anatomic patterns of particle retention and lung tissue reactions in rats may not be predictive of retention patterns and tissue responses in primates that inhale poorly soluble particles at concentrations representing high occupational exposures.


Introduction
Several chronic inhalation bioassays of pulmonary epithelial hyperplasia, metaplasia, poorly soluble, nonfibrous particles in rats, and tumors in response to chronically Syrian hamsters, and mice have shown that inhaled particles at high exposure concenrats are generally more sensitive than mice trations (1). In addition, there is limited or hamsters to the induction of persistent evidence for the carcinogenicity of coal dust in rats (2), yet coal dust exposure alone does not significantly increase the risk for lung cancer in humans (3).
The rate of particle clearance from the alveolar region differs among species. Rats and mice clear particles from the lung relatively quickly, whereas monkeys and humans clear particles more slowly (4). Anatomical differences between the fasterclearing and slower-clearing species could affect particle deposition, retention, and clearance. Because mice and rats lack respiratory bronchioles, they have simple acini. Macaque monkeys and humans have similar numbers of respiratory bronchiole generations between the terminal bronchiole and alveolar ducts (5,6), and have larger alveoli and alveolar ducts than rats (7). Therefore, monkeys and humans have more complex, larger acini than rats. The amount of alveolar septal connective tissue in the lung also differs; small rodents have less and primates more (8)(9)(10). Lastly, rats have thin pleura and relatively few pleural lymphatics; nonhuman primates have thicker pleura with more lymphatics than rat pleura; and humans have thick pleura and relatively abundant pleural lymphatics (11)(12)(13)(14).
The scientific and regulatory communities are currently debating the usefulness of pulmonary carcinogenicity data from rats exposed to high concentrations of particles for the prediction of lung cancer risk in humans exposed to much lower environmental or occupational concentrations. The purpose of the investigation (15) summarized in this paper was to directly compare patterns of particle retention and lung tissue responses of rats and monkeys exposed chronically to diesel exhaust, coal dust, or diesel exhaust combined with coal dust, at exposure concentrations equivalent to the current permissible airborne concentration in underground coal mines in the United States (2 mg respirable particulate/m3).

Materials and Methods
Lung tissues examined were from a 2-year bioassay by Lewis et al. (16) of cynomolgus monkeys and F344 rats exposed to filtered, conditioned, ambient air (FA) as controls, or to one of three exposures to particulate material: diluted whole diesel exhaust (DE) at a target particle concentration of 2 mg/m3; coal dust (CD) aerosolized in air at a target respirable (particles < 7 pm) particle concentration of 2 mg/m3; or a combination of 1 mg/m3 diesel soot with the same gaseous or vapor airborne Environmental Health Perspectives * Vol 105, Supplement 5 * September 1997 concentrations as in the diesel exhaust atmosphere and 1 mg/m3 respirable coal dust (DECD). Male cynomolgus (Macaca fascicularis) monkeys (4-5 kg when obtained) and male F344 rats (8-10 weeks of age when exposures initiated) were exposed in whole-body chambers 7 hr/day, 5 days/week, for 24 months, and sacrificed 1 day after the end of the exposure. The animal care, exposures and exposure atmospheres, and results of many parameters examined have been described in detail (16).
Hematoxylin and eosin-stained lung sections from 14 FA-, 14 CD-, 15 DE-, and 15 DECD-exposed monkeys and from 15 rats in each group were obtained from the U.S. National Institute for Occupational Safety and Health and examined histopathologically. A standard lesion terminology (17) was used for both species, and the severity of lesions was scored.
Lungs that had been inflated at a constant hydrostatic pressure of 20 to 25 cm with formalin (monkeys) or 20 cm with Karnovsky's fixative (rats) from eight animals per exposure group from each species were used for morphometry. Digitized images of the lung were captured and projected onto a computer monitor screen at a final magnification of x 1280. A 64-point grid (Stereology Toolbox, Davis, CA) was superimposed over each image, and the number of points hitting particulate material and the location of each point were recorded.
The volume percentage of the total particulate material in the lumens of alveoli and alveolar ducts and in interstitial compartments was calculated for each animal from the number of points hitting particulate material in these compartments divided by the total number of points hitting particulate material x 100%. These data are estimates of the anatomic distribution of the retained particulate material.
The criterion for statistical significance was p < 0.05 for all analyses. The pathology severity scores were analyzed using polychotomous logistic regression (18). The estimates of the volume percentages of the total particulate material in the lumens of alveoli and alveolar ducts and in interstitial compartments were assumed to be binomially distributed, and logistic regression was used for the statistical analysis. Variation among animals was accounted for in the analysis by using a scaling factor greater than one to multiply the sampling variance (18).

Histopathology
The lungs of DE-, CD-, and DECDexposed rats exhibited the same histopathology. Overall, the particles were observed mainly within multifocal collections of alveolar macrophage(s) (AM). Most commonly these macrophage aggregates were located within centriacinar alveoli, but the alveoli immediately adjacent to the pleura was another common location. A lesser portion of the retained particles was located in the interstitium.
The characteristic tissue response to densely aggregated AM was alveolar epithelial hyperplasia. Other responses were particle-associated inflammation and a local septal fibrotic reaction.
Most of the control monkey lungs contained nonsoot, noncoal dust particulate material. This material consisted of endogenous pigments, materials inhaled and retained during the lifetime of the animal, and, in some monkeys, debris from pulmonary mites. The incidence of pulmonary acariasis was the same across all exposure groups. Soot and coal dust were also present in exposed monkey lungs. The predominant sites of particle retention and the characteristic tissue responses were the same in DE-, CD-, and DECD-exposed monkeys. The retained partides had a multifocal distribution in the monkey lungs, but, in contrast to the rat, more of the particulate material was located in the interstitium than in the alveoli. Most commonly, interstitial particle-laden macrophages were a) within the alveolar septa; b) within interstitium of respiratory bronchioles; c) within the adventitia and lymphatic capillaries surrounding arterioles and veins within the pulmonary parenchyma; and d) in the pleura. The interstitial particulate material did not seem to elicit a tissue response. The portion of the particulate material within intralumenal collections of AM was smaller than in rats, and the aggregates of particle-laden macrophages elicited much less of a tissue response in the monkeys than in the rats. Tables 1 and 2 show the incidences and average severity scores of key categories of lesions in rats and monkeys, respectively. Rats had a significantly greater alveolar epithelial hyperplastic response to particle exposure than monkeys (p<0.001). Alveolar epithelial hyperplasia was significantly greater in particle exposed than in control rats (p< 0.00 1), but there was no difference in the hyperplastic response due to type of particle exposure (p = 0.3). The Table 1. Incidences and average severity scores of key particle associated or possibly particle-associateda histopathologic diagnoses in rats 7 (1.1) 4 (1.0) 4 (1.0) 8Histopathologic diagnoses whose incidence or severity suggested a possible particle association in any exposure group in either rats or monkeys. bRats were exposed to FA as controls or DE, CD, or DECD at target concentrations of 2-mg respirable particulate material/m.3 cFirst number is the number of rats with the diagnosis. The number in parenthesis is the average severity score calculated as sum of severity scores/number of rats with the diagnosis. Lesions were scored as 1, slight; 2, minimal; 3, mild; 4, moderate; 5, marked. Table 2. Incidences and average severity scores of key particle associated or possibly particle-associateda histopathologic diagnoses in monkeys.
Exposure groupb Diagnosis Filtered air Diesel exhaust Coal dust DECD Number of monkeys examined 14 15 14 15 Alveolar epithelial hyperplasia 2 (1. 8Histopathologic diagnoses whose evidence or severity suggested a possible particle association in any exposure group in either rats or monkeys. bMonkeys were exposed to FA as controls or DE, CD, or a DECD at target concentrations of 2-mg respirable particulate material/mi3. cFirst number is the number of monkeys with the diagnosis.
The number in parenthesis is the average severity score calculated as sum of severity scores/number of rats with the diagnosis. Lesions were scored as 1, slight; 2, minimal; 3, mild; 4, moderate; 5, marked.
Environmental Health Perspectives * Vol i 05, Supplement 5 * September 1997 monkeys did not have a significant alveolar epithelial hyperplastic response to particle exposure (p = 0.4). Rats had a significantly greater inflammatory response to particles than monkeys (p = 0.02). Particle-exposed rats had significantly greater inflammation than control rats (p< 0.00 1). There was no significant difference in the inflammatory response due to the type of particle exposure in rats (p = 0.5). In monkeys, there was no significant effect of particle exposure (p = 0.1).
The septal fibrotic reaction in rats occurred as collagen increased within alveolar septa in foci of AM aggregation, alveolar epithelial hyperplasia, and particleassociated inflammation. The fibrosis did not occur independently of these other reactions. In monkeys, a septal fibrotic reaction was not associated with diesel soot or CD particles. Rats had a significantly greater septal fibrotic reaction to particles than monkeys (p = 0.006). In rats, this reaction was significantly greater in particle exposed than in control rats (p=0.001). There was no significant difference in the septal fibrotic reaction due to the type of particle exposure in rats (p = 0.15). There was less septal fibrotic reaction in particle exposed than in control monkeys (p = 0.02).

Morphometry
Approximately 73 and 43% of the particulate material in exposed rats and monkeys, respectively, was in the lumens of alveoli and 80] T alveolar ducts (Figu 27 and 52% of the exposed rats and n were in the inters Figure 1, particles in and connective tissi the interstitial part Interspecies compar cantly greater volu total particulate mat alveoli and alveolar than in exposed n Conversely, a signif percentage of the to was in the interstitiu than in exposed rat each species, there w ences between DE, mals for the volume particulate material i and alveolar ducts or

Discussion
The morphometry s] between rats and mc nant sites of particle than 70% of the pa retained in the lume. lar ducts. In monkey rial was almost equ these lumenal compz stitium, but more of be in the interstitiun the predominant sit did not vary by ex Approximately present findings, combined with the known particulate material in anatomical differences and data showing ionkeys, respectively, that primates clearly deposit particles more titium. As shown in slowly than rats (4), suggest that a greater the pleural lymphatics proportion of particles or particle-laden ue were grouped with macrophages penetrate the airway epithe--icles for this analysis. lium and enter the interstitium in primates rison showed a signifithan in rats and that the particles in the me percentage of the interstitium may be cleared more slowly terial in the lumens of than those cleared via mucociliary action. If ducts in exposed rats the presence of respiratory bronchioles, the nonkeys (p < 0.001). amount of interstitial and pleural tissue, icantly greater volume and the thickness of the alveolar septa are tal particulate material important determinants of the sites of partiim of exposed monkeys cle retention, the differences between rats ts (p< 0.001). Within and humans might be even greater than the ,ere no statistical differ-differences between rats and monkeys. CD, and DECD ani-Human lungs have more extensive interlobpercentage of the total ular septa, thicker pleura, and wider interin the lumens of alveoli stitial spaces in the alveolar septa than in the interstitium. monkeys (12,19,20). The response to particles, including alveolar epithelial hyperplasia, inflammahowed clear differences tion, and focal septal fibrosis, was signifi-)nkeys in the predomicantly greater in rats than monkeys. retention. In rats, more Epithelial hyperplasia concomitant with irticulate material was the aggregation of particle-laden macrons of alveoli and alveo-phages in alveolar lumens is a characteristic rs, the particulate materesponse to many poorly soluble particles Lally divided between in the rat lung, both at exposure concentraartments and the intertions that result in lung tumors (17,(21)(22)(23) the material tended to and at exposure concentrations below n. Within each species, those resulting in lung tumors (21)(22)(23)(24). :e of particle retention Hyperplasia of the surrounding epithelium posure material. The in response to accumulation of particulate material in focal aggregates of AM was not characteristic of the response to diesel soot or CD in monkeys in this investigation Rat LU (15), nor is it characteristic of coal workers' RatIP pneumoconiosis (3,25,26), silicosis (27), -Monkey LU or talc pneumoconiosis (28) in humans.

MonkeylP
If human lungs respond to poorly soluble particles in a manner more like monkey lungs than rat lungs, perhaps the pulmonary response of rats to particles may not be predictive of the response in human lungs at concentrations representing high occupational exposures. Consideration should also be given to the question of whether carcinogenicity data from rats exposed to high concentrations of particles, which greatly exceed expected human exposure concentrations, should be used to quantitatively predict carcinogenicity in humans exposed at lower rates. Particle-CD DECD induced inflammatory and epithelial proliferative responses that seem critical to carcinogenicity in rats may not occur in olar ducts (LU) versus intersti-primate lungs exposed at environmental or exposed to DE, CD, or DECD. occupational concentrations.