Kristen J. Nikula

Comparative Pulmonary Toxicities and Carcinogenicities of Chronically Inhaled Diesel Exhaust and Carbon Black in F344 Rats

Abstract Diesel exhaust (DE) is a known pulmonary carcinogen in rats, and the carcinogenic response is known to require the presence of soot. Many estimates of human lung cancer risk from inhaled DE have been developed from rat bioassay data or from the comparative mutagenic potencies of DE soot extract and known human chemical carcinogens. To explore the importance of the DE soot-associated organic compounds in the lung tumor response of rats, male and female F344 rats were exposed chroni cally to diluted whole DE or aerosolized carbon black (CB) 16 hr/day, 5 days/week at target particle concentrations of 2.5 mg/m3 (LDE, LCB) or 6.5 mg/m3 (HDE, HCB) or to filtered air. The CB served as a surrogate for the elemental carbon matrix of DE soot. Considering both the mass fraction of solvent-extractable matter and its mutagenicity in the Ames Salmonella assay, the mutagenicity in revertants per unit particle mass of the CB was three orders of magnitude less than that of the DE soot. Both DE soot and CB particles accumulated progressively in the lungs of exposed rats, but the rate of accumulation was higher for DE soot. In general, DE and CB caused similar, dose-related, nonneoplastic lesions. CB and DE caused significant, exposure concentration-related increases, of similar magnitudes, in the incidences and prevalences of the same types of malignant and benign lung neoplasms in female rats. The incidences of neoplasms were much lower in males than females, and the mci dences were slightly higher among DE- than CB-exposed males. Survival was shortened in the CB-exposed males, and the short ened survival may have suppressed the expression of carcinoge nicity as measured by crude incidence. Logistic regression mod eling did not demonstrate significant differences between the carcinogenic potencies of CB and DE in either gender. The re sults suggest that the organic fraction of DE may not play an important role in the carcinogenicity of DE in rats.

Lung Tissue Responses and Sites of Particle Retention Differ between Rats and Cynomolgus Monkeys Exposed Chronically to Diesel Exhaust and Coal Dust

Several chronic inhalation bioassays of poorly soluble, nonfibrous particles have resulted in an increased incidence of lung tumors in rats, no increase in lung tumors in Syrian hamsters, and inconsistent results in mice. These results have raised concerns that rats may be more prone than other species to develop persistent pulmonary epithelial hyperplasia, metaplasia, and tumors in response to the accumulation of inhaled particles. In addition, particle deposition and the rate of particle clearance from the lung differ between rats and primates, as does the anatomy of the centriacinar region. For these reasons, 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 purpose of this investigation was to directly compare 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 examined histopathologically. The relative volume density of particulate material and the volume percentage of the total particulate material in defined pulmonary compartments were determined morphometrically to assess the relative amount and the anatomic distribution of retained particulate material. In all groups, relatively more particulate material was retained in monkey than in rat lungs. After adjustment for differences between rat and monkey controls, the coal dust- and the combined diesel exhaust and coal dust-exposed monkeys retained more particulate material than the coal dust- and the combined diesel exhaust and coal dust-exposed rats, respectively. There was no significant difference in the relative amount of retained particulate material between diesel exhaust-exposed monkeys and rats. Within each species, the sites of particle retention and lung tissue responses were the same for diesel soot, coal dust, and the combined material. Rats retained a greater portion of the particulate material in lumens of alveolar ducts and alveoli than monkeys. Conversely, monkeys retained a 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 intrapulmonary particle retention patterns and tissue reactions in rats may not be predictive of retention patterns and tissue responses in primates exposed to poorly soluble particles at concentrations representing high occupational exposures.

EFFECTS OF CIGARETTE SMOKE EXPOSURE AND CESSATION ON INFLAMMATORY CELLS AND MATRIX METALLOPROTEINASE ACTIVITY IN MICE

B6C3F1 female mice were exposed to cigarette smoke (CS) (250 mg/m3 total particulate material) or filtered air (FA), 6 hours/day, 5 days/week, for 6, 7, or 10 weeks, or to CS for 6 weeks, then FA for 1 or 4 additional weeks. Exposure to CS increased macrophages, neutrophils, lymphocytes, and matrix metalloproteinase (MMP)-2 and MMP-9 content in bronchoalveolar lavage fluid. Partial recovery of most lavage parameters (except lymphocytes) was observed 1 week after cessation of CS exposure with further reductions after 4 weeks, but interstitial inflammation persisted longer. These results support a role for MMPs in CS-induced emphysema and indicate that smoking cessation allows restoration toward normal homeostasis.

Failure of cigarette smoke to induce or promote lung cancer in the A/J mouse

A six-month bioassay in A/J mice was conducted to test the hypothesis that chronically inhaled mainstream cigarette smoke would either induce lung cancer or promote lung carcinogenicity induced by the tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Groups of 20 female A/J mice were exposed to filtered air (FA) or cigarette smoke (CS), injected with NNK, or exposed to both CS and NNK. At 7 weeks of age, mice were injected once with NNK; 3 days later, they were exposed to CS for 6 h/day, 5 days/week, for 26 weeks at a mean 248 mg total particulate matter/m3 concentration. Animals were sacrificed 5 weeks after exposures ended for gross and histological evaluation of lung lesions. No significant differences in survival between exposure groups was observed. A biologically significant level of CS exposure was achieved as indicated by CS-induced body weight reductions, lung weight increases, and carboxyhemoglobin levels in blood of about 17%. Crude tumor incidences, as determined from gross observation of lung nodules, were similar between the CS-exposed and FA groups, and the NNK and CS + NNK groups. Incidences in either of these latter groups were greater than either the CS or FA groups. Furthermore, tumor multiplicity in tumor-bearing animals was not significantly different among any of the three groups (FA, NNK, CS + NNK) in which tumors were observed. Thus, CS exposure neither induced lung tumors nor promoted NNK-induced tumors. Because the CS exposure concentration was probably near the maximally tolerable level, longer exposures should be evaluated to potentially establish a CS-induced model of lung carcinogenesis in the A/J mouse.

RAT LUNG TUMORS INDUCED BY EXPOSURE TO SELECTED POORLY SOLUBLE NONFIBROUS PARTICLES

Rodent bioassays have been used to assess the carcinogenicity of several inhaled, poorly soluble, nonfibrous particles that vary in toxicity and carcinogenic potency. There is substantial published information from chronic inhalation bioassays of diesel exhaust, carbon black, titanium dioxide, talc, and coal dust. This review summarizes data from studies with exposures for 2 yr or more using these 5 materials. The review has four objectives: (1) to summarize the current information available from these bioassays concerning exposure-dose-carcinogenic response in rats, (2) to summarize the pathologic and phenotypic features of the neoplastic response in rats, (3) to examine possible strain- and gender-related differences, and (4) to compare the neoplastic responses of rat to those of other species exposed to these materials.

Animal models of emphysema and their relevance to studies of particle-induced disease.

Emphysema is a pulmonary disease that may be exacerbated by inhaled particles. Over the years, many animal models of emphysema have been developed that may be useful in studying the effects of inhaled particles on humans with emphysema. Models have been described in many species, and many approaches have been described for inducing emphysema. Emphysema in humans is a parenchymal component of chronic obstructive pulmonary disease and frequently coexists in a complex with disease of the airways such as bronchitis. Animal models of emphysema usually recapitulate only one or a few aspects of this complex disease. Thus, the emphysema model must be selected carefully in order to answer specific questions about the interactive effects of particles and emphysema.Emphysema is a pulmonary disease that may be exacerbated by inhaled particles. Over the years, many animal models of emphysema have been developed that may be useful in studying the effects of inhaled particles on humans with emphysema. Models have been described in many species, and many approaches have been described for inducing emphysema. Emphysema in humans is a parenchymal component of chronic obstructive pulmonary disease and frequently coexists in a complex with disease of the airways such as bronchitis. Animal models of emphysema usually recapitulate only one or a few aspects of this complex disease. Thus, the emphysema model must be selected carefully in order to answer specific questions about the interactive effects of particles and emphysema.

Animal models of beryllium-induced lung disease.

The inhalation Toxicology Research Institute (ITRI) is conducting research to improve the understanding of chronic beryllium disease (CBD) and beryllium-induced lung cancer. Initial animal studies examined beagle dogs that inhaled BeO calcined at either 500 or 1000 degrees C. At similar lung burdens, the 500 degrees C BeO induced more severe and extensive granulomatous pneumonia, lymphocytic infiltration into the lung, and positive Be-specific lymphocyte proliferative responses in vitro than the 1000 degrees C BeO. However, the progressive nature of human CBD was not duplicated. More recently, Strains A/J and C3H/Hej mice were exposed to Be metal by inhalation. This produced a marked granulomatous pneumonia, diffuse infiltrates, and multifocal aggregates of interstitial lymphocytes with a pronounced T helper component and pulmonary in situ lymphocyte proliferation. With respect to lung cancer, at a mean lung burden as low as 17 micrograms Be/g lung, inhaled Be metal induced benign and/or malignant lung tumors in over 50% of male and female F344 rats surviving > or = 1 year on study. Substantial tumor multiplicity was found, but K-ras and p53 gene mutations were virtually absent. In mice, however, a lung burden of approximately 60 micrograms (-300 micrograms Be/g lung) caused only a slight increase in crude lung tumor incidence and multiplicity over controls in strain A/J mice and no elevated incidence in strain C3H mice. Taken together, this research program constitutes a coordinated effort to understand beryllium-induced lung disease in experimental animal models.

The Health Effects of Diesel Exhaust: Laboratory and Epidemiologic Studies

Publisher Summary Over 40 studies provide estimates of the risk of lung cancer associated with occupational exposure to diesel exhaust. The studies of occupational exposure to diesel exhaust and lung cancer have consistently observed elevated lung cancer rates among exposed workers, which cannot be readily attributed to known sources of bias or confounding. Unfortunately, however, no study provides quantitative estimates of the past exposure of study subjects to any constituent of diesel exhaust; therefore, the dose-response relation cannot be estimated with great accuracy from the available epidemiologic data. The most frequently studied occupational groups have been railroad workers and truck drivers. The studies of truck drivers show a 20–50% excess incidence and/or mortality from lung cancer, which persists when cigarette smoking is accounted for in data analysis. Some studies included only small numbers of subjects, and therefore, they offer imprecise estimates of the relative risk as indicated by the width of the 95% confidence intervals. However, the upper bounds of the 95% confidence intervals indicate that few of the results are consistent with more than a tripling of risk and the larger studies are consistent with less than a doubling.

Carcinogenic Responses of Transgenic Heterozygous p53 Knockout Mice to Inhaled 239PuO2 or Metallic Beryllium

The transgenic heterozygous p53 +/- knockout mouse has been a model for assessing the tumorigenicity of selected carcinogens administered by noninhalation routes of exposure. The sensitivity of the model for predicting cancer by inhaled chemicals has not been examined. This study addresses this issue by acutely exposing p53 +/- mice of both sexes by nose-only inhalation to either air (controls), or to 1 of 2 levels of 239PuO2 (500 or 100 Bq 239Pu) or beryllium (Be) metal (60 or 15 μg). Additional wild-type p53 +/- mice were exposed by inhalation to either 500 Bq of 239PuO2 or 60 μg of Be metal. These carcinogens were selected because they operate by differing mechanisms and because of their use in other pulmonary carcinogenesis studies in our laboratory. Four or 5 of the 15 mice per sex from each group were sacrificed 6 mo after exposure, and only 2 pulmonary neoplasms were observed. The remainder of the mice were held for life-span observation and euthanasia as they became moribund. Survival of the p53 +/- knockout mice was reduced compared to the p53 +/+ wild-type mice. No lung neoplasms were observed in p53 +/- mice exposed to air alone. Eleven of the p53 +/- mice inhaling 239PuO2 developed pulmonary neoplasms. Seven p53 +/+ mice exposed to 239PuO2 also developed pulmonary neoplasms, but the latency period for pulmonary neoplasia was significantly shorter in the p53 +/- mice. Four pulmonary neoplasms were observed in p53 +/- mice exposed to the higher dose of Be, whereas none were observed in the wild-type mice or in the heterozygous mice exposed to the lower dose of Be. Thus, both p53 +/- and p53 +/+ mice were susceptible to 239Pu-induced carcinogenesis, whereas the 53 +/- but not the p53 +/+ mice were susceptible to Be-induced carcinogenesis. However, only 2 pulmonary neoplasms (1 in each of the 239PuO2 exposure groups) were observed in the 59 p53 +/- mice that were sacrificed or euthanatized within 9 mo after exposure, indicating that the p53 +/- knockout mouse might not be appropriate for a 6-mo model of carcinogenesis for these inhaled carcinogens.

Apoptosis is a pathway responsible for the resolution of endotoxin-induced alveolar type II cell hyperplasia in the rat.

Previous studies showed that intratracheal instillation of endotoxin induces transient type II cell hyperplasia in the rat lung and described some of the mechanisms involved in the proliferative response of type II cells. The purpose of the present study was to investigate how long the type II cell hyperplasia persists and how it is resolved. The portion of epithelial cells in hyperplastic lesions of the rat lung expressing cyclin D1, an indicator for cells in the G1 phase of the cell cycle, was greatest at 3 d post instillation and decreased after 4 and 6 d. The fate of the proliferating epithelial cells was traced by injecting the rats with 5‐bromo‐2′deoxy uridine (BrdU) 2 d post instillation, the peak time point for maximum incorporation of BrdU. Exfoliated BrdU‐positive epithelial cells were detected in the alveolar spaces in tissue sections from rats 4, 5, and 6 d post instillation. BrdU‐positive epithelial cells showed flattened nuclei at 6 and 10 d post instillation. Expression of the 116 kD poly(ADP‐ribose) polymerase (PARP) was low in type II cells from control rats, and was increased at 3, 4, and 6 d post instillation. In cells obtained by lavage, only a 35 kD cleavage product of PARP was detected, which is an indicator of necrotic cell death. In isolated type II cells from rats 3, 4, and 6 d post endotoxin instillation, progressive cleavage of the PARP to its 89 kD residual fragment was detected, which is a direct evidence for the activation of caspases. Furthermore, apoptotic epithelial cells with condensed nuclei were identified by electron microscopy in rats 4 d post instillation. These results indicate that apoptosis is an additional mechanism for the resolution of endotoxin‐induced lung epithelial hyperplasias.