Effects of Subchronically Inhaled Carbon Black in Three Species. I. Retention Kinetics, Lung Inflammation, and Histopathology

Alison Elder Alison_Elder@urmc 2 3 Robert Gelein 2 3 Jacob N. Finkelstein 0 2 Kevin E. Driscoll 1 2 Jack Harkema 2 4 Gu nter Oberdo rster 2 3 0 Department of Pediatrics, University of Rochester , Rochester, New York 14642 1 Proctor and Gamble Co. , Cincinnati, Ohio 45202 2 mental Medicine. University of Rochester , 575 Elmwood Ave., Box 850, Rochester, NY 14642. Fax: (585) 256-2631 3 Department of Environmental Medicine, University of Rochester , Rochester, New York 14642 4 Department of Pathobiology and Diagnostic Investigation, Michigan State University , East Lansing, Michigan 48824 - Exposure to high concentrations of carbon black (Cb) produces lung tumors in rats, but not mice or hamsters, presumably due to secondary genotoxic mechanisms involving persistent lung inflammation and injury. We hypothesized that the lung inflammation and injury induced by subchronic inhalation of Cb are more pronounced in rats than in mice and hamsters. Particle retention kinetics, inflammation, and histopathology were examined in female rats, mice, and hamsters exposed for 13 weeks to high surface area Cb (HSCb) at doses chosen to span a no observable adverse effects level (NOAEL) to particle overload (0, 1, 7, 50 mg/m3, nominal concentrations). Rats were also exposed to low surface area Cb (50 mg/m3, nominal; LSCb). Retention and effects measurements were performed immediately after exposure and 3 and 11 months post-exposure; retention was also evaluated after 5 weeks of exposure. Significant decreases in body weight during exposure occurred only in hamsters exposed to high-dose HSCb. Lung weights were increased in high-dose Cb-exposed animals, but this persisted only in rats and mice up to the end of the study period. Equivalent or similar mass burdens were achieved in rats exposed to high-dose HSCb and LSCb, whereas surface area burdens were equivalent for mid-dose HSCb and LSCb. Prolonged retention was found in rats exposed to mid- and high-dose HSCb and to LSCb, but LSCb was cleared faster than HSCb. Retention was also prolonged in mice exposed to mid- and high-dose HSCb, and in hamsters exposed to high-dose HSCb. Lung inflammation and histopathology were more severe and prolonged in rats than in mice and hamsters, and both were similar in rats exposed to mid-dose HSCb and LSCb. The results show that hamsters have the most efficient clearance mechanisms and least severe responses of the three species. The results from rats also show that particle surface area is an important determinant of target tissue dose and, therefore, effects. From these results, a subchronic NOAEL of 1 mg/m3 respirable HSCb (Printex 90) can be assigned to female rats, mice, and hamsters. Key Words: Carbon black; particle retention; species comparison; particle overload; inflammation; histopathology. Several studies have shown that inhaled carbon black (Cb) particles, when they are administered at doses that cause particle overload in the lungs as well as chronic inflammation and epithelial hyperplasia, induce lung tumors in rats but not in other rodent species (Driscoll et al., 1996; Heinrich et al., 1995; Mauderly et al., 1994; Rausch et al., 2004). Cb is not directly mutagenic (Kirwin et al., 1981); therefore, tumor formation in rats must occur via a secondary mechanism. Impaired clearance plays a role, but it is unlikely to be solely responsible for the observed tumors because rats, mice, and hamsters all demonstrate particle overload when the dose of Cb is high enough (Oberdo rster, 1995a). Studies by Driscoll et al. (1997) demonstrated a connection between the severity of inflammation and ex vivo mutations by co-incubating lung lavage inflammatory cells from Cb-exposed rats with rat lung epithelial cells. When the percentage of neutrophils in the lavage fluid was 50%, the hprt mutation rate increased significantly, possibly related to the generation and release of reactive oxygen species and/or a depletion in antioxidants. Indeed, the mutagenic response was inhibited in the presence of an antioxidant (catalase). Thus, the persistence of particles in the lung, the overall lung burden, the severity of the inflammatory response, and the release of oxidants all likely combine to produce tumors in rats, but the reasons why mice and hamsters do not develop tumors under the same high Cb load conditions is not entirely clear. We designed a species-comparative study to test the overarching hypothesis that rat-specific tumor responses are related to species differences in particle clearance kinetics, lung inflammation and injury, antioxidant cellular defense mechanisms, and secondary genotoxicity. In this study, rats, mice, and hamsters were exposed for 13 weeks to inhaled Cb such that equivalent lung burdens in terms of retained mass were achieved. We measured particle accumulation and clearance kinetics, cytotoxicity, inflammation, cell proliferation, lung morphology, oxidant and antioxidant production, ex vivo mutations, and genotoxicity as a function of time post-exposure. The Author 2005. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please email: This article addresses the specific hypothesis that retention, inflammation, and lung injury as a consequence of inhaled Cb exposure are more severe and persistent in rats than in mice and hamsters, and it details the results of particle lung burden analyses and the accompanying changes in body and lung weights, lavage inflammatory cells, and cell proliferation and histopathology. The details of pro- and anti-inflammatory mediator production, oxidant stress responses, and hprt mutations will be presented in a forthcoming article. Animals. All animals were obtained at 5 weeks of age and were specific pathogenfree. A total of 320 female F-344 rats were obtained from Harlan (Indianapolis, IN); 670 female B6C3F1 mice were purchased from Charles River (Wilmington, MA); and 276 F1B Syrian golden female hamsters were purchased from BioBreeders (Watertown, MA). Several extra animals from each species were exposed to allow replacement of any animals that died unexpectedly. Additional sentinels were euthanized throughout the study to monitor pathogen status. Females were chosen because they were previously shown to be more sensitive to the induction of lung tumors by poorly soluble, low toxicity particles than males (Oberdorster, 1995b). All animals were acclimatized for at least 2 weeks prior to the beginning of the studies and were uniquely identified with a subcutaneous transponder chip (BioMedic Data Systems, Seaford, DE). They were fed Purina rodent chow and water ad libitum and were housed in an AAALAC-accredited barrier facility with 12-h light/ dark cycles. The hamster diet was supplemented with rolled oats (as suggested by a veterinarian at BioBreeders) during the post-exposure phase of the study to increase fiber content and avoid the development of diarrhea. Body weights were obtai (...truncated)