Research Subchronic Pulmonary Pathology, Iron Overload, and Transcriptional Activity after Libby Amphibole Exposure in Rat Models of Cardiovascular Disease Jonathan H. Shannahan,1 Abraham Nyska,2 Mark Cesta,3 Mette C.J. Schladweiler,4 Beena D. Vallant,5 William O. Ward,6 Andrew J. Ghio,7 Stephen H. Gavett,4 and Urmila P. Kodavanti 4 1Curriculum in Toxicology, University of North Carolina School of Medicine, Chapel Hill, North Carolina, USA; 2Sackler School of Medicine, Tel Aviv University, Timrat, Israel; 3Cellular and Molecular Pathology Branch, National Institute of Environmental Health Sciences, National Institute of Health, Research Triangle Park, North Carolina, USA; 4Cardiopulmonary and Immunotoxicology Branch, Environmental Public Health Division, 5Genomics Core, and 6Biostatistics Core, Research Core Units, National Health and Environmental Effects Research Laboratory, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina, USA; 7Clinical Research Branch, Environmental Public Health Division, National Health and Environmental Effects Research Laboratory, Office of Research and Development, U.S. Environmental Protection Agency, Chapel Hill, North Carolina, USA Background : Surface-available iron (Fe) is proposed to contribute to asbestos-induced toxicity through the production of reactive oxygen species. o Bjectiv : Our goal was to evaluate the hypothesis that rat models of cardiovascular disease with coexistent Fe overload would be increasingly sensitive to Libby amphibole (LA)-induced subchronic lung injury. Methods : Male healthy Wistar Kyoto (WKY), spontaneously hypertensive (SH), and SH heart failure (SHHF) rats were intratracheally instilled with 0.0, 0.25, or 1.0 mg LA (with saline as the vehicle). We examined bronchoalveolar lavage fluid (BALF) and histological lung sections after 1 week, 1 month, or 3 months for pulmonary biomarkers and pathology. SHHF rats were also assessed at 6 months for pathological changes. r esult : All animals developed concentration- and time-dependent interstitial fibrosis. Time- dependent Fe accumulation occurred in LA-laden macrophages in all strains but was exacerbated in SHHF rats. LA-exposed SHHF rats developed atypical hyperplastic lesions of bronchiolar epi- thelial cell origin at 3 and 6 months. Strain-related baseline differences existed in gene expression at 3 months, with persistent LA effects in WKY but not SH or SHHF rats. LA exposure altered genes for a number of pathways, including inflammation, immune regulation, and cell-cycle control. Cell- cycle control genes were inhibited after LA exposure in SH and SHHF but not WKY rats, whereas tumor suppressor genes were induced only in WKY rats. The inflammatory gene expression also was apparent only in WKY rats. c onclusi : These data show that in Fe-overload conditions, progressive Fe accumulation occurs in fiber-laden macrophages within LA-induced lesions. Fe overload does not appear to contribute to chronic inflammation, and its role in hyperplastic lesion development requires further examination. k ey words : cardiovascular disease, iron overload, Lilly amphibole, pulmonary pathology. Environ Health Perspect 120:85–91 (2012). http://dx.doi.org/10.1289/ehp.1103990 [Online 6 October 2011] During vermiculite mining, residents and miners in Libby, Montana (USA), were exposed to a mixture of amphibole-type asbestos fibers containing winchite, richerite, tremolite, and other minerals (Moolgavkar et al. 2010). Epidemiological studies have shown a dose-dependent correlation between Libby amphibole (LA) exposure and the inci- dences of asbestos-induced diseases, including asbestosis, lung cancer, and mesothelioma (Moolgavkar et al. 2010). Animal studies have shown that the toxicity of amphibole fibers is possibly due to their biopersistence within the lung, causing prolonged inflammation and oxidative stress (Bernstein et al. 2003). Asbestos produces reactive oxygen spe- cies (ROS) through Fenton reactions likely facilitated by surface-available metals such as iron (Fe). Asbestos and silica can associate with endogenous Fe after inhalation, form- ing Fe-rich asbestos bodies on fiber surfaces (Ghio et al. 2004). After binding, this Fe is postulated to enhance the ability of asbes- tos to produce ROS, thereby exacerbating its toxicity (Hardy and Aust 1995). We have shown that LA can bind Fe and that this Fe is redox active in an acellular environment (Shannahan et al. 2011a). However, Fe loading of LA or cells inhibits the acute pro- inflammatory response in vitro in cells and in vivo in rats (Shannahan et al. 2011a). The role of Fe in the progression of the diseases induced by LA and other asbestos materials is unknown. Increased tissue Fe overload may increase bioactive and protein-bound Fe levels and affect the inflammatory response after an envi- ronmental insult. Individuals with chronic diseases such as thalassemia, diabetes, arthri- tis, cancer, and cardiovascular disease (CVD) have been shown to have systemic Fe over- load (Abdalla et al. 2011; Sen et al. 2011; Toyokuni 2011). The systemic Fe overload, associated inflammation, and oxidative stress in CVD (Kruszewski 2004) may influence host susceptibility to asbestos-induced diseases. We have shown that the spontaneously hyper- tensive (SH) and SH heart failure (SHHF) Environmental Health Perspectives • volume 120 | number 1 | January 2012 rats exist in a state of pulmonary Fe overload (as demonstrated by increased pulmonary fer- ritin and transferrin), inflammation, and oxi- dative stress compared with control Wistar Kyoto (WKY) rats (Shannahan et al. 2010). The hypertensive state of both the SH and SHHF rats has been linked to genetic anoma- lies in the rennin angiotensin system (Leckie 2001) and the β-adrenergic system (Remmers et al. 1993); however, the role of other genetic factors remains unknown. Previous work has shown that an acute exposure to LA in these rat models does not exacerbate inflammation despite increases in Fe-binding proteins and gene expression (Shannahan et al. 2011b). High baseline inflammation together with dysregulated Fe homeostasis might diminish the acute inflammogenic response to toxicants. In the present study, we hypothesized that the subchronic presence of LA in the lungs of SH and SHHF rats will cause further accumula- tion of fiber-associated Fe and inhibit inflam- mation. Furthermore, we presumed that examining the transcriptional activity of the whole lung genome might provide additional insights into the role of Fe in the progression of asbestos-induced lung disease. Address correspondence to U. Kodavanti, MD B105-02, National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, NC 27711 USA. Telephone: (919) 541-4963. Fax: (919) 541-0026. E-mail: Kodavanti.Urmila@epa.gov Supplemental Material is available online (http:// dx.doi.org/10.1289/ehp.1103990). This work was supported by U.S. Environmental Protection Agency/University of North Carolina Toxicology Research Program training agreement CR833237, with the Curriculum in Toxicology, University of North Carolina at Chapel Hill, and in part by the Intramural Research Program of the National Institute of Environmental Health Sciences, National Institutes of Health. The research described in this article has been reviewed by the National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, and approved for publication. Approval does not signify that the contents necessar- ily reflect the views and the policies of the agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use. The authors declare they have no actual or potential competing financial interests. Received 25 May 2011; accepted 6 October 2011. 85