Shannahan et al. overload. Only SHHF rats developed atypi- cal hyperplastic lesions of bronchiolar origin in response to LA. The overall transcriptional activity supports the development of hyper- plastic lesions in SHHF rats after LA exposure as well as the baseline chronic inflammation. However, the lack of persistence in LA-induced inflammatory gene expression despite Fe accu- mulation in SHHF rats is not consistent with its involvement in LA-induced hyperplastic lesions. Thus, CVD patients with Fe overload might not exhibit significant inflammation with asbestos exposure, while they might be susceptible to developing hyperplastic lesions. Persistent systemic inflammation and Fe overload are hallmark features of chronic dis- eases in humans and in animals; however, it is not known if they are consequences of the dis- ease or causal factors (Yao and Rahman 2011). We have shown that SH and SHHF rats have increased baseline pulmonary inflammation and Fe overload that relates to the severity of disease (SHHF > SH) (Shannahan et al. 2010). Genes involved in inflammatory processes mediated by NFκB and other signaling pathways were generally up-regulated at baseline in both SH and SHHF rats compared with WKY rats, along with several metabolic and pathological processes. Thus, systemic chronic inflammation and Fe overload coexist in CVD at baseline. Gene expression analysis revealed that the LA effect on inflammatory genes that regulate innate immunity is still evident at 3 months postexposure in WKY rats but less so in SHHF and SH rats. Although inflammatory genes were up-regulated at baseline in SH and SHHF rats, many of the genes regulating acquired immunity were generally suppressed (Figure 4, cluster 1). This pattern of response suggests that, at 3 months after LA exposure, SH and SHHF rats are unable to sustain increased inflamma- tory gene expression despite their elevated base- line lung neutrophils. This may be related to their already high baseline expression and an inability to further up-regulate inflammatory genes after LA exposure. Persistent inflammatory response in the case of asbestos exposure leads to pulmonary fibrosis at the terminal bronchiolar region, coinciding with the site of fiber deposition (Brody et al. 1981). Despite gene expression pattern differences between strains, our study WKY Saline WKY LA SH Saline SH LA SHHF Saline SHHF LA 1 r e t s u l C 2 r e t s u l C 3 r e t s u l C 90 demonstrates similar degrees of fibrotic lesions in the lung. Even though the LA-induced neu- trophilic influx had subsided in all three strains at 3 months, the inflammatory gene expression still remained induced in WKY rats compared with SH and SHHF rats, which express high baseline levels of these genes. Increased inflam- matory gene expression may be necessary for progression to fibrosis in all strains regardless of whether gene expression is increased at baseline or after LA exposure. LA-induced inflamma- tion may have been dampened because of accu- mulated Fe within fiber-laden macrophages in Fe-overloaded SHHF rats. Underlying genetic differences may also be involved in differential regulation of inflammation. Unlike long thin fibers that remain interca- lated at the terminal bronchiolar region, shorter fibers are likely to be readily phagocytosed by alveolar macrophages (Bernstein et al. 2010). In vitro studies have shown that even epithe- lial cells can take up relatively short LA fibers (Duncan et al. 2010). In the present study, light microscopy examinations with phase con- trast revealed the presence of LA fibers within alveolar macrophages at 3 months, suggest- ing that these fibers are taken up by macro- phages and not cleared from the lung within this time frame. Amosite fibers similar in length to LA have been shown to be engulfed by macrophages in vivo (Bernstein et al. 2010). Additionally, the half-life of these amosite fibers has been shown to be > 90 days, suggesting that some of these fibers will remain in the lung for the lifetime of the animal (Bernstein et al. 2010). The ability of alveolar macrophages to engulf but not clear the LA fibers may result in chronic inflammation and disease progression. The exacerbated progressive accumulation of Fe within fiber-laden alveolar macrophages in the SHHF rats supports the hypothesis that the increased availability of cellular Fe leads to accumulation of Fe at the site of fiber deposi- tion within the lung. Coating of endogenous Fe on the surface of fibers has been observed in mice and in humans after exposure (Ghio et al. 2006, 2009). Furthermore, our previous study with LA (Shannahan et al. 2011a) and another study with quartz (Ghiazza et al. 2011) dem- onstrated that the bioavailability of catalyti- cally active Fe may reduce the inflammatory response after fiber exposure. Consequently, the Fe overload in SH and SHHF rats might reduce the inflammatory gene induction caused by LA due to accumulated Fe at the site of fiber deposition. Surprisingly, only the SHHF rats devel- oped atypical hyperplastic lesions at 3 months after exposure to LA. These proliferative lesions likely originated from bronchiolar epithelial cells and did not involve squamation. Unlike typical hyperplastic changes seen in epithe- lial cells after asbestos-induced lung fibrosis (Robledo et al. 2000), the hyperplastic changes Figure 5. Clustering of cell-cycle control–specific pulmonary genes comparing WKY, SH, and SHHF rats 3 months after exposure of 1.0 mg LA. DEGs related to cell-cycle control were assembled from KEGG pathways 4115 and 5200. All genes were hierarchically clustered, median centered, and viewed using Treeview as indicated in Figure 4. ovlume 120 | number 1 | January 2012 • Environmental Health Perspectives