Shannahan et al. One important observation made in 3-month H&E-stained slides of LA-exposed SHHF rats was the presence of focal atypical hyperplastic lesions (Figure 2A). These lesions appeared to be concentration dependent [saline, 0/6 lesion incidence (no. lesions/no. rats from which lung sections were stained with H&E); 0.25 mg LA, 2/6 lesion incidence; 1 mg LA, 4/6 lesion incidence]. The lesions were located at or near terminal bronchioles and were char- acterized by stacked, multiple layers of epithelial cells lining alveolar ducts or alveolar walls. To determine the further progression of hyperplas- tic changes over time, additional SHHF rats instilled at the same dose levels were examined 3 and 6 months after LA exposure. This experi- ment verified the presence of atypical hyper- plastic lesions in SHHF rats at 3 and 6 months after LA exposure; however, the lesions did not progress in severity from 3 to 6 months (data not shown). Immunohistochemical evaluation. In order to determine the cellular origin of these hyperplastic lesions and the possible pres- ence of squamation, the tissue sections were immunohistochemically evaluated using sev- eral cytokeratin and epithelial cell markers in SHHF rats. These lesions were negative for the squamous epithelial markers cytokeratin 10 and 14, as indicated by a lack of brown H & E Cytokeratin 10 Cytokeratin 14 Cytokeratin 19 Surfactant protein A Clara cell 10 Figure 2. Immunohistochemical characterization of atypical hyperplastic lesions in SHHF rats at 3 months after a single instillation of 1.0 mg LA. (A) H&E-stained lung section. Asterisks indicate the locations of hyperplastic lesions. (B, C) Lung sections immunohistochemically stained for the squamous cell markers cytokeratin 10 (B) and cytokeratin 14 (C). The hyperplastic changes (arrows) did not stain for cytokeratins 10 and 14. (D) Lung sections stained for the bronchiolar cell marker cytokeratin 19 (brown staining shown by arrows). (E, F) Lung sections stained for surfactant protein A (E), a marker of alveolar type II and Clara cells, and the Clara cell marker Clara cell 10 (F). Arrows indicate locations of epithelial cells; asterisks indi- cate areas of staining in macrophages. Negative control antibodies did not stain positive for any marker. Magnification, 20×. 88 ovlume 120 | number 1 | January 2012 • Environmental Health Perspectives staining (Figure 2B,C). However, lesions stained positive (brown stained cells) for a) the bronchiolar epithelial cell marker cyto- keratin 19 (Figure 2D), b) the Clara cell and alveolar type II cell marker surfactant protein A (Figure 2E), and c) the Clara cell–specific marker Clara cell 10 (Figure 2F). These results are consistent with a bronchiolar epithelial cell origin of the hyperplastic lesions. Gene expression analysis. We compared the gene expression profiles of controls with those of rats treated with 1.0 mg LA in each strain to determine which genes and pathways differed at baseline and remained altered at 3 months. Baseline strain differences primarily involved genes related to immune regulation, inflammation, and metabolic disease condi- tion. It was apparent from the Venn diagram [see Supplemental Material, Figure 2 (http:// dx.doi.org/10.1289/ehp.1103990)] that nearly 2,000 genes had changed in the WKY and SH rats at 3 months after LA exposure, whereas minimal changes (180 genes) occurred in the SHHF rats. Only 15 genes were modified in common among all three strains at 3 months after LA exposure. The SH rats demonstrated the highest total number of genes changed after LA exposure (SH > WKY > SHHF), with more genes having down-regulated compared with the WKY and SHHF rats. In order to understand the relative strain differences and LA effects in parallel, we com- pared the mean expression values of WKY saline control rats with those of WKY LA, SH saline, SH LA, SHHF saline, and SHHF LA rats and determined relative fold differences. Although we found marked intensity differences after LA exposure, most of the baseline gene changes seen in SH and SHHF rats compared with WKY rats were in the same direction (up or down) as those seen in WKY rats. The path- ways significantly altered because of baseline strain differences or LA exposure, based on the list of DEGs for each contrast, are depicted in the heat map shown in Figure 3. Several KEGG pathways, including those involving lipid and steroid metabolism, glyco- lysis, inflammation, leukocyte migration, cell- cycle control, oxidative phosphorylation, and diabetes, were found altered at baseline and at 3 months after exposure to LA (Figure 3). The glutathione metabolism pathway was signifi- cantly changed only in WKY rats exposed to LA. Similarly, only WKY rats exposed to LA demonstrated an effect on pathways related to oxidative phosphorylation and tumor suppres- sion (p53) signaling. SH rats exposed to LA demonstrated an effect on pathways assigned to cancer, whereas SHHF rats had this path- way affected significantly at baseline as well as after LA exposure (Figure 3). SHHF rats exposed to LA also had other pathways related to cell proliferation induced, including basal cell carcinoma.