“Methods” (http://dx.doi.org/10.1289/ ehp.1103990)]. Statistical analysis of BALF and pathology data. Data are expressed as mean ± SE (WKY, n = 8–12/group; SH and SHHF, n = 6/group). SigmaStat (version 3.5; Systat Software, Point Richmond, CA, USA) was used to determine statistical comparisons via a two-way analysis of variance (ANOVA) followed by a post hoc comparison using the Holm-Sidak method (p ≤ 0.05). Subjective numerical scoring of Perls blue and trichrome staining assigned to each animal were added together for each group and statistically analyzed using Fisher’s exact test. Statistical significance was deter- mined when p ≤ 0.05. Gene array data analysis. The CEL files were loaded into Rosetta Resolver (ver- sion 7.2; Rosetta Inpharmatics, Seattle, WA, USA) and normalized with the Rosetta error model. Differentially expressed probe sets were determined using a one-way ANOVA with a Benjamini-Hochberg multiple-test correc- tion (false discovery rate set at 0.05). Each of the five treatments (including SH saline and SHHF saline) was contrasted with WKY saline. The resulting five lists of differentially expressed genes (DEGs) were combined to generate a master list. A Venn diagram was generated from the contrast of LA-induced changes relative to control for each strain using Rosetta Resolver. Functional analysis of gene array data set. For a global assessment of affected Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways (Bioinformatics Center, Institute of Clincial Research, Kyoto University, Kyoto, Japan; http://www.genome.jp/kegg/), the mas- ter list of DEGs relative to WKY saline was sub- mitted to DAVID (Database for Annotation, Visualization and Integrated Discovery; http:// david.abcc.ncifcrf.gov/) Huang et al. 2009a, 2009b) for analysis. p-Values using a Fisher exact test indicate overrepresentation of DEGs in a pathway and were used to generate a heat map of KEGG pathways. Functional lists of genes were constructed from KEGG pathway genes for inflammation and cell-cycle control [see Supplemental Material, “Methods” (http:// dx.doi.org/10.1289/ehp.1103990)]. Each func- tional list is depicted in a heat map, where each gene was median centered considering indi- vidual animals in all six groups and clustered based on commonality of directional changes. Eisen’s cluster analysis was used for clustering genes (Eisen et al. 1998) and the results were viewed using Treeview [Cluster version 3.0; Michiel de Hoon (mdehoon@ims.utokyo. ac.jp); University of Tokyo, Human Genome Center, Tokyo, Japan]. Results Lung injury markers, histopathological evalu- ation, and ferric Fe staining. We noted base- line strain-related differences in BALF markers Libby amphibole exposure in rat models of cardiovascular disease of inflammation and lung injury; however, no LA-inducible changes were apparent at 3 months after exposure [see Supplemental Material, Figure 1 (http://dx.doi.org/10.1289/ ehp.1103990)]. BALF changes and lung his- tological analysis for the 1-day, 1-week, and 1-month time points have been reported recently (Shannahan et al. 2011b). In the present study, we examined pathological lung lesions from 3-month H&E slides and also performed trichrome staining for collagen and Perls blue staining for ferric Fe at 1 week, 1 month, and 3 months in order to understand the time course of the development of fibrosis and accumulation of ferric Fe. All strains dem- onstrated interstitial fibrosis after exposure to LA (Table 1). More intense fibrotic foci were apparent in terminal bronchiolar areas where fibers likely accumulate (Figure 1A,B). No strain-related differences could be ascertained 1 week from analyzing the trichrome staining. BALF and histological lung sections from control WKY and SH rats did not stain for ferric Fe, whereas a few alveolar macrophages stained positive in control SHHF rats (Table 1). No discernible changes in Perls blue staining were apparent in LA-exposed WKY rats at any time point. At 1 month and 3 months, only a few macrophages stained positive for fer- ric Fe in SH rats exposed to 1 mg LA. The SHHF rats had markedly greater numbers of macrophages that stained positive for Fe com- pared with WKY and SH rats (SHHF > SH > WKY; Table 1). These Fe-positive macro- phages were observed around the lesions of interstitial fibrosis (Figure 1C,D). Alveolar macrophages appeared to internalize LA by 1 week after exposure, and fibers remained vis- ible in macrophages at 3 months after expsoure (Figure 1E,F). 3 months e m o r h c i r T s l r e P e k a t p u r e b ﬁ e g a h p o r c a m r a l o e v l A Figure 1. Time-related collagen deposition, ferric Fe accumulation, and alveolar macrophage fiber uptake in the lung after a single intratracheal instillation of LA in SHHF rats: photomicrographs of lung tissue sections 1 week (left column) and 3 months (right column) after exposure to 1.0 mg LA. (A,B) Stained for collagen using Masson’s trichrome. Arrows indicate blue staining in fibrotic lesions. Magnification, 20×. (C,D) Stained for ferric Fe (arrows) using Perls blue. The asterisks indicate unstained fibrotic areas. Magnification, 60×. (E,F) Perls blue–stained sections of lung using phase-contrast microscopy to focus on alveolar macrophage uptake of LA fibers. Arrows indicate fiber locations. Magnification, 40×. Environmental Health Perspectives • volume 120 | number 1 | January 2012 87