Shannahan et al. Materials and Methods Animals. Male, 11- to 12-week-old, healthy WKY, SH, and obese SHHF rats were pur- chased from Charles River Laboratories (Raleigh, NC, USA). Rats were main- tained in an Association for Assessment and Accreditation of Laboratory Animal Care– approved animal facility at 21 ± 1°C, 50 ± 5% relative humidity, and 12/12-hr light/dark cycle. Animals received standard rat chow (Rat Chow 5001; Purina Mills, Brentwood, MO, USA) and water ad libitum. The Institutional Animal Care and Use Committee of the U.S. Environmental Protection Agency’s National Health Environmental Effects Research Laboratory approved the protocol. The ani- mals were treated humanely and with regard for alleviation of suffering. Intratracheal instillation of Libby amphi- bole. The Libby amphibole (LA) sample col- lected from the Rainy Creek complex near Libby, Montana, in 2007 was size fractionated to isolate a rat respirable fraction (particulate matter ≤ 2.5 mμ in aerodynamic diameter (i.e., PM2.5). Rats (WKY, n = 12/time point; SH, n = 6/time point; SHHF, n = 6/time point) were intratracheally instilled with saline or 0.25 or 1.0 mg of LA (with saline as the vehicle) as described previously (Shannahan et al. 2011a) [also see “Methods” in Supplemental Material (http://dx.doi.org/10.1289/ehp.1103990)]. Doses were chosen to assure a response in the lung, allowing for a comparative analysis between strains. Theoretically, a rat will deposit 0.07 mg fibers during 6 hr of inhalation at 10 mg/m3, assuming that minute volume is 200 mL and the deposition fraction to pulmo- nary region is 0.10. The WKY sample size was increased to twice that of SH and SHHF rats so that data from WKY rats with nonpathological cardiac hypertrophy could be excluded for comparisons (Shannahan et al. 2010). An additional experiment was also performed in which 36 SHHF rats were exposed to saline or 0.25 or 1.0 mg LA in saline and examined at 3 or 6 months for bronchoalveolar lavage fluid (BALF) and lung pathology. Necropsy, sample collection, and analysis. Rats were anesthetized with sodium pentobarbi- tal (Virbac AH, Fort Worth, TX, USA; 50–100 mg/kg, intraperitoneal) 1 week, 1 month, or 3 months after instillation of LA. Blood was removed from the vasculature of the lung by perfusion via the pulmonary artery to avoid Table 1. Histological evaluation of relative staining intensities for ferric Fe and collagen after exposure to LA in WKY, SH, and SHHF rats: average score and number of animals affected by exposure. Average score (no. of animals affected by exposure) LA concentration (mg/rat) Animals/group Perls blue staining for ferric Fe Trichrome staining for collagen 0.0 0.25 1.0 0.0 0.25 1.0 0.0 0.25 1.0 0.0 0.25 1.0 0.0 0.25 1.0 0.0 0.25 1.0 0.0 0.25 1.0 0.0 0.25 1.0 0.0 0.25 1.0 5 6 6 6 6 6 6 6 6 5 5 5 6 6 5 6 6 6 5 6 5 6 6 6 6 5 6 0.0 0.0 0.0 0.0 0.3 (2) 0.3 (2) 0.0 0.8 (4)*# 0.8 (4)* 0.0 0.0 0.0 0.0 0.0 1.0 (4)*# 0.3 (2) 1.5 (6)*# 1.7 (6)*# 0.0 0.0 0.4 (2) 0.0 0.5 (2) 0.8 (3)* 0.3 (2) 0.6 (2) 1.7 (6)*# 0.0 1.0 (6)* 1.0 (6)* 0.2 (1) 1.0 (6)* 1.0 (6)* 0.0 1.0 (6)* 1.2 (6)* 0.0 1.0 (5)* 0.8 (4)* 0.0 0.8 (5)* 1.0 (6)* 0.0 1.0 (6)* 1.2 (6)* 0.0 1.0 (6)* 1.7 (5)* 0.0 0.8 (5)* 1.5 (6)* 0.0 0.8 (5)* 2.0 (6)* Time point One week WKY One month WKY SH SHHF SH SHHF WKY SH SHHF 86 Three months interference in data analysis involving Fe-binding proteins. The right lung lobes were lavaged with Ca2+/Mg2+-free phosphate-buffered saline and stored at –80°C for later analysis. The left lung was fixed by instillation of 10% neutral buffered formalin through the trachea. Cell differential and analysis. Aliquots of BALF were taken for total cell counts (Coulter, Miami, FL, USA). Differential cell determinations of cytospin preparations (Shandon, Pittsburgh, PA, USA) were made using LeukoStat staining (Fisher Scientific, Pittsburgh, PA, USA). Macrophages and neu- trophils were counted under light microscopy. Cell-free BALF was analyzed for total protein, albumin, ferritin, and transferrin (Shannahan et al. 2010) [see Supplemental Material, “Methods” (http://dx.doi.org/10.1289/ ehp.1103990)]. Lung histopathology. The left lung was embedded in paraffin, sectioned to a thick- ness of 3 μ m (transverse), and stained with hematoxylin and eosin (H&E), Perls Prussian blue, or Masson’s trichrome (Experimental Pathology Labs, Durham, NC, USA). Histopathological changes in the tissues were scored using semiquantitative grading at five levels (0, normal; 1, minimal; 2, mild; 3, moderate; 4, severe) taking into consider- ation the degree of severity and the type of lesion [see Supplemental Material, “Methods” (http://dx.doi.org/10.1289/ehp.1103990)]. In an additional experiment, lung tissues from SHHF rats were stained with H&E and exam- ined for lesions at 3- and 6-month time points. Immunohistochemistry. The cellular ori- gin of hyperplasic changes in SHHF rats at 3 months was examined immunohistochemi- cally. Five-micrometer-thick lung sections from rats exposed to 0.0, 0.25, or 1.0 mg LA for 3 months were immunohistochemi- cally stained for several epithelial cell mark- ers [see Supplemental Material, “Methods” (http://dx.doi.org/10.1289/ehp.1103990)]. Sections were assessed for surfactant protein A (sc-7699), Clara cell protein 10 (sc-9772), cytokeratin 10 (sc-58720), and cytokeratin 14 (sc-53253) using antibodies from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and cytokeratin 19 (CRC946) from Cell Marque (Rocklin, CA, USA). Gene array. Total RNA was isolated from caudal lung lobes using RNeasy Mini Kits (Qiagen, Valencia, CA, USA). RNA integ- rity was assessed by the RNA 6000 LabChip® (Caliper Life Sciences, Hopkinton, MA, USA) kit using an Agilent 2100 bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). Changes in global gene expression were exam- ined at the 3-month time point in each strain for saline control and LA (1.0 mg/rat) groups (n = 6) using the Affymetrix platform (Rat Genome 230 2.0 Array; Affymetrix, Santa Clara, CA, USA) [see Supplemental Material, WKY, SH, and SHHF rats exposed to saline (control) or LA (0.25 or 1.0 mg/rat) were examined for pulmonary collagen deposition and ferric Fe accumulation using Masson’s trichrome and Perls blue staining, respectively. Values represent average severity score, from 0 to 4 in increasing severity. *p < 0.05, Fisher’s exact test, within strain compared with saline control. #p < 0.05, strain difference compared with WKY at the same exposure concentration. ovlume 120 | number 1 | January 2012 • Environmental Health Perspectives