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. 2016 Apr 15;13:17.
doi: 10.1186/s12989-016-0130-z.

Persistent Effects of Libby Amphibole and Amosite Asbestos Following Subchronic Inhalation in Rats

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Free PMC article

Persistent Effects of Libby Amphibole and Amosite Asbestos Following Subchronic Inhalation in Rats

Stephen H Gavett et al. Part Fibre Toxicol. .
Free PMC article

Abstract

Background: Human exposure to Libby amphibole (LA) asbestos increases risk of lung cancer, mesothelioma, and non-malignant respiratory disease. This study evaluated potency and time-course effects of LA and positive control amosite (AM) asbestos fibers in male F344 rats following nose-only inhalation exposure.

Methods: Rats were exposed to air, LA (0.5, 3.5, or 25.0 mg/m(3) targets), or AM (3.5 mg/m(3) target) for 10 days and assessed for markers of lung inflammation, injury, and cell proliferation. Short-term results guided concentration levels for a stop-exposure study in which rats were exposed to air, LA (1.0, 3.3, or 10.0 mg/m(3)), or AM (3.3 mg/m(3)) 6 h/day, 5 days/week for 13 weeks, and assessed 1 day, 1, 3, and 18 months post-exposure. Fibers were relatively short; for 10 mg/m(3) LA, mean length of all structures was 3.7 μm and 1% were longer than 20 μm.

Results: Ten days exposure to 25.0 mg/m(3) LA resulted in significantly increased lung inflammation, fibrosis, bronchiolar epithelial cell proliferation and hyperplasia, and inflammatory cytokine gene expression compared to air. Exposure to 3.5 mg/m(3) LA resulted in modestly higher markers of acute lung injury and inflammation compared to AM. Following 13 weeks exposure, lung fiber burdens correlated with exposure mass concentrations, declining gradually over 18 months. LA (3.3 and 10.0 mg/m(3)) and AM produced significantly higher bronchoalveolar lavage markers of inflammation and lung tissue cytokines, Akt, and MAPK/ERK pathway components compared to air control from 1 day to 3 months post-exposure. Histopathology showed alveolar inflammation and interstitial fibrosis in all fiber-exposed groups up to 18 months post-exposure. Positive dose trends for incidence of alveolar epithelial hyperplasia and bronchiolar/alveolar adenoma or carcinoma were observed among LA groups.

Conclusions: Inhalation of relatively short LA fibers produced inflammatory, fibrogenic, and tumorigenic effects in rats which replicate essential attributes of asbestos-related disease in exposed humans. Fiber burden, inflammation, and activation of growth factor pathways may persist and contribute to lung tumorigenesis long after initial LA exposure. Fiber burden data are being used to develop a dosimetry model for LA fibers, which may provide insights on mode of action for hazard assessment.

Keywords: Adenoma; Amosite; Asbestos; Carcinoma; Dosimetry; Fibrosis; Inflammation; Inhalation; Libby amphibole; Risk assessment.

Figures

Fig. 1
Fig. 1
Short-term inhalation study: lung inflammation markers in bronchoalveolar lavage fluid (BALF) immediately after final exposure to AM or LA for 10 days (5 days/week for 2 weeks; 6 h/day). Numbers in legend represent target mass concentrations (mg/m3). Values represent mean ± SE of BALF macrophages and neutrophils (top panels) and concentrations of ALP, LDH, protein, and NAG in BALF supernatant (middle and lower panels) (n = 7 rats/group). P < 0.05 † vs. all other groups; * vs. air control; # vs. LA 0.5; ‡ vs. AM 3.5
Fig. 2
Fig. 2
Short-term inhalation study: transcriptional markers of apoptosis and inflammation in lung samples after final exposure to AM or LA for 10 days. Results show relative mean values ± SE of lung tissue mRNA for inflammasome pathway components (Nlrp3, Pycard, Casp1), downstream cytokines (Il1b and Il18), and other pro-inflammatory cytokines (Il6, Tnfa, Cxcl2) as determined by RT-qPCR (n = 7 rats per group). P < 0.05 † vs. all other groups; * vs. air control; # vs. LA 0.5; ‡ vs. AM 3.5
Fig. 3
Fig. 3
Short-term inhalation study: histopathologic effects and terminal bronchiolar epithelial cell proliferation 4 days after final exposure to AM or LA for 10 days. ac, Representative images of normal terminal bronchiole and surrounding alveoli from air control (Con) group (a), mixed cell alveolar inflammation, predominantly macrophages and neutrophils (b), and bronchiolar epithelial hyperplasia (c). Slides were stained with hematoxylin and eosin. df, Immunostaining of cells with BrdU. Representative images of terminal bronchioles from Con (d) and high concentration LA (25 mg/m3 target) (e) groups. Positively labeled cells are indicated by brown nuclei; background staining used hematoxylin. f, Percentage of positive BrdU-labeled cells (labeling index, LI), shown as individual LI values (circles), mean values (horizontal bars), and standard error (vertical bars) for each group. *P < 0.05 and **P < 0.01 compared to air control (Con) group; ^ P < 0.01 by linear trend test among LA dose groups. Images were taken at 20x (a, c) or 40x (b, d, e) objective magnification
Fig. 4
Fig. 4
Long-term stop-exposure inhalation study: lung fiber burden. Numbers of all asbestos structures (any structure with L ≥0.2 μm; top panel) and WHO fibers (L ≥5 μm, L/D ≥3; bottom panel) recovered in five lung lobes (LRT) of rats exposed to AM or LA for 13 weeks (5 days/week for 13 weeks; 6 hr/day) and evaluated at 1 day, 1 month, 3 months, or 18 months after the end of the exposure. Values in legend represent target mass concentrations (mg/m3). Results show mean values ± SE (n = 4 rats per group). P < 0.05 † vs. all other groups; # vs. LA 1.0 group
Fig. 5
Fig. 5
Long-term stop-exposure inhalation study: lung inflammation markers in BALF. Rats were exposed for 13 weeks to AM or LA and evaluated at 1 day, 1, 3, and 18 months post-exposure. Values in legend represent target mass concentrations (mg/m3). Results show means ± SE of BALF macrophages, neutrophils, and lymphocytes, and concentrations of protein, LDH, and ALP (Alk Phos) in BALF supernatant (n = 8 rats/group). P < 0.05 † vs. all other groups; * vs. air control; # vs. LA 1.0; ‡ vs. AM 3.3; § vs. LA 3.3
Fig. 6
Fig. 6
Long-term stop-exposure inhalation study: lung pro-inflammatory cytokines and markers of Akt and ERK pathway activation from right cranial lung lobe tissue. Rats were exposed for 13 weeks to AM or LA and evaluated at 1 day, 1, and 3 months after the end of the exposure. Values in legend represent target mass concentrations (mg/m3). Results show mean values ± SE (n = 6–7 rats per group). a Cytokine levels expressed relative to total protein in sample. b Phosphorylated components of Akt pathway (pAkt, pS6RP) and MAPK/ERK cascade (pMEK1/2, pSTAT3) expressed relative to levels in air control group. P < 0.05 † vs. all other groups; * vs. air control; # vs. LA 1.0; ‡ vs. AM 3.3; § vs. LA 3.3
Fig. 7
Fig. 7
Stop-exposure inhalation study: persistent effects on histopathology 18 months after exposure to AM or LA for 13 weeks. All images shown are from 10 mg/m3 LA target mass concentration except panel E (3.3 mg/m3 LA). Images represent a alveolar inflammation (macrophages) with intracytoplasmic fibers (arrowheads), b interstitial fibrosis (indicated by blue-stained collagen), c subpleural fibrosis (lower image) compared to normal pleura (upper image), d alveolar epithelial hyperplasia (indicated by cuboidal type II pneumocytes lining alveolar walls), e bronchiolar/alveolar (B/A) adenoma, and f B/A carcinoma. Slides were stained with either hematoxylin and eosin (a, df) or Masson’s trichrome (b, c). Images were taken at 60x (a), 40x (b, c), or 20x (df) objective magnification

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