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Cellular and inflammatory responses in bronchoalveolar lavage and lungs in rats after intratracheal instillation of Libby amphibole or amosite asbestos
Citation:
Padilla-Carlin, D. J., M. SCHLADWEILER, U. P. KODAVANTI, J. H. Shannahan, J. H. Richards, A. Nyska, A. Bern, H. Lowers, G. Meeker, AND S. H. GAVETT. Cellular and inflammatory responses in bronchoalveolar lavage and lungs in rats after intratracheal instillation of Libby amphibole or amosite asbestos. Presented at north Carolina Society of Toxicology Fall meeting, RTP, NC, October 13, 2009.
Impact/Purpose:
This abstract gives an update on research progress on acute pulmonary toxicological effects of Libby asbestos vs. amosite asbestos as a positive control.
Description:
The high incidence of asbestos-related disease in residents of Libby, Montana, is associated with the mining of asbestos-contaminated vermiculite, but the etiology of disease related to Libby amphibole asbestos (LA) exposure is unclear. In this study, water elutriation was used to prepare LA and amosite (positive control) asbestos equivalent to airborne particulate matter less that 2.5 um mass median aerodynamic diameter (PM2.5). Male Fisher 344 rats (~250 g) were exposed to a single bolus (250 ul) of either saline (SAL), amosite (0.65 mg/rat), or LA (0.65 or 6.5 mg/rat) by intratracheal instillation (n = 8/group). The time-course of pulmonary inflammation and injury were analyzed in bronchoalveolar lavage (BAL) fluid and histopathological evaluation in rats euthanized at 1, 3, 7 d, 2 wk, and 3 mo after exposure. BAL from animals after asbestos instillation had increased parameters of lung toxicity when compared to SAL. Total cell counts (TCC) were higher in the asbestos-exposed groups at 1 d with respect to SAL, but by 7 d, TCC was comparable to SAL. Although long amosite and relatively short LA fibers were found intercalated within lavagable alveolar macrophages, no consistent changes in macrophage numbers occurred in the asbestos-exposed animals at the various timepoints. Neutrophil numbers were sharply increased at 1 d in the high-dose LA and amosite groups (SAL: ~3,800/ml; amosite: ~397,000/ml; low LA: ~81,700/ml; high LA: ~234,000/ml) but then decreased from 3 d to 3 mo after exposure, though neutrophil counts were significantly elevated in the high dose LA group at all time points with respect to SAL. Other BAL lung injury parameters such as total protein, albumin, lactate dehydrogenase, N-Acetyl-B-Dglucosaminidase, and y-glutamyl transferase were elevated at all time points in the high-dose LA and amosite-exposed groups, but no consistent pattern was noted for total antioxidant status. Furthermore, the iron-binding proteins, ferritin and transferrin, were detected at most timepoints in BAL of the amosite and high-dose LA groups. Unsaturated iron-binding capacity in BAL did not demonstrate a consistent pattern among the groups at each timepoint. Inflammatory cytokines (i.e., TNF α, IL-1B, and IL-6) were also evaluated in BAL. TNF-α was elevated at 1d, IL-6 was lower at 3 and 7 d, and IL-lB remained lower at all timepoints in the high-dose LA group. Furthermore, histopathologic evaluation of the lungs was conducted (Figure 1). In the amosite group, a prominent presence of intra-alveolar and intra-bronchial macrophages and neutrophils were found only 1 d after exposure. At subsequent timepoints, the lungs exhibited interstitial accumulation of macrophages, very large multinucleated giant cells, and fibroblastic cell proliferation, resulting in granulomatous lesions. Intra-alveolar accumulation of macrophages and neutrophils and alveolar epithelial hyperplasia surrounding the terminal and respiratory bronchioles, which is indicative of granulomatous inflammation, was demonstrated at all timepoints following LA exposure. The inflammation concomitant with fibrosis occurred only in the high dose LA group. At the later timepoints (e.g. 7 d), multifocal intra-alveolar and interstitial granulomatous inflammation occurred, but accumulation of macrophages within the alveoli was slightly reduced at 3 mo. In conclusion, a single intratracheal instillation exposure to amosite or high-dose LA in rats evokes a potent inflammatory response and persistent lung injury (up to 3 mo). Future studies will consist of 1) determination ofthe relationship between short term exposure and disease related to LA and 2) a comparative investigation between surface area and fibers/mass ofamosite and LA and how these parameters relate to their toxicity. Funding: EPA/ONC CR833237. This abstract does not reflect USEPA policy. Control (Saline) Amosite (0.65 mglrat; 3 mol LA (0.65 mgfrat: 3 mol LA (6.5 mgfrat; 3 mol Figure 1. Histopathology (H & E stain) of lung parenchyma from F344 rats treated with saline (Control; A), Amosite (0.65 mglrat; B 8. E), or Libby amphibole (LA; 0.65 or 6.5 mglrat; C,D, F, 8. G) asbestos and necropsied at 1d and 3 mo after a single intratraCheal instillation (magnification =40 X). (A) Control: no histopathological changes. (B) Amosite (0.65 mglrat; 1 d): presence of intra-alveolar and intra-bronchial (arrows) macrophages and polymorphonuclear (PMN) cells. (e) LA (O.65mgfrat; 1 d): mild presence of intra-alVeolar macrophages with PMN cells (arrows). (D) LA (6.5 mg/rat; 1 d): marked presence of intraalveolar macrophages Yoith polymorphonuclear cells (arrows). (E) Amosite (0.65 mgfrat; 3 mol: multiple nodules of