Am. J. Respir. Cell Mol. Biol., Volume 20, Number 6, June, 1999 1310-1318

1,1-Dichloroethylene-Induced Clara Cell Damage Is Associated with In Situ Formation of the Reactive Epoxide
Immunohistochemical Detection of Its Glutathione Conjugate

Poh-Gek Forkert

Department of Anatomy and Cell Biology, Queen's University, Kingston, Ontario, Canada

Pulmonary Clara cells are selectively damaged in mice given 1,1-dichloroethylene (DCE), a chemical used in the plastics industry. The cytotoxicity is attributed to formation of a reactive metabolite believed to be the DCE-epoxide, which was detected in vitro. We have undertaken in vivo studies to test the hypothesis that in situ formation of the DCE-epoxide within Clara cells mediates the cell-specific injury manifested after DCE exposure. Formation of the epoxide was estimated by trapping of the metabolite with glutathione (GSH) and identifying the conjugated products as 2-(S-glutathionyl) acetyl glutathione ([B]) and 2-S-glutathionyl acetate ([C]). High-pressure liquid chromatographic analyses showed that conjugates [B] and [C] were both detected in lung cytosol isolated from mice treated in vivo with [¹⁴C]DCE. Epoxide levels in the cytosol, as estimated by the total amount of conjugates formed, were dose-dependent at DCE doses ranging from 25 to 225 mg/kg. Pretreatment of mice with buthionine sulfoximine (BSO) decreased sulfhydryl levels and significantly inhibited the formation of the GSH conjugates. Epoxide levels were also reduced by pretreatment with diallyl sulfone (DASO₂), an inhibitor of the P450 isozyme CYP2E1. A polyclonal antibody was developed that is specific for conjugate [C] and that recognizes an antigen consisting of the conjugate epoxide-GSH-glutaraldehyde-bovine serum albumin. Immunohistochemical studies with this antibody revealed staining in Clara cells of mice treated with DCE. Staining was also present in Clara cells of mice treated with both BSO and DCE, but at slightly reduced levels. Reduction of this staining was more pronounced in Clara cells of mice treated with both DASO₂ and DCE. These results show that the DCE-epoxide is formed in vivo, is localized in Clara cells, and correlates with the cytotoxicity manifested in this cell type.

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