Covalent binding of acrylonitrile to specific rat liver glutathione S-transferases in vivo.
Acrylonitrile (AN) is an industrial vinyl monomer that is acutely toxic. When administered to rats, AN covalently binds to tissue proteins in a dose-dependent but nonlinear manner [Benz, F. W., Nerland, D. E., Li, J., and Corbett, D. (1997) Fundam. Appl. Toxicol. 36, 149-156]. The nonlinearity in covalent binding stems from the fact that AN rapidly depletes liver glutathione after which the covalent binding to tissue proteins increases disproportionately. The identity of the tissue proteins to which AN covalently binds is unknown. The experiments described here were conducted to begin to answer this question. Male Sprague-Dawley rats were injected subcutaneously with 115 mg/kg (2.2 mmol/kg) [2,3-(14)C]AN. Two hours later, the livers were removed, homogenized, and fractionated into subcellular components, and the radioactively labeled proteins were separated on SDS-PAGE. One set of labeled proteins was found to be glutathione S-transferase (GST). Specific labeling of the mu over the alpha class was observed. Separation of the GST subunits by HPLC followed by scintillation counting showed that AN was selective for subunit rGSTM1. Mass spectral analysis of tryptic digests of the GST subunits indicated that the site of labeling was cysteine 86. The reason for the high reactivity of cysteine 86 in rGSTM1 was hypothesized to be due to its potential interaction with histidine 84, which is unique in this subunit.
Nerland
D E
DE
Department of Pharmacology & Toxicology, University of Louisville Medical School, Louisville, Kentucky 40292, USA. d.nerland@louisville.edu
Cai
J
J
Pierce
W M
WM
Benz
F W
FW
D000181:Acrylonitrile / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002273:Carcinogens / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D005982:Glutathione Transferase / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D007553:Isotope Labeling; D008099:Liver / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D008297:Male; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D051381:Rats; D017207:Rats, Sprague-Dawley
D002273:Carcinogens; D005982:Glutathione Transferase; D000181:Acrylonitrile