Arsenicals inhibit thioredoxin reductase in cultured rat hepatocytes

Thioredoxin reductase (TR), an NADPH-dependent flavoenzyme that catalyzes t he reduction of many disulfide-containing substrates, plays an important ro le in the cellular response to oxidative stress. Trivalent arsenicals, espe cially methyl As that contains trivalent arsenic (MAsIII), are potent nonco mpetitive inhibitors of TR purified from mouse liver. Because MAsIII is pro duced in the biomethylation of As, it was postulated that the extent of inh ibition of TR in cultured rat hepatocytes would correlate with the intracel lular concentration of methyl As. Exposure of cultured hepatocytes to inorg anic As-III (iAs(III)), MAsIII, or aurothioglucose (ATG, a competitive inhi bitor of TR activity) for 30 min caused a concentration-dependent reduction in TR activity. The estimated IC50 was much greater than 100 muM for iAS(I II), similar to 10 muM for ATG, and similar to3 muM for MAsIII. In hepatocy tes exposed to 1 muM MAsIII for up to 24 h, the inhibition of TR activity w as maximal (similar to 40%) after exposure for 15 min. After exposure for 3 h [when most MAsIII has been converted to dimethyl As (DMAs)], TR activity in these cells had returned to control levels. Notably, exposure of the ce ll to 50 muM DMAsIII did not affect TR activity. In hepatocytes exposed to 10 muM iAs(III) for up to 24 h, the inhibition of TR activity was progressi ve; at 24 h, activity was reduced similar to 35%. Following exposure to iAs III or MAsIII, the extent of inhibition of TR activity correlated strongly with the intracellular concentration of MAs. Taken together, these results suggest that arsenicals formed in the course of cellular metabolism of As a re potent inhibitors of TR activity. In particular, MAsIII, an intermediate in the metabolic pathway, is an especially potent inhibitor of TR. Hence, the capacity of cells to produce or consume the intermediates in the pathwa y for As methylation may be an important determinant of susceptibility to t he toxic effects of As.