Arsenicals inhibit thioredoxin reductase in cultured rat hepatocytes

Thioredoxin reductase (TR), an NADPH-dependent flavoenzyme that catalyzes the reduction of many disulfide-containing substrates, plays an important role in the cellular response to oxidative stress. Trivalent arsenicals, especially methyl As that contains trivalent arsenic (MAsIII), are potent noncompetitive inhibitors of TR purified from mouse liver. Because MAsIII is produced in the biomethylation of As, it was postulated that the extent of inhibition of TR in cultured rat hepatocytes would correlate with the intracellular concentration of methyl As. Exposure of cultured hepatocytes to inorganic As-III (iAs(III)), MAsIII, or aurothioglucose (ATG, a competitive inhibitor 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(III), similar to 10 muM for ATG, and similar to3 muM for MAsIII. In hepatocytes exposed to 1 muM MAsIII for up to 24 h, the inhibition of TR activity was 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 cell 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 progressive; at 24 h, activity was reduced similar to 35%. Following exposure to iAsIII 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 are 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 pathway for As methylation may be an important determinant of susceptibility to the toxic effects of As.