Methylarsenicals and Arsinothiols Are Potent Inhibitors of Mouse Liver Thioredoxin Reductase

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Abstract
Thioredoxin reductase (TR, EC 1.6.4.5) was purified 5800-fold from the livers of adult male B6C3F1 mice. The estimated molecular mass of the purified protein was about 57 kDa. The activity of the purified enzyme was monitored by the NADPH-dependent reduction of 5,5‘-dithiobis(2-nitrobenzoic acid) (DTNB); this activity was fully inhibited by 1 μM aurothioglucose. Arsenicals and arsinothiols, complexes of AsIII-containing compounds with l-cysteine or glutathione, were tested as inhibitors of the DTNB reductase activity of the purified enzyme. Pentavalent arsenicals were much less potent inhibitors than trivalent arsenicals. Among all the arsenicals, CH3AsIII was the most potent inhibitor of TR. CH3AsIII was found to be a competitive inhibitor of the reduction of DTNB (Ki ∼ 100 nM) and a noncompetitive inhibitor of the oxidation of NADPH. The inhibition of TR by CH3AsIII was time-dependent and could not be reversed by the addition of a dithiol-containing molecule, 2,3-dimercaptosuccinic acid, to the reaction mixture. The inhibition of TR by CH3AsIII required the simultaneous presence of NADPH in the reaction mixture. However, unlike other pyridine nucleotide disulfide oxidoreductases, there was no evidence that mouse liver TR was inactivated by exposure to NADPH. Treatment with CH3AsIII did not increase the NADPH oxidase activity of the purified enzyme. Thus, CH3AsIII, a putative intermediate in the pathway for the biomethylation of As, is a potent and irreversible inhibitor of an enzyme involved in the response of the cell to oxidative stress.

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