Reduction of the Pentavalent Arsenical Dimethylarsinic Acid and the GSTO1 Substrate S-(4-Nitrophenacyl)glutathione by Rat Liver Cytosol: Analyzing the Role of GSTO1 in Arsenic Reduction.

Abstract

Dimethylarsinic acid (DMAs(V)) is the major urinary metabolite of inorganic arsenic. The relatively atoxic DMAs(V) is reduced in the body to the much more toxic and thiol-reactive dimethylarsinous acid (DMAs(III)). Glutathione S-transferase omega 1 (GSTO1) can catalyze this toxification step; however, its role in the reduction of DMAs(V) in vivo or by tissue extracts is unclear. Therefore, we assessed the role of GSTO1 in the reduction of DMAs(V) to DMAs(III) by rat liver cytosol. The experiments revealed that glutathione (GSH) supported the cytosolic DMAs(V) reduction specifically and that GSH analogues and GSH conjugates, such as S-alkylglutathiones and S-(4-nitrophenacyl)glutathione (4-NPG; a GSTO1 specific substrate), inhibited the formation of DMAs(III). Observations in line with the view that GSTO1 catalyzes the cytosolic reduction of DMAs(V) include (i) findings pointing to the presence of a GSH-binding site on the DMAs(V)-reducing cytosolic enzyme, (ii) identical responsiveness of the DMAs(V)- and 4-NPG-reducing activities in rat liver cytosol to the GSTO1 specific inhibitors KT53 and chloromethylfluorescein diacetate, and (iii) perfect coelution of the two activities during affinity and anion exchange chromatography of cytosolic proteins. Other observations appear ambiguous as to the role of GSTO1 in the cytosolic reduction of DMAs(V). These include the different sensitivities of the DMAs(V)-reducing and GSTO1 activities to aurothioglucose, trivalent antimony, and zinc ions, as well as the preserved GSTO1 activity in cytosols whose DMAs(V)-reducing activity was lost due to spontaneous thiol oxidation. These disparate findings may be reconciled by assuming that GSTO1 catalyzes the reduction of both DMAs(V) and 4-NPG in rat liver cytosol; however, the enzyme employs different sites and/or mechanisms when reducing these substrates.