Glutathione Modulates Recombinant Rat Arsenic (+3 Oxidation State) Methyltransferase-Catalyzed Formation of Trimethylarsine Oxide and Trimethylarsine

Abstract

Humans and other species enzymatically convert inorganic arsenic (iAs) into methylated metabolites. Although the major metabolites are mono- and dimethylated arsenicals, trimethylated arsenicals have been detected in urine following exposure to iAs. The AS3MT gene encodes an arsenic (+3 oxidation state) methyltransferase, which catalyzes both the oxidative methylation of trivalent arsenicals and the reduction of pentavalent arsenicals. In reaction mixtures containing recombinant rat AS3MT (rrAS3MT) and radiolabeled arsenite, mono- and dimethylated arsenicals and a third radiolabeled product can be resolved by thin-layer chromatography. Hydride generation atomic absorption spectrometry and electrospray ionization mass spectrometry identified the third reaction product as trimethylarsine oxide. The addition of glutathione to reaction mixtures containing radiolabeled arsenite and rrAS3MT increased the yield of methylated and dimethylated arsenicals but suppressed the formation of trimethylarsine oxide. Although a dimethylarsenic-glutathione complex was rapidly converted to trimethylarsine oxide, the addition of a molar excess of glutathione to dimethylarsenic suppressed the production of trimethylarsine oxide. The nonquantitative recovery of radioarsenic from reaction mixtures suggested that AS3MT catalyzed the formation of a volatile arsenical. This volatile species was identified as trimethylarsine. Thus, AS3MT catalyzes the formation of all products in a reaction sequence leading from an inorganic to a volatile methylated arsenical. The regulation of this pathway by intracellular glutathione may be an important determinant of the pattern and extent of formation of arsenicals.

Commonalities in Metabolism of Arsenicals

“…2). [6,7] In human uroepithelial cells that neither express AS3MT nor methylate iAs, heterologous expression of recombinant rat AS3MT confers methylation capacity and modulates cytotoxicity of arsenicals. [8] Intermediates and products in this pathway differ in reactivity and toxicity.…”

“…As noted above, trimethylarsine oxide is a liver carcinogen in rats. [10] AS3MT catalyzes conversion of trimethylarsine oxide to trimethylarsine, [7] a volatile genotoxic species. [22] Chickens treated with arsenocholine exhale a strong garlic-like odor consistent with trimethylarsine expiration; [23] thus, the chicken orthologue of mammalian AS3MT gene (J. Li et al, in preparation) may catalyze the final reductive step in this pathway.…”

Oxidation state specific generation of arsines from methylated arsenicals based on l-cysteine treatment in buffered media for speciation analysis by hydride generation-automated cryotrapping-gas chromatography-atomic absorption spectrometry with the multiatomizer

“…One of the possible analytical approaches is an oxidation state selective hydride generation followed by a cryotrapping and gas chromatography (HG-CT), i.e., trapping of volatile arsines under liquid nitrogen and their subsequent volatilization by gradual heating and separation according to boiling points and chromatographic properties of the trap [1], with the detection by methods of analytical atomic spectroscopy. The HG-CT can be used for analysis of arsenicals which produce volatile arsines upon reaction with sodium borohydride, including all known metabolites of iAs found in mammalian species [1,9]. Another advantage of HG-CT as compared to a chromatography separation of As species prior to detection approach usually by inductively coupled plasma-mass spectrometry (ICP MS) or hydride generation -atomic fluorescence [7] is that the analysis in important complex biological matrices such as urine or cell lysates can be performed directly with minimum sample pretreatment.…”

“…The first dimension, the cryotrapping and separation of arsines generated from iAs, MAs, DMAs and TMAs(V)O species, regardless of the oxidation state of As, has been well developed and documented [1,9,12,13] and reviewed [14]. The main drawback that has prevented a widespread application of the HG-CT based methods is the labor intensiveness associated with the switching from cooling to heating of the cryogenic trap, which is often controlled manually.…”

“…This approach has widely been used for the analysis of iAs(III) and iAs(V) [21,22]. Only recent work has shown that the pH-specific HG can also been applied for the analysis of methylated tri-and pentavalent arsenicals [1,9,10].…”