Styblo M, Yamauchi H, Thomas D J
Curriculum in Toxicology, University of North Carolina at Chapel Hill 27514, USA.
Toxicol Appl Pharmacol. 1995 Dec;135(2):172-8. doi: 10.1006/taap.1995.1220.
The time course and extent of methylation of 1 microM arsenite (iAsIII), arsenate (iAsV), methylarsenite (MeAsIII), methylarsenate (MeAsV), and MeAsIII-diglutathione complex (MeAsIII(GS)2) were examined in an in vitro assay system that contained rat liver cytosol. Precursor arsenicals and methylated metabolites were analyzed by thin-layer chromatography (TLC) or by hydride generation-atomic absorption spectrophotomoetry (HG-AAS). More than 90% of iAsIII was converted to a dimethylated species (Me2As) during a 90-min incubation at 37 degrees C; the amount of monomethylated metabolite was maximal at 15 min. In contrast, only 40% of iAsV was dimethylated during a 90-min incubation. Comparison of the yields of methylated species in the whole in vitro assay system as determined by HG-AAS and in an ultrafiltrate prepared from the in vitro assay system as determined by TLC indicated that nearly 70% of the dimethylated metabolite (possibly Me2AsIII) that was produced during a 90-min incubation was bound to proteins (> 10 kDa). The percentage of protein-bound arsenic in the assay system incubated at 0 degree C with trivalent arsenicals was three-to fivefold greater than the binding of corresponding pentavalent species. This indicated that both iAsIII and trivalent organoarsenicals interact avidly with proteins. Both MeAsIII prepared by metabisulfite-thiosulfate reduction of MeAsV and a MeAsIII(GS)2 were quantitatively converted to Me2As during 90-min incubation. In contrast, only 3% of MeAsV was dimethylated during this interval. These results suggest that trivalent arsenicals are preferred substrates for methylation reactions and that the reduction of As from pentavalent to trivalent states may be a critical step in the control of the rate of metabolism of As.
在含有大鼠肝细胞溶胶的体外分析系统中，研究了1微摩尔亚砷酸盐（iAsIII）、砷酸盐（iAsV）、甲基亚砷酸盐（MeAsIII）、甲基砷酸盐（MeAsV）和MeAsIII - 二谷胱甘肽复合物（MeAsIII(GS)2）的甲基化时间进程和程度。通过薄层色谱法（TLC）或氢化物发生 - 原子吸收分光光度法（HG - AAS）分析前体砷化合物和甲基化代谢物。在37℃孵育90分钟期间，超过90%的iAsIII转化为二甲基化产物（Me2As）；单甲基化代谢物的量在15分钟时达到最大值。相比之下，在90分钟孵育期间，只有40%的iAsV发生二甲基化。通过HG - AAS测定的整个体外分析系统中甲基化产物的产率与通过TLC测定的由体外分析系统制备的超滤液中甲基化产物的产率比较表明，在90分钟孵育期间产生的近70%的二甲基化代谢物（可能是Me2AsIII）与蛋白质（>10 kDa）结合。在0℃与三价砷化合物孵育的分析系统中，蛋白质结合砷的百分比比相应五价物种的结合高三到五倍。这表明iAsIII和三价有机砷化合物都与蛋白质有强烈的相互作用。通过偏亚硫酸氢盐 - 硫代硫酸盐还原MeAsV制备的MeAsIII和MeAsIII(GS)2在90分钟孵育期间都定量转化为Me2As。相比之下，在此期间只有3%的MeAsV发生二甲基化。这些结果表明，三价砷化合物是甲基化反应的优选底物，并且将砷从五价还原为三价状态可能是控制砷代谢速率的关键步骤。
