Kinetics of Arsenic Methylation by Freshly Isolated B6C3F1 Mouse Hepatocytes

Abstract The toxic and carcinogenic effects of arsenic may be mediated by both inorganic and methylated arsenic species. The methylation of arsenic III is thought to take place via sequential oxidative methylation and reduction steps to form monomethylarsenic (MMA) and dimethylarsenic (DMA) species, but recent evidence indicates that glutathione complexes of arsenic III can be methylated without oxidation. The kinetics of arsenic methylation were determined in freshly isolated hepatocytes from male B6C3F1 mice. Hepatocytes (>90% viability) were isolated by collagenase perfusion and suspended in Williams’ Medium E with various concentrations of arsenic III (sodium m -arsenite). Aliquots of the lysed cell suspension were analyzed for arsenic species by hydride generation-atomic absorption spectrometry. The formation of MMA III from sodium arsenite (1 μM) was linear with respect to time for >90 min. DMA III formation did not become significant until 60 min. MMA V and DMA V were not consistently observed in the incubations. These results suggest that the glutathione complex mechanism of methylation plays an important role in arsenic biotransformation in mouse hepatocytes. Metabolism of arsenic V was not observed in mouse hepatocytes, consistent with inhibition of arsenic V active cellular uptake by phosphate in the medium. The formation of MMA III increased with increasing arsenic III concentrations up to approximately 2 μM and declined thereafter. The concentration dependence is consistent with a saturable methylation reaction accompanied by uncompetitive substrate inhibition of the reaction by arsenic III . Kinetic analysis of the data suggested an apparent K M of approximately 3.6 μM arsenic III , an apparent V max of approximately 38.9 μg MMA III formed/L/h/million cells, and an apparent K I of approximately 1.3 μM arsenic III . The results of this study can be used in the physiologically based pharmacokinetic model for arsenic disposition in mice to predict the concentration of MMA III in liver and other tissues.