Kinetics of arsenic methylation by freshly isolated B6C3F1 mouse hepatocytes

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 microM) 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 microM 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 microM arsenic(III), an apparent V(max) of approximately 38.9 microg MMA(III) formed/L/h/million cells, and an apparent K(I) of approximately 1.3 microM 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.