Comparative Methylation of Arsenic in Primary Hepatocytes from CC003 and CC0057 Humanized Mouse Models Expressing the Human Arsenic Methyltransferase

Inorganic arsenic (iAs), a naturally occurring metalloid found commonly in drinking water, has been linked to a variety of adverse health outcomes. In humans, detoxification of iAsIII occurs through methylation carried out by the enzyme arsenic (+3 oxidation state) methyltransferase (AS3MT). AS3MT converts iAs into monomethyl arsenic (MAs) and dimethyl arsenic (DMAs).  Humanized mice expressing the human AS3MT gene closely mimic human iAs metabolism and thus provide a better research model than wild-type mice, which methylate iAs far more efficiently. Previous studies of iAsIII methylation have largely relied on humanized mouse strains with limited genetic diversity. The present study aimed to compare iAs metabolism using genetically diverse humanized (Hu) Collaborative Cross (CC) mouse strains. The goal was to determine if differences in hepatocyte methylation capacity contribute to the observed variation in the urinary arsenic metabolite profiles of two humanized CC strains, CC003.129S6-Isr(HSA10;BORCS7-AS3MT)1Bhk/PmvMmnc (Hu CC003) and CC057.129S6-Isr(HSA10;BORCS7-AS3MT)1Bhk/UncMmnc (Hu CC057).  To do this, cultured primary hepatocytes from males in both strains were exposed to iAs or Mas and time- and concentration-dependent production of methylated metabolites was examined. Media and cell lysates were harvested to assess arsenic metabolite yields and to calculate Michaelis-Menten constants, Km and Vmax. We found no statistically significant strain-dependent differences in Km or Vmax in hepatocytes exposed to either iAs or MAs. In contrast, comparison of iAs and Mas methylation kinetics revealed that Km and Vmax were significantly higher for MAs than for iAsfor both strains. These results are consistent with lower affinity for Mas but higher capacity to methylate Mas as compared to iAs. The fact that no differences in the methylation kinetics were found between the strains may suggest that factors other than the capacity of hepatocytes to methylate iAs are responsible for the differences in arsenic metabolite profiles in urines of CC003 and CC057 mice.