Direct analysis and stability of methylated trivalent arsenic metabolites in cells and tissues

Abstract

Chronic ingestion of water containing inorganic arsenic (iAs) has been linked to a variety of adverse health effects, including cancer, hypertension and diabetes. Current evidence suggests that the toxic methylated trivalent metabolites of iAs, methylarsonous acid (MAs^III) and dimethylarsinous acid (DMAs^III) play a key role in the etiology of these diseases. Both MAs^III and DMAs^III have been detected in urine of subjects exposed to iAs. However, the rapid oxidation of DMAs^III and, to a lesser extent, MAs^III in oxygen-rich environments leads to difficulties in the analysis of these metabolites in samples of urine collected in population studies. Results of our previous work indicate that MAs^III and DMAs^III are relatively stable in a reducing cellular environment and can be quantified in cells and tissues. In the present study, we used the oxidation state-specific hydride generation-cryotrapping-atomic absorption spectroscopy (HG-CT-AAS) to examine the presence and stability of these trivalent metabolites in the liver of mice and in UROtsa/F35 cells exposed to iAs. Tri- and pentavalent metabolites of iAs were analyzed directly (without chemical extraction or digestion). Liver homogenates prepared in cold deionized water and cell culture medium and lysates were stored at either 0 °C or −80 °C for up to 22 days. Both MAs^III and DMAs^III were stable in homogenates stored at −80 °C. In contrast, DMAs^III in homogenates stored at 0 °C began to oxidize to its pentavalent counterpart after 1 day; MAs^III remained stable for at least 3 weeks under these conditions. MAs^III and DMAs^III generated in UROtsa/F35 cultures were stable for 3 weeks when culture media and cell lysates were stored at −80 °C. These results suggest that samples of cells and tissues represent suitable material for the quantitative, oxidation state-specific analysis of As in laboratory and population studies examining the metabolism or toxic effects of this metalloid.