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

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 (MAsIII) and dimethylarsinous acid (DMAsIII) play a key role in the etiology of these diseases. Both MAsIII and DMAsIII have been detected in urine of subjects exposed to iAs. However, the rapid oxidation of DMAsIII and, to a lesser extent, MAsIII 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 MAsIII and DMAsIII 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 degrees C or -80 degrees C for up to 22 days. Both MAsIII and DMAsIII were stable in homogenates stored at -80 degrees C. In contrast, DMAsIII in homogenates stored at 0 degrees C began to oxidize to its pentavalent counterpart after 1 day; MAsIII remained stable for at least 3 weeks under these conditions. MAsIII and DMAsIII generated in UROtsa/F35 cultures were stable for 3 weeks when culture media and cell lysates were stored at 80 degrees 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.