Blakely M. Adair

Arsenic (+3 Oxidation State) Methyltransferase and the Methylation of Arsenicals

Metabolic conversion of inorganic arsenic into methylated products is a multistep process that yields mono-, di-, and trimethylated arsenicals. In recent years, it has become apparent that formation of methylated metabolites of inorganic arsenic is not necessarily a detoxification process. Intermediates and products formed in this pathway may be more reactive and toxic than inorganic arsenic. Like all metabolic pathways, understanding the pathway for arsenic methylation involves identification of each individual step in the process and the characterization of the molecules which participate in each step. Among several arsenic methyltransferases that have been identified, arsenic (+3 oxidation state) methyltransferase is the one best characterized at the genetic and functional levels. This review focuses on phylogenetic relationships in the deuterostomal lineage for this enzyme and on the relation between genotype for arsenic (+3 oxidation state) methyltransferase and phenotype for conversion of inorganic arsenic to methylated metabolites. Two conceptual models for function of arsenic (+3 oxidation state) methyltransferase which posit different roles for cellular reductants in the conversion of inorganic arsenic to methylated metabolites are compared. Although each model accurately represents some aspects of enzyme’s role in the pathway for arsenic methylation, neither model is a fully satisfactory representation of all the steps in this metabolic pathway. Additional information on the structure and function of the enzyme will be needed to develop a more comprehensive model for this pathway.

Tissue distribution and urinary excretion of inorganic arsenic and its methylated metabolites in C57BL6 mice following subchronic exposure to arsenate in drinking water.

The relationship of exposure and tissue concentration of parent chemical and metabolites over prolonged exposure is a critical issue for chronic toxicities mediated by metabolite(s) rather than parent chemical alone. This is an issue for AsV because its trivalent metabolites have unique toxicities and relatively greater potency compared to their pentavalent counterparts for many endpoints. In this study, dose-dependency in tissue distribution and urinary excretion for inorganic arsenic and its methylated metabolites was assessed in female C57Bl/6 mice exposed to 0, 0.5, 2, 10 or 50 ppm arsenic (as arsenate, AsV) in their drinking water for 12 weeks. No adverse effects were observed and body weight gain did not differ significantly among groups. Urinary excretion of arsenite monomethylarsonous acid (MMA(III)), dimethylarsinous acid (DMA(III)), dimethylarsinic acid (DMAV), and trimethylarsine oxide (TMAO) increased linearly with dose, whereas AsV and monomethylarsonic acid (MMAV) excretion was non-linear with respect to dose. Total tissue arsenic accumulation was greatest in kidney > lung > urinary bladder >>> skin > blood > liver. Monomethyl arsenic (MMA, i.e. MMA(III)+MMAV) was the predominant metabolite in kidney, whereas dimethylarsenic (DMA, i.e., DMA(III)+DMAV) was the predominant metabolite in lung. Urinary bladder tissue had roughly equivalent levels of inorganic arsenic and dimethylarsenic, as did skin. These data indicate that pharmacokinetic models for arsenic metabolism and disposition need to include mechanisms for organ-specific accumulation of some arsenicals and that urinary metabolite profiles are not necessarily reflective of target tissue dosimetry.

Comprehensive analysis of arsenic metabolites by pH-specific hydride generation atomic absorption spectrometry

In a variety of biological systems, inorganic arsenic (iAs) is metabolized to yield methylated arsenicals that contain arsenic in +5 or +3 oxidation states. Atomic absorption spectrometry (AAS) coupled with a pH-specific generation of arsines has been used for selective analysis of trivalent and pentavalent inorganic, mono-, and dimethylated arsenicals in biological matrices. We have optimized this method to permit simultaneous detection and quantification of all relevant metabolites of iAs, including trimethylarsine oxide (TMAsVO). The optimization includes increasing the density of the chromatographic adsorbent used for cold-trapping of generated arsines and modification of the temperature gradient for release of arsines from the cold trap. These modifications improve the boiling-point separation of arsine, methylarsine, dimethylarsine, and trimethylarsine before the detection by AAS. Arsines from trivalent arsenicals and from TMAsVO are selectively generated at pH 6. At pH 1, arsines are generated from both tri- and pentavalent arsenicals. Thus, the optimized technique permits analysis of arsenite (iAsIII), arsenate (iAsV), monomethylarsonic acid (MAsV), monomethylarsonous acid (MAsIII), dimethylarsinic acid (DMAsV), dimethylarsinous acid (DMAsIII), and TMAsVO. The detection limits range from 0.14 ng As (for TMAsVO) to 0.40 ng As (for iAsV). Calibration curves are linear over the concentration range of 0.5–100 ng As. Recoveries vary between 85 and 124%. The precision of the method in various biological matrices ranges from 1.0 to 14.5%. Using the optimized technique, both trivalent and pentavalent methylated and dimethylated arsenicals, but not TMAsVO, have been detected in cultured primary human hepatocytes exposed to iAsIII. In contrast, TMAsVO was detected as the final product of in vitro methylation of iAsIII by rat AsIII-methyltransferase, cyt19. TMAsVO was also detected in the urine of mice treated with MAsV or DMAsV. Thus, the optimized method improves the efficiency of arsenic speciation analysis in biological matrices, providing a more comprehensive picture of the role of metabolism in the disposition and action of iAs.

Impact of life stage and duration of exposure on arsenic-induced proliferative lesions and neoplasia in C3H mice

Epidemiological studies suggest that chronic exposure to inorganic arsenic is associated with cancer of the skin, urinary bladder and lung as well as the kidney and liver. Previous experimental studies have demonstrated increased incidence of liver, lung, ovary, and uterine tumors in mice exposed to 85 ppm (approximately 8 mg/kg) inorganic arsenic during gestation. To further characterize age susceptibility to arsenic carcinogenesis we administered 85 ppm inorganic arsenic in drinking water to C3H mice during gestation, prior to pubescence and post-pubescence to compare proliferative lesion and tumor outcomes over a one-year exposure period. Inorganic arsenic significantly increased the incidence of hyperplasia in urinary bladder (48%) and oviduct (36%) in female mice exposed prior to pubescence (beginning on postnatal day 21 and extending through one year) compared to control mice (19 and 5%, respectively). Arsenic also increased the incidence of hyperplasia in urinary bladder (28%) of female mice continuously exposed to arsenic (beginning on gestation day 8 and extending though one year) compared to gestation only exposed mice (0%). In contrast, inorganic arsenic significantly decreased the incidence of tumors in liver (0%) and adrenal glands (0%) of male mice continuously exposed from gestation through one year, as compared to levels in control (30 and 65%, respectively) and gestation only (33 and 55%, respectively) exposed mice. Together, these results suggest that continuous inorganic arsenic exposure at 85 ppm from gestation through one year increases the incidence and severity of urogenital proliferative lesions in female mice and decreases the incidence of liver and adrenal tumors in male mice. The paradoxical nature of these effects may be related to altered lipid metabolism, the effective dose in each target organ, and/or the shorter one-year observational period.

Commonalities in Metabolism of Arsenicals

Environmental Context. Health effects associated with inorganic arsenic include various cancers and increased risk of diabetes. Millions of people in Bangladesh and India are at risk through use of contaminated drinking water. When humans ingest inorganic arsenic, it is rapidly converted to methylated metabolites. Although this methylation process is largely understood, the metabolism of other arsenicals (e.g. arsenosugars to dimethylarsenic) is very unclear. Connections among pathways for metabolism of various arsenicals are now being elucidated. Commonalities and differences in these pathways may be important determinants of the risk associated with exposure to these agents. Abstract. Elucidating the pathway of inorganic arsenic metabolism shows that some of methylated arsenicals formed as intermediates and products are reactive and toxic species. Hence, methylated arsenicals likely mediate at least some of the toxic and carcinogenic effects associated with exposure to arsenic. Trimethylarsonium compounds and arsenosugars are two other classes of arsenicals to which humans are routinely exposed and there is evidence that both classes are metabolized to produce methylated arsenicals. Here, we review evidence for production of methylated metabolism and consider the challenges posed in unraveling a complex web for metabolism of arsenicals in humans.