Tissue distribution and urinary excretion of inorganic arsenic and its methylated metabolites in C57BL6 mice following subchronic exposure to arsenate in drinking water
Introduction
Inorganic arsenic (iAs) in both pentavalent (AsV, arsenate) and trivalent (AsIII, arsenite) forms is ubiquitous at varying levels in water, soil, air, and food. Human exposure to naturally occurring high levels of iAs in drinking water is known to cause cancers of the skin, lung, urinary bladder and other internal organs, as well as cardiovascular and peripheral vascular disease, diabetes and other non-cancer health effects (Yoshida et al., 2004, Navas-Acien et al., 2005). In recent years, growing recognition of the magnitude of arsenic-induced disease risk has prompted both the U.S. EPA and international organizations to recommend and enact lower exposure limits for inorganic arsenic in drinking water (IPCS, 2001, USEPA, 2001b).
Metabolism of AsV is generally accepted to proceed via a series of sequential reduction and oxidative methylation reactions to form mono-, di- and trimethylated products. A single enzyme, arsenic (3+ oxidation state) methyltransferase (AS3MT), has been identified which is able to catalyze all of these steps (Thomas et al., 2007). Reduction of pentavalent arsenicals can be catalyzed by other enzymes including glutathione-S-transferase omega (Zakharyan et al., 2005, Chowdhury et al., 2006) and may also occur non-enzymatically in the presence of appropriate physiological conditions (pH, oxygen tension) and endogenous reductants such as glutathione and thioredoxin (Thomas et al., 2004). The overall contribution of various enzymatic and non-enzymatic transformation mechanisms to arsenic metabolism and distribution has yet to be established. Alternative methylation pathways have also been proposed that involve glutathione conjugates as intermediates, but the in vivo relevance of these pathways is unclear (Hayakawa et al., 2005).
Identification of the active metabolite(s) for various arsenic-induced adverse effects remains elusive. Previously, iAs methylation had been thought to be solely a deactivation reaction, because it facilitates excretion of arsenic primarily in urine, and the pentavalent methylated forms of arsenic (monomethylarsonic acid, MMAV; dimethylarsinic acid, DMAV) are relatively non-toxic following acute exposure compared to either AsV or arsenite (AsIII) (Kaise et al., 1989). However, certain more recent evidence has documented that MMAIII and DMAIII have potent cytotoxic and genotoxic activity relative to their pentavalent counterparts (Kligerman et al., 2003, Wang et al., 2007). In addition DMAV has been unequivocally established as a complete bladder carcinogen in rats and multi-organ tumor promoter in rodents (Cohen et al., 2006). Cohen et al. (2002) have presented evidence to suggest that the DMAIII formed in vivo from DMAV is the active agent in bladder cell cytotoxicity and cell proliferation observed in rats exposed to DMAV. The finding that both MMAIII and DMAIII are excreted in urine of rodents and humans exposed to iAs suggests that these toxic metabolites are sufficiently stable to be distributed to other tissues (Mandal et al., 2001, Valenzuela et al., 2005).
In female B6C3F1 mice administered a single oral dose of arsenate or arsenite, it has been observed that urinary arsenic speciation is not necessarily reflective of tissue distribution (Kenyon et al., 2005a, Kenyon et al., 2005b). The time course of distribution of arsenicals in either case is both tissue-specific and dose-dependent. Overall blood levels of all arsenicals are lower compared to other tissues. Among tissues, both liver and kidney achieve the highest total arsenic levels and the predominant metabolite is iAs. The liver and kidney differ in that levels of MMA achieved are 5-to 10- fold higher in kidney. In the case of lung, DMA is the predominant metabolite both in terms of maximal concentration achieved (Cmax) and area under the curve (AUC). Similar trends were observed following 9 days repeated dosing with 0.5 mg As/kg as AsV, i.e., arsenical distribution was tissue-specific and changed with repeated exposure. This study also demonstrated that the tissue burden of MMA in blood, kidney and liver was not reflective of the profile of arsenicals excreted in urine. In contrast, the distribution of metabolites in bladder tissue was reflective of urinary excretion after nine days of repeated dosing (Hughes et al., 2003).
The objective of this study was to examine the relationship between subchronic exposure to iAs (as AsV) in drinking water and the tissue distribution and urinary excretion of iAs and its methylated metabolites. This is a critical to understanding and extrapolating the risk from prolonged exposure to iAs because earlier studies indicate that there are differing potentials among organs to accumulate various arsenicals and evidence suggests that the deleterious effects of arsenic may also be due to the action of multiple arsenicals at different stages in the process of eliciting a toxic or carcinogenic response.
Section snippets
Chemicals
Sodium arsenate was obtained from Sigma Chemical Co. (St. Louis, MO). The following arsenicals were used as standards for atomic absorption spectrometry analysis: sodium arsenite (99% pure; Sigma-Aldrich, St. Louis, MO), sodium arsenate (96%, Sigma), MMAV, disodium salt (98%, Chem Service, West Chester, PA), DMAV, and sodium salt (98%, Strem Chemicals, Inc., Newburyport, MA). MMAIII, DMAIII and TMAO were synthesized by Professor William Cullen with purities of arsenicals exceeding 95% (Cullen
Results
No signs of overt toxicity were observed in the mice throughout the duration of the study. Body weight gain was not significantly affected by arsenic exposure (Fig. 1). The calculated average daily arsenic intake determined weekly and averaged over the entire study period was 0.083 ± 0.005, 0.35 ± 0.04, 1.89 ± 0.11 and 7.02 ± 0.61 mg As/kg body weight/day, for the 0.5, 2, 10 and 50 ppm groups, respectively. Water from all treatment groups analyzed by NAA was within 2.5% of the target concentration.
Discussion
This study is unique in that we report speciated arsenic metabolite accumulation in target and non-target tissues, as well as urinary excretion, across a wide dose range under conditions of subchronic exposure to AsV in drinking water. Mice subchronically exposed to AsV accumulate arsenic in kidney, urinary bladder and lung to a much greater extent compared to blood, liver and skin. Although kidney, bladder and lung tissues preferentially accumulate arsenic, the distribution of metabolites
Disclaimer
Portions of this work were presented at the Society of Toxicology 2008 meeting. This manuscript has been reviewed in accordance with the policy of the National Health and Environmental Effects Research Laboratory, U. S. Environmental Protection Agency, and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the Agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use.
Acknowledgments
We thank Brenda Edwards and Carol Mitchell of the U.S. EPA for their assistance with animal handling and analytical work.
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