Differential Selectivity of Human and Mouse ABCC4/Abcc4 for Arsenic Metabolites

Abstract

Millions of people globally are exposed to the proven human carcinogen arsenic at unacceptable levels in drinking water. In contrast, arsenic is a poor rodent carcinogen, requiring >100-fold higher doses for tumor induction, which may be explained by toxicokinetic differences between humans and mice. The human ATP-binding cassette subfamily C (ABCC) transporter hABCC4 mediates the cellular efflux of a diverse array of metabolites, including the glutathione (GSH) conjugate of the highly toxic monomethylarsonous acid (MMA^III), monomethylarsenic diglutathione [MMA(GS)₂], and the major human urinary arsenic metabolite dimethylarsinic acid (DMA^V). Our objective was to determine if mouse Abcc4 (mAbcc4) protected against and/or transported the same arsenic species as hABCC4. The anti-ABCC4 antibody M₄I-10 epitope was first mapped to an octapeptide (⁴¹¹HVQDFTA⁴¹⁸F) present in both hABCC4 and mAbcc4, enabling quantification of relative amounts of hABCC4/mAbcc4. mAbcc4 expressed in human embryonic kidney (HEK)293 cells did not protect against any of the six arsenic species tested [arsenite, arsenate, MMA^III, monomethylarsonic acid, dimethylarsinous acid, or DMA^V], despite displaying remarkable resistance against the antimetabolite 6-mercaptopurine (>9-fold higher than hABCC4). Furthermore, mAbcc4-enriched membrane vesicles prepared from transfected HEK293 cells did not transport MMA(GS)₂ or DMA^V despite a >3-fold higher transport activity than hABCC4-enriched vesicles for the prototypic substrate 17β-estradiol-17-(β-D-glucuronide). Abcc4^(+/+) mouse embryonic fibroblasts (MEFs) were ∼3-fold more resistant to arsenate than Abcc4^(-/-) MEFs; however, further characterization indicated that this was not mAbcc4 mediated. Thus, under the conditions tested, arsenicals are not transported by mAbcc4, and differences between the substrate selectivity of hABCC4 and mAbcc4 seem likely to contribute to arsenic toxicokinetic differences between human and mouse. SIGNIFICANCE STATEMENT: Toxicokinetics of the carcinogen arsenic differ among animal species. Arsenic methylation is known to contribute to this, whereas arsenic transporters have not been considered. Human ATP-binding cassette subfamily C member 4 (hABCC4) is a high-affinity transporter of toxicologically important arsenic metabolites. Here we used multiple approaches to demonstrate that mouse Abcc4 does not protect cells against or transport any arsenic species tested. Thus, differences between hABCC4 and mAbcc4 substrate selectivity likely contribute to differences in human and mouse arsenic toxicokinetics.