Multidrug Resistance Protein 1 (MRP1/ABCC1)-Mediated Cellular Protection and Transport of Methylated Arsenic Metabolites Differs between Human Cell Lines.

The ATP-binding cassette (ABC) transporter multidrug resistance protein 1 (MRP1/ABCC1) protects cells from arsenic (a proven human carcinogen) through the cellular efflux of arsenic triglutathione [As(GS)3] and the diglutathione conjugate of monomethylarsonous acid [MMA(GS)2]. Previously, differences in MRP1 phosphorylation (at Y920/S921) and N-glycosylation (at N19/N23) were associated with marked differences in As(GS)3 transport kinetics between HEK293 and HeLa cell lines. In the current study, cell line differences in MRP1-mediated cellular protection and transport of other arsenic metabolites were explored. MRP1 expressed in HEK293 cells reduced the toxicity of the major urinary arsenic metabolite dimethylarsinic acid (DMAV), and HEK-WT-MRP1-enriched vesicles transported DMAV with high apparent affinity and capacity (Km 0.19 µM, Vmax 342 pmol⋅mg-1protein⋅min-1). This is the first report that MRP1 is capable of exporting DMAV, critical for preventing highly toxic dimethylarsinous acid formation. In contrast, DMAV transport was not detected using HeLa-WT-MRP1 membrane vesicles. MMA(GS)2 transport by HeLa-WT-MRP1 vesicles had a greater than threefold higher Vmax compared with HEK-WT-MRP1 vesicles. Cell line differences in DMAV and MMA(GS)2 transport were not explained by differences in phosphorylation at Y920/S921. DMAV did not inhibit, whereas MMA(GS)2 was an uncompetitive inhibitor of As(GS)3 transport, suggesting that DMAV and MMA(GS)2 have nonidentical binding sites to As(GS)3 on MRP1. Efflux of different arsenic metabolites by MRP1 is likely influenced by multiple factors, including cell and tissue type. This could have implications for the impact of MRP1 on both tissue-specific susceptibility to arsenic-induced disease and tumor sensitivity to arsenic-based therapeutics.

Characterization of arsenic hepatobiliary transport using sandwich-cultured human hepatocytes.

Arsenic is a proven human carcinogen and is associated with a myriad of other adverse health effects. This metalloid is methylated in human liver to monomethylarsonic acid (MMA(V)), monomethylarsonous acid (MMA(III)), dimethylarsinic acid (DMA(V)), and dimethylarsinous acid (DMA(III)) and eliminated predominantly in urine. Hepatic basolateral transport of arsenic species is ultimately critical for urinary elimination; however, these pathways are not fully elucidated in humans. A potentially important human hepatic basolateral transporter is the ATP-binding cassette (ABC) transporter multidrug resistance protein 4 (MRP4/ABCC4) that in vitro is a high-affinity transporter of DMA(V) and the diglutathione conjugate of MMA(III) [MMA(GS)(2)]. In rats, the related canalicular transporter Mrp2/Abcc2 is required for biliary excretion of arsenic as As(GS)(3) and MMA(GS)(2). The current study used sandwich cultured human hepatocytes (SCHH) as a physiological model of human arsenic hepatobiliary transport. Arsenic efflux was detected only across the basolateral membrane for 9 out of 14 SCHH preparations, 5 had both basolateral and canalicular efflux. Basolateral transport of arsenic was temperature- and GSH-dependent and inhibited by the MRP inhibitor MK-571. Canalicular efflux was completely lost after GSH depletion suggesting MRP2-dependence. Treatment of SCHH with As(III) (0.1-1 µM) dose-dependently increased MRP2 and MRP4 levels, but not MRP1, MRP6, or aquaglyceroporin 9. Treatment of SCHH with oltipraz (Nrf2 activator) increased MRP4 levels and basolateral efflux of arsenic. In contrast, oltipraz increased MRP2 levels without increasing biliary excretion. These results suggest arsenic basolateral transport prevails over biliary excretion and is mediated at least in part by MRPs, most likely including MRP4.

A novel pathway for arsenic elimination: human multidrug resistance protein 4 (MRP4/ABCC4) mediates cellular export of dimethylarsinic acid (DMAV) and the diglutathione conjugate of monomethylarsonous acid (MMAIII).

Hundreds of millions of people worldwide are exposed to unacceptable levels of arsenic in drinking water. This is a public health crisis because arsenic is a Group I (proven) human carcinogen. Human cells methylate arsenic to monomethylarsonous acid (MMA(III)), monomethylarsonic acid (MMA(V)), dimethylarsinous acid (DMA(III)), and dimethylarsinic acid (DMA(V)). Although the liver is the predominant site for arsenic methylation, elimination occurs mostly in urine. The protein(s) responsible for transport of arsenic from the liver (into blood), ultimately for urinary elimination, are unknown. Human multidrug resistance protein 1 (MRP1/ABCC1) and MRP2 (ABCC2) are established arsenic efflux pumps, but unlike the related MRP4 (ABCC4) are not present at the basolateral membrane of hepatocytes. MRP4 is also found at the apical membrane of renal proximal tubule cells, making it an ideal candidate for urinary arsenic elimination. In the current study, human MRP4 expressed in HEK293 cells reduced the cytotoxicity and cellular accumulation of arsenate, MMA(III), MMA(V), DMA(III), and DMA(V) while two other hepatic basolateral MRPs (MRP3 and MRP5) did not. Transport studies with MRP4-enriched membrane vesicles revealed that the diglutathione conjugate of MMA(III), monomethylarsenic diglutathione [MMA(GS)(2)], and DMA(V) were the transported species. MMA(GS)(2) and DMA(V) transport was osmotically sensitive, allosteric (Hill coefficients of 1.4 ± 0.2 and 2.9 ± 1.2, respectively), and high affinity (K0.5 of 0.70 ± 0.16 and 0.22 ± 0.15 µM, respectively). DMA(V) transport was pH-dependent, with highest affinity and capacity at pH 5.5. These results suggest that human MRP4 could be a major player in the elimination of arsenic.

Monomethylarsenic diglutathione transport by the human multidrug resistance protein 1 (MRP1/ABCC1).

The ATP-binding cassette (ABC) transporter protein multidrug resistance protein 1 (MRP1; ABCC1) plays an important role in the cellular efflux of the high-priority environmental carcinogen arsenic as a triglutathione conjugate [As(GS)(3)]. Most mammalian cells can methylate arsenic to monomethylarsonous acid (MMA(III)), monomethylarsonic acid (MMA(V)), dimethylarsinous acid (DMA(III)), and dimethylarsinic acid (DMA(V)). The trivalent forms MMA(III) and DMA(III) are more reactive and toxic than their inorganic precursors, arsenite (As(III)) and arsenate (As(V)). The ability of MRP1 to transport methylated arsenicals is unknown and was the focus of the current study. HeLa cells expressing MRP1 (HeLa-MRP1) were found to confer a 2.6-fold higher level of resistance to MMA(III) than empty vector control (HeLa-vector) cells, and this resistance was dependent on GSH. In contrast, MRP1 did not confer resistance to DMA(III), MMA(V), or DMA(V). HeLa-MRP1 cells accumulated 4.5-fold less MMA(III) than HeLa-vector cells. Experiments using MRP1-enriched membrane vesicles showed that transport of MMA(III) was GSH-dependent but not supported by the nonreducing GSH analog, ophthalmic acid, suggesting that MMA(III)(GS)(2) was the transported form. MMA(III)(GS)(2) was a high-affinity, high-capacity substrate for MRP1 with apparent K(m) and V(max) values of 11 µM and 11 nmol mg(-1)min(-1), respectively. MMA(III)(GS)(2) transport was osmotically sensitive and inhibited by several MRP1 substrates, including 17ß-estradiol 17-(ß-D-glucuronide) (E(2)17ßG). MMA(III)(GS)(2) competitively inhibited the transport of E(2)17ßG with a K(i) value of 16 µM, indicating that these two substrates have overlapping binding sites. These results suggest that MRP1 is an important cellular protective pathway for the highly toxic MMA(III) and have implications for environmental and clinical exposure to arsenic.

Comparative toxicity of arsenic metabolites in human bladder cancer EJ-1 cells.

The human bladder is one of the primary target organs for arsenic-induced carcinogenicity, and arsenic metabolites in urine have been suspected to be directly involved in carcinogenesis. Thioarsenicals are commonly found in human and animal urine and are also considered to be highly toxic arsenic metabolites. The present study was performed to gain insight into the toxicity and accumulation of arsenic species found in urine, including arsenate (iAs(V)), arsenite (iAs(III)), monomethylarsonic acid (MMA(V)), monomethylmonothioarsonic acid (MMMTA(V)), dimethylarsinic acid (DMA(V)), dimethylarsinous acid (DMA(III)), dimethylmonothioarsinic acid, (DMMTA(V)), and dimethyldithioarsinic acid (DMDTA(V)) in human bladder cancer EJ-1 cells. The order of cytotoxicity of these arsenic compounds in EJ-1 human bladder cancer cells was DMA(III), DMMTA(V) > iAs(III) ≫ iAs(V) > MMMTA(V) > MMA(V), DMA(V), and DMDTA(V), indicating that the sulfur-containing DMMTA(V) was among the most toxic arsenic compounds similar to trivalent DMA(III). We further characterized the DNA damage, generation of highly reactive oxygen species (hROS), and expression of proteins p21 and p53 in cells after exposure to iAs(III), DMA(III), and DMMTA(V). Cellular exposure to DMMTA(V) resulted in reduced protein expression of p53 and p21, increased DNA damage, and increased intracellular hROS (hydroxyl radical). In contrast, iAs(III) significantly increased the protein expression of p21 and p53 and did not increase the hROS at the IC(50). Intracellular glutathione (GSH) was reduced by 60% after exposure to DMA(III) or DMMTA(V), suggesting that DMMTA(V) causes cell death through oxidative stress. In contrast, GSH levels increased in cells exposed to iAs(III), and hROS only increased after a long exposure to iAs(III). Our findings demonstrate that DMMTA(V) may be one of the most toxicologically potent arsenic species, relevant to arsenic-induced carcinogenicity in the urinary bladder.

Selenium-dependent and -independent transport of arsenic by the human multidrug resistance protein 2 (MRP2/ABCC2): implications for the mutual detoxification of arsenic and selenium.

Simultaneous exposure of lab animals to toxic doses of the human carcinogen arsenic (As) and the essential trace element selenium (Se) results in a remarkable mutual detoxification. A likely basis for this is the in vivo formation and biliary excretion of seleno-bis(S-glutathionyl) arsinium ion [(GS)(2)AsSe](-); however, the transport protein responsible for the biliary efflux of [(GS)(2)AsSe](-) has not been identified. The multidrug resistance protein 2 (MRP2/ABCC2) is an adenosine triphosphate (ATP)-binding cassette transporter expressed at the canalicular membrane of hepatocytes. Rat Mrp2 is known to excrete the As glutathione (GSH/GS-) conjugates arsenic triglutathione [As(GS)(3)] and monomethyl arsenic diglutathione [CH(3)As(GS)(2)] into bile, and in vitro studies have established As(GS)(3) as a substrate for human MRP2. In the present study, membrane vesicles prepared from human embryonic kidney (HEK293T) cells transfected with human MRP2 were used to demonstrate that MRP2 transports [(GS)(2)AsSe](-). In addition, the characteristics of MRP2 transport of As(GS)(3) and [(GS)(2)AsSe](-) were investigated. As(GS)(3) and [(GS)(2)AsSe](-) are chemically labile and have the potential to dissociate. However, arsenite (As(III)) +/- selenite (Se(IV)) transport was not detected in the absence of GSH or in the presence of the non-reducing GSH analog, ophthalmic acid, suggesting that the conjugates are the transported forms. The apparent K(m) values for [(GS)(2)AsSe](-) and As(GS)(3) were 1.7 and 4.2 microM, respectively, signifying high relative affinities. Membrane vesicles prepared from human erythrocytes, which express the MRP2-related MRP1/ABCC1, MRP4/ABCC4 and MRP5/ABCC5, transported As(GS)(3) in an MRP1- and ATP-dependent manner but did not transport [(GS)(2)AsSe](-). These results have important implications for the Se-dependent and -independent disposition of As.