Elaine M. Leslie

Arsenic-glutathione conjugate transport by the human multidrug resistance proteins (MRPs/ABCCs).

Millions of people world-wide are chronically exposed to inorganic forms of the environmental toxicant arsenic in drinking water. This has led to a public health crisis because arsenic is a human carcinogen, and causes a myriad of other adverse health effects. In order to prevent and treat arsenic-induced toxicity it is critical to understand the cellular handling of this metalloid. A large body of literature describes the importance of the cellular tripeptide glutathione (γ-Glu-Cys-Gly,GSH/GS) in the excretion of arsenic. The triglutathione conjugate of arsenite [As(III)(GS)(3)] and the diglutathione conjugate of monomethylarsonous acid [MMA(III)(GS)(2)] have been isolated from rat bile and mouse urine, and account for the majority of excreted arsenic, suggesting these are important transportable forms. The ATP-binding cassette (ABC) transporter proteins, multidrug resistance protein 1 (MRP1/ABCC1) and the related protein MRP2 (ABCC2), are thought to play an important role in arsenic detoxification through the cellular efflux of arsenic-GSH conjugates. Current knowledge on the cellular handling of arsenic with a special emphasis on the transport pathways of the arsenic-GSH conjugates As(III)(GS)(3), MMA(III)(GS)(2), and dimethylarsenic glutathione DMA(III)(GS), as well as, the seleno-bis(S-glutathionyl) arsinium ion [(GS)(2)AsSe](-) are reviewed.

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.

Character and Temporal Evolution of Apoptosis in Acetaminophen-Induced Acute Liver Failure

Objective:To evaluate the role of hepatocellular and extrahepatic apoptosis during the evolution of acetaminophen-induced acute liver failure. Design and Setting:A prospective observational study in two tertiary liver transplant units. Patients:Eighty-eight patients with acetaminophen-induced acute liver failure were recruited. Control groups included patients with nonacetaminophen-induced acute liver failure (n = 13), nonhepatic multiple organ failure (n = 28), chronic liver disease (n = 19), and healthy controls (n = 11). Measurements:Total and caspase-cleaved cytokeratin-18 (M65 and M30) measured at admission and sequentially on days 3, 7, and 10 following admission. Levels were also determined from hepatic vein, portal vein, and systemic arterial blood in seven patients undergoing transplantation. Protein arrays of liver homogenates from patients with acetaminophen-induced acute liver failure were assessed for apoptosis-associated proteins, and histological assessment of liver tissue was performed. Main Results:Admission M30 levels were significantly elevated in acetaminophen-induced acute liver failure and non-acetaminophen induced acute liver failure patients compared with multiple organ failure, chronic liver disease, and healthy controls. Admission M30 levels correlated with outcome with area under receiver operating characteristic of 0.755 (0.639–0.885, p < 0.001). Peak levels in patients with acute liver failure were seen at admission then fell significantly but did not normalize over 10 days. A negative gradient of M30 from the portal to hepatic vein was demonstrated in patients with acetaminophen-induced acute liver failure (p = 0.042) at the time of liver transplant. Analysis of protein array data demonstrated lower apoptosis-associated protein and higher catalase concentrations in acetaminophen-induced acute liver failure compared with controls (p < 0.05). Explant histological analysis revealed evidence of cellular proliferation with an absence of histological evidence of apoptosis. Conclusions:Hepatocellular apoptosis occurs in the early phases of human acetaminophen-induced acute liver failure, peaking on day 1 of hospital admission, and correlates strongly with poor outcome. Hepatic regenerative/tissue repair responses prevail during the later stages of acute liver failure where elevated levels of M30 are likely to reflect epithelial cell death in extrahepatic organs.

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 (MMAIII), monomethylarsonic acid (MMAV), dimethylarsinous acid (DMAIII), and dimethylarsinic acid (DMAV). 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, MMAIII, MMAV, DMAIII, and DMAV while two other hepatic basolateral MRPs (MRP3 and MRP5) did not. Transport studies with MRP4-enriched membrane vesicles revealed that the diglutathione conjugate of MMAIII, monomethylarsenic diglutathione [MMA(GS)2], and DMAV were the transported species. MMA(GS)2 and DMAV 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). DMAV 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.

Cellular arsenic transport pathways in mammals

Natural contamination of drinking water with arsenic results in the exposure of millions of people world-wide to unacceptable levels of this metalloid. This is a serious global health problem because arsenic is a Group 1 (proven) human carcinogen and chronic exposure is known to cause skin, lung, and bladder tumors. Furthermore, arsenic exposure can result in a myriad of other adverse health effects including diseases of the cardiovascular, respiratory, neurological, reproductive, and endocrine systems. In addition to chronic environmental exposure to arsenic, arsenic trioxide is approved for the clinical treatment of acute promyelocytic leukemia, and is in clinical trials for other hematological malignancies as well as solid tumors. Considerable inter-individual variability in susceptibility to arsenic-induced disease and toxicity exists, and the reasons for such differences are incompletely understood. Transport pathways that influence the cellular uptake and export of arsenic contribute to regulating its cellular, tissue, and ultimately body levels. In the current review, membrane proteins (including phosphate transporters, aquaglyceroporin channels, solute carrier proteins, and ATP-binding cassette transporters) shown experimentally to contribute to the passage of inorganic, methylated, and/or glutathionylated arsenic species across cellular membranes are discussed. Furthermore, what is known about arsenic transporters in organs involved in absorption, distribution, and metabolism and how transport pathways contribute to arsenic elimination are described.

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) (0.1-1u2009µM) 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.

Polymorphic variants of MRP4/ABCC4 differentially modulate the transport of methylated arsenic metabolites and physiological organic anions

Broad inter-individual variation exists in susceptibility to arsenic-induced tumours, likely involving differences in the ability of individuals to eliminate this metalloid. We recently identified human multidrug resistance protein 4 (MRP4/ABCC4) as a novel pathway for the cellular export of dimethylarsinic acid (DMAV), the major urinary arsenic metabolite in humans, and the diglutathione conjugate of the highly toxic monomethylarsonous acid [MMA(GS)2]. These findings, together with the basolateral and apical membrane localization of MRP4 in hepatocytes and renal proximal tubule cells, respectively, suggest a role for MRP4 in the urinary elimination of hepatic arsenic metabolites. Accordingly, we have now investigated the influence of non-synonymous single nucleotide polymorphisms (SNPs) on MRP4 levels, cellular localization, and arsenical transport. Of eight MRP4 variants (C171G-, G187W-, K304N-, G487E-, Y556C-, E757K-, V776I- and C956S-MRP4) characterized, two (V776I- and C956S-MRP4) did not localize appropriately to the plasma membrane of HEK293T and LLC-PK1 cells. Characterization of the six correctly localized mutants revealed that MMA(GS)2 transport by C171G-, G187W-, and K304N-MRP4 was 180%, 73%, and 30% of WT-MRP4 activity, respectively, whereas DMAV transport by K304N- and Y556C-MRP4 was 30% and 184% of WT-MRP4, respectively. Transport of the prototypical physiological MRP4 substrates prostaglandin E2 and 17β-estradiol 17-(β-d-glucuronide) by the six variants was also differentially affected. Thus, MRP4 variants have differing abilities to transport arsenic and endogenous metabolites through both altered function and membrane localization. Further investigation is warranted to determine if genetic variations in ABCC4 contribute to inter-individual differences in susceptibility to arsenic-induced (and potentially other) diseases.

Detection of Ophthalmic Acid in Serum from Acetaminophen-Induced Acute Liver Failure Patients Is More Frequent in Non-Survivors

Background/Aim Acetaminophen (APAP) hepatotoxicity is related to the formation of N-acetyl-p-benzoquinone imine (NAPQI), which is detoxified through conjugation with reduced glutathione (GSH). Ophthalmic acid (OA) is an analogue of GSH in which cysteine is replaced with 2-aminobutyrate. Metabolomics studies of mice with APAP-induced acute liver failure (APAP-ALF) identified OA as a marker of oxidative stress and hepatic GSH consumption. The aim of the current study was to determine whether OA is detectable in APAP-ALF human patients either early (day 2) or late (day 4) and whether OA levels were associated with in-hospital survival in the absence of liver transplant. Methods Serum samples from 130 APAP-ALF patients (82 survivors, 48 non-survivors) were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and correlated with clinical data from the United States Acute Liver Failure Study Group (US ALFSG) Registry (2004–2011). Results Survivors had significantly lower admission bilirubin (4.2 vs. 5.7 mg/dl) and lactate levels (3.3 vs. 6.5 μmol/l, p<0.05 for all). During the first 7 days of the study, survivors were less likely to require mechanical ventilation (55% vs. 88%), vasopressor support (9.8% vs. 67%) or renal replacement therapy (26% vs. 63%, p< 0.001 for all). Non-survivors were more likely to have detectable OA levels early (31% vs. 15%, p = 0.034) and late (27% vs. 11%, p = 0.02). However there were no significant differences in mean OA levels between non-survivors and survivors (early 0.48 vs. 0.36, late 0.43 vs. 0.37, P > 0.5 for all). Conclusion OA was detectable more frequently in APAP-ALF non-survivors but mean OA levels were not associated with survival. The routine clinical administration of N-acetyl cysteine could replenish GSH levels and prevent OA production.

Arsenic Triglutathione [As(GS)3] Transport by Multidrug Resistance Protein 1 (MRP1/ABCC1) Is Selectively Modified by Phosphorylation of Tyr920/Ser921 and Glycosylation of Asn19/Asn23

The ATP-binding cassette (ABC) transporter multidrug resistance protein 1 (MRP1/ABCC1) is responsible for the cellular export of a chemically diverse array of xenobiotics and endogenous compounds. Arsenic, a human carcinogen, is a high-affinity MRP1 substrate as arsenic triglutathione [As(GS)3]. In this study, marked differences in As(GS)3 transport kinetics were observed between MRP1-enriched membrane vesicles prepared from human embryonic kidney 293 (HEK) (Km 3.8 µM and Vmax 307 pmol/mg per minute) and HeLa (Km 0.32 µM and Vmax 42 pmol/mg per minute) cells. Mutant MRP1 lacking N-linked glycosylation [Asn19/23/1006Gln; sugar-free (SF)-MRP1] expressed in either HEK293 or HeLa cells had low Km and Vmax values for As(GS)3, similar to HeLa wild-type (WT) MRP1. When prepared in the presence of phosphatase inhibitors, both WT- and SF-MRP1-enriched membrane vesicles had a high Km value for As(GS)3 (3–6 µM), regardless of the cell line. Kinetic parameters of As(GS)3 for HEK-Asn19/23Gln-MRP1 were similar to those of HeLa/HEK-SF-MRP1 and HeLa-WT-MRP1, whereas those of single glycosylation mutants were like those of HEK-WT-MRP1. Mutation of 19 potential MRP1 phosphorylation sites revealed that HEK-Tyr920Phe/Ser921Ala-MRP1 transported As(GS)3 like HeLa-WT-MRP1, whereas individual HEK-Tyr920Phe- and -Ser921Ala-MRP1 mutants were similar to HEK-WT-MRP1. Together, these results suggest that Asn19/Asn23 glycosylation and Tyr920/Ser921 phosphorylation are responsible for altering the kinetics of MRP1-mediated As(GS)3 transport. The kinetics of As(GS)3 transport by HEK-Asn19/23Gln/Tyr920Glu/Ser921Glu were similar to HEK-WT-MRP1, indicating that the phosphorylation-mimicking substitutions abrogated the influence of Asn19/23Gln glycosylation. Overall, these data suggest that cross-talk between MRP1 glycosylation and phosphorylation occurs and that phosphorylation of Tyr920 and Ser921 can switch MRP1 to a lower-affinity, higher-capacity As(GS)3 transporter, allowing arsenic detoxification over a broad concentration range.