Role of MRP2 and GSH in intrahepatic cycling of toxins

doi:10.1016/S0300-483X(01)00459-0

Toxicology

Volume 167, Issue 1, 5 October 2001, Pages 73-81

https://doi.org/10.1016/S0300-483X(01)00459-0 Get rights and content

Abstract

MRP2 is a canalicular transporter in hepatocytes mediating the transport of a wide spectrum of amphipathic compounds. This includes organic anions but also compounds complexed with GSH as, e.g. α-naphthylisothiocyanate (ANIT) and arsenite. These reversible complexes may fall apart in bile after MRP2-mediated transport, which induces high concentrations of the toxic compound in the biliary tree. To further investigate the role of MRP2 in transport and toxicity of both compounds, we conducted experiments in transduced polarized epithelial cells and in vivo, using the Mrp2-deficient TR⁻ rat as a model. Our results show, that in MRP2-transduced MDCK II cells both compounds induce disproportionally strong apical GSH secretion. This induction of GSH secretion was not observed in the parent cells lacking MRP2 expression. This indicated that after transport via MRP2 both complexes released GSH upon which the compound could re-enter the cells. The resulting cycling of both toxins led to concentration dependent GSH depletion of the cells. To further test our hypothesis we administered arsenite (12.5 μmol absolute i.v.) to Wistar and Mrp2-deficient TR⁻ rats and collected bile. While both arsenite and GSH secretion were absent in TR⁻ rats, the total secretion of arsenite into Wistar bile (2.91 μmol) was accompanied by a excess secretion of 24 μmol GSH, indicating that arsenite undergoes multiple cycles of GSH complexation. We also administered ANIT to both animal models and could show that TR⁻ rats are protected from ANIT induced cholestasis. This indicates that Mrp2-mediated biliary secretion of GS-ANIT is a prerequisite for development of cholestasis in rats. We hypothesize that the toxic parent compound ANIT is regenerated in the biliary tree where it can exert its toxic properties on bile duct epithelial cells.

Introduction

MRP2 (ABCC2), initially termed canalicular multispecific organic anion transporter (cMOAT), has been identified first as a transporter for organic anions at the canalicular membrane of the hepatocyte. This comprises compounds which are anionic by themselves as well as compounds which have been conjugated to glucuronic acid, sulfate or glutathione to facilitate excretion. However, it has been shown now, that also uncharged compounds can be transported by MRP transporters. This concerns aflatoxin B1 (Loe et al., 1997) and vincristine (Loe et al., 1998) for MRP1 and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) for MRP2 (Dietrich et al., 2001). In these cases, it has been hypothesized that co-transport with glutathione (GSH) might play a permissive role for transport via MRP2.

Labile complexing of compounds with GSH was shown to be a detoxification mechanism for α-naphthylisothiocyanate (ANIT) (Carpenter-Deyo et al., 1991) and arsenite (Gyurasics et al., 1991). Those compounds therefore are candidates for canalicular transport by MRP2. For arsenite, exactly this has been shown very recently (Kala et al., 2000), for ANIT this has been hypothesized (Orsler et al., 1999).

ANIT is known for a long time as a cholestatic compound and has been widely used as a model compound for induction of intrahepatic cholestasis (Becker and Plaa, 1965, Capizzo and Roberts, 1970). ANIT is complexed to GS-ANIT in the hepatocyte and secreted as such into bile. This GS-ANIT complex is labile at pH 7.4 and falls apart within 15 min after transport into bile (Carpenter-Deyo et al., 1991). It is still under debate whether the biliary epithelium or the hepatocytes are the primary target of ANIT-mediated toxicity. For both cell types toxic effects, visualized as morphological changes, have been demonstrated (Connolly et al., 1988, Kossor et al., 1993, Orsler et al., 1999), but the relative contribution of these respective effects on overall toxicity is still unknown. A first aim of our study was to show that MRP2 mediates extrusion of GS-ANIT out of the hepatocyte. Furthermore it might be important for mechanistic elucidation of toxicity to determine whether cholestatic incidents already occur with ANIT in the hepatocyte or whether biliary secretion of ANIT is a prerequisite for that. To test this we compared the effect of ANIT in normal Wistar rats and Mrp2-deficient TR⁻ rats by monitoring bile secretion and liver injury.

Another aim of our study was to test the hypothesis whether ANIT and arsenite are able to cycle back into cells after being secreted from these cells as GSH complexed compounds. This has been speculated for ANIT earlier, when a relationship of 1:35 between ANIT and GSH was found in bile of ANIT exposed rats (Jean et al., 1995). We now report similar findings for the reversible complex of arsenite and GSH.

Section snippets

Chemicals

ANIT, Acivicin and buthionine-sulfoximine were from Sigma, St. Louis, Missouri, USA. Sodium arsenite solution (NaAsO₂; 0.05 M), dimethylsulfoxid (DMSO) and all other chemicals used for analysis or HPLC were analytical grade and were bought from Merck, Darmstadt, Germany.

Cell experiments

Transduction of the polarized canine kidney cell line MDCK II with human MRP2 and characterization of the respective product has been described previously (Evers et al., 1998). Expression of the protein was verified using

In vitro experiments

Adding ANIT or arsenite to the basal or apical compartment of MRP2-transduced cells, grown on polycarbonate filters, resulted in concentration dependent apical secretion of GSH with concomitant GSH depletion of the cells (Fig. 1A, C, E and G). Apical GSH secretion was significantly higher in MRP2-expressing cells than in untreated transduced cells or in treated parent cells. GSH depletion in treated transduced cells at higher concentrations was significantly stronger than in treated

Discussion

Both ANIT and arsenite induce GSH secretion in MRP2-transduced MDCK II cells, while this effect is much lower or absent in untransduced parent cells. Together with earlier findings for both compounds (Carpenter-Deyo et al., 1991, Orsler et al., 1999, Kala et al., 2000), this is indicative for MRP2 mediated transport of GSH complexed forms of both compounds. While for arsenite this transport property of MRP2 was already shown earlier (Kala et al., 2000), this conclusion for ANIT can be drawn on

Acknowledgements

We thank Harry Aiking (Free University, Amsterdam) for quantifying arsenite in our bile samples.

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Citation Excerpt :
These results are consistent with studies comparing Mrp2-deficient TR- Wistar rats to their WT counterparts showing that Mrp2 is responsible for > 99% of total arsenic biliary excretion (in the forms As(GS)3 and MMA(GS)2) [11]. Rats tend to have more extensive biliary excretion than other animals [12,60,65,75], and have markedly different arsenic toxicokinetics compared to humans, thus it was critical to show this in a human model. Multiple chemical forms of selenium have been used as supplements, including purified selenite and SeMet [76,77], as well as selenium mixtures contained in selenized yeast and selenium-enriched lentils (predominantly SeMet in combination with multiple other selenium species potentially including MeSeCys) [30,31,34,78–80].
Millions of people worldwide are exposed to unacceptable levels of arsenic, a proven human carcinogen, in drinking water. In animal models, arsenic and selenium are mutually protective through formation and biliary excretion of seleno-bis (S-glutathionyl) arsinium ion [(GS)₂AsSe]⁻. Selenium-deficient humans living in arsenic-endemic regions are at increased risk of arsenic-induced diseases, and may benefit from selenium supplementation. The influence of selenium on human arsenic hepatobiliary transport has not been studied using optimal human models. HepaRG cells, a surrogate for primary human hepatocytes, were used to investigate selenium (selenite, selenide, selenomethionine, and methylselenocysteine) effects on arsenic hepatobiliary transport. Arsenite + selenite and arsenite + selenide at different molar ratios revealed mutual toxicity antagonism, with the latter being higher. Significant levels of arsenic biliary excretion were detected with a biliary excretion index (BEI) of 14 ± 8%, which was stimulated to 32 ± 7% by selenide. Consistent with the formation and biliary efflux of [(GS)₂AsSe]⁻, arsenite increased the BEI of selenide from 0% to 24 ± 5%. Arsenic biliary excretion was lost in the presence of selenite, selenomethionine, and methylselenocysteine. Sinusoidal export of arsenic was stimulated ∼1.6-fold by methylselenocysteine, but unchanged by other selenium forms. Arsenic canalicular and sinusoidal transport (±selenide) was temperature- and GSH-dependent and inhibited by MK571. Knockdown experiments revealed that multidrug resistance protein 2 (MRP2/ABCC2) accounted for all detectable biliary efflux of arsenic (±selenide). Overall, the chemical form of selenium and human MRP2 strongly influenced arsenic hepatobiliary transport, information critical for human selenium supplementation in arsenic-endemic regions.
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¹: Present address: Medizinische Klinik III, Universitätsklinikum der RWTH, Pauwelsstr. 30, 52057 Aachen, Germany.

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Role of MRP2 and GSH in intrahepatic cycling of toxins

Abstract

Introduction

Section snippets

Chemicals

Cell experiments

In vitro experiments

Discussion

Acknowledgements

Toxicol. Appl. Pharmacol.

Toxicol. Appl. Pharmacol.

Biochem. Pharmacol.

Toxicol. Appl. Pharmacol.

Biochem. Pharmacol.

Biochem. Pharmacol.

J. Biol. Chem.

Toxicol. Appl. Pharmacol.

Toxicol. Appl. Pharmacol.

Biochim. Biophys. Acta

Anal. Biochem.