Differential regulation of canalicular multispecific organic anion transporter (cMOAT) expression by the chemopreventive agent oltipraz in primary rat hepatocytes and in rat liver
Arnaud Courtois,
Léa Payen,
Laurent Vernhet,
Fabrice Morel,
André Guillouzo and
Olivier Fardel1
INSERM U456 `Détoxication et Réparation Tissulaire', Faculté de Pharmacie, 2 avenue du Pr L. Bernard, 35043 Rennes Cedex, France
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Abstract
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Expression of the canalicular multispecific organic anion transporter (cMOAT), an efflux pump involved in biliary secretion of xenobiotics, was investigated in rat hepatocytes exposed to the chemopreventive agent oltipraz. Northern blotting indicated that this compound increased cMOAT mRNA levels in primary cultured hepatocytes. Such an induction of cMOAT transcripts was demonstrated to be dose-dependent and started as early as 4 h treatment; in addition, western blotting showed increased levels of 190 kDa cMOAT in oltipraz-treated primary rat hepatocytes when compared with their untreated counterparts. In contrast, administration of oltipraz to rats failed to enhance hepatic cMOAT mRNA and protein amounts whereas it was found to induce liver expression of glutathione S-transferase P1, a well-known oltipraz-regulated drug metabolizing enzyme. These data therefore suggest that cMOAT up-regulation occurring in rat hepatocytes in response to oltipraz may be restricted to in vitro situations and is therefore unlikely to be directly involved in the in vivo chemopreventive properties of oltipraz.
Abbreviations: cMOAT, canalicular multispecific organic anion transporter; GST, glutathione S-transferase; MRP, multidrug resistance-associated protein; P-gp, P-glycoprotein; RTPCR, reverse transcriptionpolymerase chain reaction.
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Introduction
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The canalicular multispecific organic anion transporter (cMOAT), also termed multidrug resistance-associated protein (MRP2) or canalicular multidrug resistance-associated protein (cMRP), is a membrane transporter predominantly found at the biliary pole of hepatocytes (1,2). This 190 kDa protein mediates the passage of various types of organic anions, including glucuronate, sulphate and glutathione conjugates, from hepatocytes into the bile (3). The hepatobiliary secretion of such compounds is therefore strongly reduced in mutant rat strains lacking cMOAT expression such as the TR rat and the EHBR rat (1,2,4). Similarly, a deficiency in human cMOAT expression due to a mutation in the gene causes decreased biliary excretion of bilirubin glucuronides, resulting in hereditary conjugated hyperbilirubinemia, known as DubinJohnson syndrome (5).
cMOAT belongs to the superfamily of ATP-binding cassette transporters that comprises other drug efflux pumps such as the 170 kDa P-glycoprotein (P-gp) and the 190 kDa multidrug resistance-associated protein (MRP, also known as MRP1) (1). MRP, initially described in tumour cell lines exhibiting a multidrug resistance phenotype (6), shares numerous substrates with cMOAT, including drug conjugates (7). In contrast to cMOAT, MRP is, however, expressed at low levels in the normal liver where it is thought to be localized at the lateral poles of the parenchymal cells as assessed by immunohistochemical analyses (8).
Hepatic formation of many compounds considered as cMOAT substrates, i.e. glutathione or glucuronate conjugates of xenobiotics, is mediated at least in part by phase II drug metabolizing enzymes such as glutathione S-transferases (GST) and UDP-glucuronosyl transferases (9). Interestingly, the expression levels of these detoxifying enzymes are well-known to be regulated in response to treatment with various chemicals (10). In particular, oltipraz, a synthetic derivative of the plant product 1,2-dithiole-3-thione originally developed as an anti-schistosomal compound, has been demonstrated to increase expression of conjugating enzymes such as GST A1/2 and GST P1 (11,12). Such an effect on detoxication enzymes is thought to result in enhanced metabolic inactivation of chemical carcinogens and therefore has been postulated to account, at least in part, for the well-known chemopreventive properties of oltipraz towards chemical carcinogenesis (13). Whether this compound may also alter hepatic levels of drug transporters, in particular those of cMOAT, remains, however, unknown, but may be relevant to determine since cMOAT may mediate the secretion into the bile of many compounds metabolized by oltipraz-induced detoxifying enzymes. The present study was therefore designed to analyse the expression of cMOAT in response to treatment with oltipraz. This work was conducted using primary rat hepatocyte cultures, which have been proposed as suitable models for investigating the effects of chemopreventive agents on hepatic detoxication pathways (11,14), and using oltipraz-treated rats.
Hepatocytes were isolated by two-step perfusion of the liver of male SpragueDawley or Wistar rats (weighing 180200 g) with a collagenase solution as previously described (15). Cells were then plated at a density of 105 cells/cm2 in plastic dishes in Williams' medium supplemented with 0.2 mg/ml bovine serum albumin, 10 µg/ml bovine insulin and 10% (v/v) fetal calf serum. After a 4 h cell attachment period, the medium was discarded and replaced by serum-free Williams' medium containing 100 nM dexamethasone. Oltipraz, dissolved in dimethylsulphoxide, was added with the latter medium. Control cultures received the same concentration of solvent, which did not exceed 0.2% (v/v). In agreement with previous data (11), oltipraz, at the concentrations used, was not found to have any obvious cytotoxic effects towards hepatocytes as assessed by careful light microscopic examination of the cultures.
For animal treatment, male Wistar rats (weighing 180200 g) were fed a diet with oltipraz added at a final concentration of 0.075% (w/w) for 2 or 4 days. At the end of treatment, rats were killed and livers were immediately removed and kept at 80°C until use.
For northern blotting, total RNA was extracted from cells or liver fragments using the guanidium thiocyanate/cesium chloride method (16) as modified by Raymondjean et al. (17). Total RNA (10 µg) was then subjected to electrophoresis in a denaturing formaldehydeagarose gel and transferred onto Hybond-N+ sheets (Amersham, Les Ulis, France). The membrane sheets were hybridized overnight at 65°C with 32P-labelled probes; cMOAT mRNAs were detected with a 860 bp cMOAT cDNA fragment (18) whereas GST P1 mRNAs were analysed with the pGSTr7 cDNA probe (19). After hybridization, the sheets were washed, dried and autoradiographed at 80°C. Equal RNA loading onto the gel and the efficiency of transfer were checked by rehybridizing the blots with an 18S rRNA probe (20).
For reverse transcriptionpolymerase chain reaction (RTPCR) assays, total RNA (0.5 µg) was first reverse transcribed using 200 U of Moloney murine leukemia virus transcriptase (Gibco BRL, Cergy-Pontoise, France). An amount of cDNA representing 25 ng of RNA was then subjected to PCR using 0.2 U of Taq polymerase (Eurogentec, Seraing, Belgium) and 250 ng of cMOAT primers previously described (21). RTPCR products obtained were then subjected to electrophoresis in a 1% agarose gel and visualized by staining the gel with ethidium bromide.
Western blot analyses were performed using crude membranes prepared from primary hepatocytes by differential centrifugation as reported by Germann et al. (22) and using whole cell lysates of liver fragments. Protein samples were separated on 7.5% (for cMOAT analysis) or 12.5% (for GST P1 analysis) polyacrylamide gels and electrophoretically transferred to a nitrocellulose sheet. The sheets were then blocked for 2 h with Tris-buffered saline containing 3% bovine serum albumin and 1% milk. Polyclonal anti-cMOAT antibodies EAG15 (2) (kindly provided by Dr D.Keppler, Deutsches Krebsforschungszentrum, Heidelberg, Germany) or RM2 (directed against the C-terminal sequence of rat cMOAT) and polyclonal anti-GST P1 antibody (Biotrin, Dublin, Ireland) were then applied to the membranes for 1 h at room temperature. After washing, the blots were incubated with peroxidase-conjugated anti-rabbit antibody (Valbiotech, Evry, France) and were further developed by chemiluminescence using the Amersham ECL detection system.
Alkaline phosphatase activity in crude membrane fractions was measured spectrophotometrically at 410 nm using a standard method as previously described (23).
Northern blot analysis showed that oltipraz induced cMOAT mRNA expression in primary SpragueDawley rat hepatocytes in a dose-dependent manner (Figure 1A
). An increase in cMOAT mRNA levels was evident in hepatocytes treated for 48 h with oltipraz at 18.75 µM, whereas higher concentrations of oltipraz (50 and 100 µM) produced maximal induction of cMOAT transcripts. In contrast, cultured hepatocytes exposed to a lower dose of oltipraz (12.5 µM) displayed amounts of cMOAT transcripts similar to those found in their untreated counterparts. The time course of cMOAT mRNA induction by 50 µM oltipraz was then analysed. As indicated in Figure 1B
, up-regulation of cMOAT transcripts in primary hepatocytes started as soon as 4 h treatment and was maximal for longer exposures (1248 h) to oltipraz, whereas shorter treatment (1 h) failed to alter cMOAT mRNA levels. As previously reported (2), several cMOAT transcripts of different sizes, especially a major one of 5.5 kb, were evident on the northern blots; their relative proportions were, however, not obviously altered in response to oltipraz treatment. Such cMOAT mRNAs probably represent alternative mRNA splicing variants with different 3'-untranslated region lengths (24).

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Fig. 1. Dose dependence (A) and time course (B) of cMOAT mRNA induction in response to oltipraz treatment in primary rat hepatocytes. Each well contains 10 µg total RNA isolated from primary cultured SpragueDawley rat hepatocytes untreated (UNT) or exposed to various doses of oltipraz (OPZ) (6.25100 µM) for 48 h (A) or to 50 µM oltipraz for various lengths of time (148 h) (B). RNAs were then transferred to Hybond-N+ sheets and hybridized with cMOAT and 18S probes. The results shown are representative of two independent experiments.
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The up-regulation of cMOAT mRNA levels evident in oltipraz-treated primary hepatocytes in northern blotting experiments was also demonstrated using RTPCR assays with specific cMOAT primers (data not shown). In addition, such regulation, described in primary cultures of SpragueDawley rat hepatocytes, was also found to occur in cultured hepatocytes obtained from Wistar rats and exposed to 50 µM oltipraz for 48 h (data not shown). These results therefore suggest that isolated hepatocytes obtained from different rat strains respond to oltipraz treatment in a similar manner with respect to cMOAT expression. In contrast, regulation of P-gp levels in primary hepatocytes exposed to 2-acetylaminofluorene has been shown to be dependent on the rat strain used for preparation of the liver cells; especially, primary SpragueDawley rat hepatocytes, unlike Wistar hepatocytes, were found not to be responsive to 2-acetylaminofluorene (25).
To determine whether the increase in cMOAT mRNA in oltipraz-treated cultured liver cells was associated with up-regulation of cMOAT at the protein level, crude membranes were further prepared from primary SpragueDawley rat hepatocytes exposed to 50 µM oltipraz and from their untreated counterparts and were analysed by western blotting using the anti-cMOAT EAG15 antibody. As indicated in Figure 2
, cells treated with oltipraz displayed enhanced amounts of a EAG15-reactive 190 kDa protein corresponding to cMOAT. This 190 kDa protein was not detected in control blots using non-immune rabbit serum as primary antibody instead of EAG15. In contrast to cMOAT, alkaline phosphatase, a membrane marker enzyme, was not up-regulated by oltipraz treatment. Indeed, alkaline phosphatase activity measured in crude membranes prepared from oltipraz-treated hepatocytes corresponded to 82 ± 16% of the value found in membranes obtained from untreated hepatocytes. This result suggests that the effect of oltipraz on cMOAT levels in primary hepatocytes is likely specific and not related to general up-regulation of membrane protein expression.

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Fig. 2. Western blot analysis of membrane proteins obtained from primary hepatocytes exposed to oltipraz. Crude membrane fractions were prepared from primary SpragueDawley rat hepatocytes either untreated (UNT) or exposed to 50 µM oltipraz (OPZ) for 72 h. Membrane proteins were then separated on 7.5% polyacrylamide gels and transferred onto nitrocellulose sheets. After incubation with EAG15 antibody raised against cMOAT, the blot was developed using an ECL chemiluminescence kit. The position of molecular mass standards in kDa is indicated on the right. The results shown are representative of three independent experiments.
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cMOAT mRNA and protein levels were further analysed in the livers of oltipraz-treated Wistar rats and of their untreated counterparts. Surprisingly, both cMOAT mRNA and cMOAT protein amounts were not obviously altered in response to treatment with oltipraz for 4 days (Figure 3A and B
); shorter exposure to oltipraz (2 days) also failed to affect cMOAT expression (data not shown). Such results do not favour the idea that cMOAT can be included in the liver detoxifying functions which are in vivo induced by oltipraz and are thereby thought to contribute to its known chemopreventive properties (13). The basis for such an absence of up-regulation of cMOAT by oltipraz in vivo, which contrasts with the in vitro situation, is rather unclear. It could be linked to inadequate experimental conditions for treating the rats. However, the dose of oltipraz and the duration of treatment used in our study have been previously shown to be sufficient to induce liver expression of oltipraz-regulated drug metabolizing enzymes (11). Besides, northern and western blotting indicated that GST P1 mRNA and protein levels were strongly enhanced in the liver of oltipraz-treated rats when compared with those observed in the liver of untreated rats (Figure 3A and B
), thus unequivocally demonstrating that the treatment that we performed was efficient in increasing expression of well-known oltipraz-regulated detoxifying enzymes such as GST P1 (11). Interestingly, a discrepancy between the in vivo and in vitro situations, similar to that found for regulation of cMOAT in response to oltipraz, has also been demonstrated with respect to the effects of the polycyclic aromatic hydrocarbon 3-methylcholanthrene on P-gp levels in rat hepatocytes (26). Indeed, treatment of primary hepatocytes with 3-methylcholanthrene resulted in strong induction of P-gp mRNA whereas administration of the chemical to rats failed to alter hepatic P-gp expression (26). Taken together, these data suggest that caution may be required when extrapolating data from in vitro systems such as primary cultures of hepatocytes to in vivo situations, especially for regulatory pathways of hepatic drug transporters. Previous studies have, however, shown a good correlation between in vivo and in vitro data with respect to the regulation of some drug metabolizing enzymes by various xenobiotics, including chemopreventive agents (11).

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Fig. 3. Effect of oltipraz treatment on cMOAT and GST P1 mRNA (A) and protein (B) levels in rat liver. Rats were either untreated (UNT) or treated with 0.075% (w/w) oltipraz (OPZ) in the diet for 4 days. (A) Total RNA was then isolated from liver, transferred to Hybond-N+ sheets and hybridized with cMOAT, GST P1 and 18S probes. (B) Cell lysates were prepared from liver fragments, separated on polyacrylamide gels and transferred onto nitrocellulose sheets. After incubation with polyclonal antibodies raised against cMOAT and GST P1, the blots were developed using an ECL chemiluminescence kit. The positions of molecular mass standards in kDa are indicated on the right. The results shown are representative of five animals in each group (control and oltipraz-treated).
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The molecular mechanism by which oltipraz induces cMOAT expression in primary hepatocytes remains to be determined. Interestingly, the range of concentrations of oltipraz acting in vitro on cMOAT expression (Figure 1A
) is close to that required for induction of oltipraz-regulated enzymes such as GST A1/2 and manganese superoxide dismutase (11,27). In addition, the time course of cMOAT mRNA up-regulation in primary oltipraz-treated rat hepatocytes (Figure 1B
) is similar to that described for GST A1/2 mRNAs (11). These data may suggest that the mechanism by which oltipraz enhances cMOAT expression in primary rat hepatocytes is similar to that involved in oltipraz-mediated overexpression of GST A1/2. For GST A1/2, the effects of oltipraz have been related to the presence of cis-acting regulatory elements in the promoter of the responsive genes (28); these elements, termed antioxidant-responsive element sequences, have, however, not been reported in the promoter of the rat cMOAT gene (29); similarly, they have not been found in the 5'-flanking regions of some oltipraz-regulated cytochromes P-450 genes such as cytochrome P450 1A and 2B (30).
In summary, our results demonstrate that, although treatment of primary rat hepatocytes with oltipraz resulted in a strong induction of cMOAT expression, hepatic cMOAT mRNA and protein levels, unlike those of GST P1, were not obviously altered in oltipraz-treated rats. Such results therefore suggest that the well-established in vivo chemopreventive properties of oltipraz probably do not involve up-regulation of cMOAT expression in the liver.
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Acknowledgments
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We would like to thank Dr V.Lecureur and Dr S.Langouet for helpful discussions. This study was supported by the Association pour la Recherche sur le Cancer and the Ligue Nationale contre le Cancer (Comité d'Ille et Vilaine). A.Courtois and L.Payen are recipients of fellowships from the Association pour la Recherche sur le Cancer and the Ligue Nationale contre le Cancer (Comité du Morbihan), respectively.
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Notes
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1 To whom correspondence should be addressed Email: olivier.fardel{at}univ-rennes1.fr 
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Received December 4, 1998;
revised September 3, 1999;
accepted September 3, 1999.