HEPATOLOGY Vol. 34, No. 4, Pt. 2, 2001 AASLD ABSTRACTS 4 7 3 A

1203

CHOLESTEROL FLUX IN PRIMARY MOUSE HEPATOCYTES. Luis A Ba- ires, Steven J Luke, Div GI & Nutrition/The Children's Hospital of Philadel- phia, Philadelphia, PA; Margery A Connelly, David L Williams, Div of Phar- maeology/SUNY, Stony Brook, NY; Anna E Bormick, Elda Favari, Genevieve W StoLIdt, Patricia G Yancey, Margarita De la Llera-Moya, David A Piccoli, George H Rothblat, Div GI & Nutrition/The Children's Hospital of Philadelphia, Phil- adelphia, PA

Background: Two HDL receptors, ATP-binding Cassette 1 (ABCA1) and scav- enger receptor-BI (SR-BI), are important in reverse cholesterol transport. SR-BI is expressed in liver and steroidogenic tissues. The purpose of this s tudy was to determine the SR-BI expression in primary mouse hepatocytes, and its role in free: cholesterol (FC) flux. Methods: Hepatocytes were isolated using collage- nase perfusion. SR-BI was quantified from Western blots using antibodies against SR-BL Efflux was determined by the release of [ 1,2-3H] cholesterol from hepatocytes to human HDL3 . Some monolayers were preincubated with 22(OH)cholesterol + 9cis retinoic acid (CRA) or cAMP before efflux. Other experiments measured flux of [1,2-3H]cholesterol between hepatocytes and HDL3 or HDL3 enriched with sphingomyelin or phospatidylcholine (PC). El- flux: and influx were measured after 4 h incubation. Results: The SR-BI expres- sion in hepatocytes incubated with 10% FBS decreased to 33% of the 24h level at 72h. Hepatocytes incubated in WiUiams E medium supplemented with DMSO and dexamethasone, +/- 5% FBS, maintained higher SR-BI levels. El- flux of [1,2-3Hlcholesterol to HDL3 was not upregulated by 22(OH)choles- terol + CRA or cAMP. The enrichment of HDL3 with PC stimulated SR-BI mediated efflux, but enrichment with sphingomyelin did not. Sphingomyelin enrichment of HDL3 reduced influx of FC into hepatocytes whereas PC en- riched did not.Conclusions: SR-BI receptors are expressed in mouse hepato- cytes, levels decreased over a 72h incubation period. The FC efflux and influx data are consistent with a role of SR-BI in mediating FC flux between hepato- cytes and HDL>

1204

THE ABC-TRANSPORTER GENES MDRI/MDR1B, MRPI, AND MRP3 ARE HIGHLY EXPRESSED IN HEPATIC PROGENITOR CELLS. Jenny E Ros, University- Hospital Groningen, Groningen Netherlands; Tania Roskams, University of Leuven, Leuven Belgium; Mariska Geuken, Rick Havinga, Han Moshage, University of Groningen, Groningen Netherlands; Bryon E Petersen, University of Florida, Gainesville, FL; Folkert Kuipers, University Hospital Groningen, Groningen Netherlands; Michael Muller, University of Wagenin- hen, Wageningen Netherlands; Peter L Jansen, University Hospital Groningen, Groningen Netherlands

Hepatic progenitor cells will proliferate when hepatocyte proliferation is suppressed after loss of liver mass. These progenitor cells are considered to be the precursors of both hepatocytes and cholangiocytes. Liver progenitor cells are characterized by expression o[ proteins like Thy-i and OV-6. We hypothesize that high expression levels of ABC-trans- porters involved in cellular protection may enable hepatic progenitor ceils to survive and proliferate during toxic liver injury. Aim: To define the expression pattern of ABC-trans- porter genes in hepatic progenitor ceils of rat and human origin. Methods: To induce the rat progenitor cell compartment, Wistar rats were treated with 2-acetylaminofluorene (2- AAF). Seven days after 2-AAF treatment 70% partial hepatectomy (PHx) was performed. Control rats received placebo and/or were sham-operated. Rats were sacrificed 9 days after PHx. In a separate experiment, a highly enriched progenitor cell population was isolated from these livers by flow cytometry using Thy-1 as a marker. RNA was isolated and mRNA levels o1 the ABC-transporters Mdrl b(Abcb l b), Mdr2(Abcb4), Bsep(Abcbl 1), Mrpl (Abccl ), Mrp2(Abcc2), and Mrp3(Abcc3) were determined by real time quantitative RT-PCR (Taq- Man), using ribosomal 18S RNA levels as internal standard. The expression of Thy-1 mRNA was used as a marker for the proliferation of progenitor ceils. Immunohistochemical stud- ies were performed on liver specimens from patients diagnosed with chronic hepatitis C and submassive liver cell necrosis and compared to expression in normal liver, using specific antibodies for MDR1 and MRP3. To study induction of ABC-transporter expres- sion in diseased liver, antibodies were diluted until the signal in normal liver was barely detectable. Results: Treatment of rats with 2-AAF alone or 2-AAF/PHx resulted in strong induction ofMdrlb mRNA levels in total liver (~80 and ~120-fold respectively, p<0.05 compared to placebo/sham treated rats). The difference between the 2-AAF and 2-AAF/ PHx groups was not statistically significant. 2-AAF/PHx treatment increased the mRNA levels of Mdr2, Mrpl, and Mrp3 --2, ~3, and -i0-fold respectively (p<0.05 compared to placebo/sham). Thyl mRNA levels were increased - 22-fold (p<0.05 compared to placebo/ sham). The mRNA expression levels of Mdrl b, Mdr2, Mrpl, and M~3 significantly corre- lated with the expression Ievels of Thy-1 mRNA (p<0.01). Bsep andMrp2 mRNA expres- sion levels did not change after 2-AAF/PHx treatment. Isolated Thy-1 positive cells expressed high levels of Mdrl b, MrpI, and Mrp3, but had low expression of the hepatocyte- specific transporters Mrp2, Mdr2, and Bsep. Liver specimens front patients diagnosed with hepatitis C and submassive necrosis showed strong expression of MDR1 (apical mem- brane) and MRP3 (basolateraI membrane) on proliferating periportal bile ducmlar celIs, the putative human hepatic progenitor cells. Conclusion: Our results demonstrate that hepatic progenitor cells in rats display high expression levels of the ABC-transporter genes Mdrlb, Mrpl, and Mrp3. These data correspond with the finding that human progenitor ceils are strongly positive for MDR1 and MRP3. High expression levels of specific ABC- transporters in progenitor ceils may contribute to a multldrug resistant phenotype which enables these cells to survive unfavourahle conditions associated with toxic liver injury.

120:5

CHEMOPREVENTIVE AGENT-MEDIATED UP-REGULATION OF MUD TIDRUG RESISTANCE-ASSOCIATED PROTEIN 2 (MRP2) IN PRIMARY HUMAN AND RAT HEPATOCYTES. Lea Payen, Aruaud Courtois, Maud Loewert, Lydie Sparfel, Andre Guiflouzo, Olivier Fardel, Inserm U456 (detox- ication et reparation tissulaire), Rennes France

Chemopreventive agents such as oltipraz or sulforaphane confer protection towards chemical carcinogens through, at least in part, altering levels and/or activity of drug metabolizing enzymes, leading to decreased activation and enhanced metabolic inactivation of carcinogenic compounds. Besides detoxi- fying enzymes, drug membrane transporters also play a major role in elimina- tion of xenobiotics and may therefore be a target of chemopreventive agents. To test this hypothesis, we have studied regulation of muhidrug resistance- associated protein 2 (MRP2), a major biliary efflux pump handling carcino- genic compounds, in primary rat and human hepatocytes exposed to oltipraz and sulforaphane. These compounds were found to enhance mRNA MRP2 levels in both human and rat hepatocytes in a dose-dependent manner; they also increased 190 kDa of the MRP2 protein expression as demonstrated by Western blotting. Features of MRP2 mRNA up-regulation by sulforaphane in rat hepatocytes, i.e. dose response and time course of the induction, were demonstrated to be similar to those found for sulforaphane-related induction of drug metabolizing enzymes such as glutathione-S-transferase A1/2 and NAD(P)H:quinone oxidoreductase. Sulforaphane effect on MRP2 transcripts was fully abolished by co-treatment with actinomycin D, suggesting it required active gene transcription. It was associated with increased cellular production of reactive oxygen species (ROS) and addition of dimethylsulfoxide, that re- duced ROS formation, also decreased MRP2 induction, suggesting involve- ment of ROS in MRP2 regulation. Such a l ink with ROS was further supported by the increase of MRP2 expression occurring in primary rat hepatocytes treated by t-butylhydroquinone known to regulate drug metabolizing enzymes through ROS formation. These data indicate that MRP2 can be classified among the detoxifying proteins that are regulated by chemopreventive agents ; this export pump may therefore contribute to their anticarcinogenic proper- ties.

1206

IDENTIFICATION OF AMINO ACID RESIDUES ESSENTIAL FOR OATP2 MEDIATED DIGOXIN TRANSPORT. Jessica Van Montfoort, Peter J Meier, Clinical Pharmacology and Toxicology, Zurich Switzerland; Bruno Hagen- bnch, Division of Clinical Pharmacology and Toxicology, Department of Med- icine, University Hospital, CH-8091 Zurich/Switzerland, Zurich Switzerland

Background: Organic anion transporting polypeptides (Oatps) mediate the uptake of a variety of organic compounds into rat hepatocytes. Despite numer- ous studies that investigated the multi-specificity of Oatps, nothing is known about the structural elements involved in substrate binding and transport. Although Oatpl and Oatp2 share 77% amino acid identity, the cardiac glyco- side digoxin is a unique substrate for Oatp2. Aim: Identification of the domains and amino acid residues that are essential for digoxin transport by rat Oatp2. Methods: Chimeric proteins were constructed between rat Oatpl and rat Oatp2 using an overlap extension PCR approach. Individual amino acids were replaced by site-directed mutagenesis. Complementary- RNAs of the resulting constructs were functionally tested in Xenopus laevis oocytes. Results: A chi- mera containing trans-membrane (TM) domains 1 to 7 of Oatpl and 8 to 12 of Oatp2 retained digoxin transport activity similar to wild-type Oatp2. This suggests that amino acid residues in the last 5 TM domains of Oatp2 are essential for digoxin transport. Using additional chimeric constructs, the im- portant region could be narrowed down to a stretch of 64 residues between amino acids 324 and 388 ranging from the end of TM domain seven to the beginning of TM domain nine. An amino acid sequence comparison revealed that Oatp 1 and Oatp2 differed in only six residues within this region. Mutation of three amino acids (A330G, V353A and V354I) did not change digoxin transport. However, individual mutations of F328V, $334K or M361S resulted in reduced digoxin transport activities. The strongest decrease in digoxin transport was observed when the polar serine at position 334 was changed to a positively charged lysine. Conclusions: Exchange of three amino acid residues of Oatp2 near the extra cellular domains between TM seven and eight with the respective residues of Oatp l results in a strong reduction of Oatp2 mediated digoxin transport. Thus, these amino acid residues are essential for either binding or transport of the cardiac glycoside digoxin by rat Oatp2.