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The well-known analgesic and antipyretic drug N-acetyl-p-aminophenol (acetaminophen; APAP) has been previously reported to affect MDR1 expression in rat liver. In this study, we have investigated the effect of subtoxic doses of APAP on MDR1 expression and activity in rat intestine and human intestinal cells. Administration of APAP at increasing doses of 0.2, 0.3, and 0.6 g/kg b.w., i.p. over three consecutive days, induced MDR1 expression in rat duodenum (+240%) and ileum (+160%) as detected by western blotting. This was accompanied by preserved localization of the protein at the surface of the villus, as detected by confocal immunofluorescence microscopy. MDR1 activity was increased by 50% in APAP treated rats, as evaluated by serosal to mucosal secretion of rhodamine 123 in everted intestinal sacs. Treatment with APAP also decreased by 65% the portal vein concentrations of digoxin found in anesthetized rats after intraduodenal administration of this drug, which is consistent with an APAP-induced increased efficacy of intestinal barrier for digoxin net absorption. Exposure of LS 174T human colon adenocarcinoma cells to subtoxic APAP concentration (5 mM) induced an increase in MDR1 mRNA expression (+46%), which was accompanied with an enhanced ability (+78%) to reduce intracellular content of rhodamine 123. Taken together these data suggest the existence of APAP-induced stimulation of MDR1 transcription in the intestinal epithelium. These findings are of clinical relevance, as co-administration of APAP with other MDR1 substrates could indirectly inhibit the net intestinal absorption of these drugs, leading to changes in their pharmacokinetics and therapeutic efficacy.

The rat hepatoma/human fibroblast hybrid cell line WIF-B9 was developed to be used in studies requiring maintained hepatocyte-like polarity. To enhance their usefulness in order to investigate hepatic phase III detoxification process, we have characterized a subline of WIF-B9 cells (WIF-B9/R) obtained by exposure to progressively increasing cisplatin concentrations (up to 10 microM) and double sub-clonal selection. As compared to WIF-B9 cells, the cytostatic effect of cisplatin and doxorubicin on WIF-B9/R cells was lower (>10-fold), whereas the ability to reduce cell loading of cisplatin, doxorubicin, rhodamine 123 and calcein was higher. As their parent cells, WIF-B9/R cells express hepatocyte-like polarity. However, they have enhanced stable expression of Mdr1, Mrp1, Mrp2, Mrp3 and BCRP, but not Bsep/BSEP, as determined by real-time quantitative RT-PCR and western blot. Differentiation to hepatocyte-like phenotype was characterized by the formation of canalicular-like structures, containing in their membranes immunocytochemically detectable Mdr1, Mrp2 and BCRP. Functionality of these ABC transporters was evaluated by using specific substrates and inhibitors. Thus, canalicular-like structures were able to concentrate calcein, rhodamine 123 and doxorubicin. Moreover, verapamil, probenecid and Hoechst-33342 inhibited doxorubicin efflux and enhanced its content in WIF-B9/R cells. Probenecid inhibited calcein efflux and increased calcein cell load, but had no effect on cell loading of rhodamine 123, which was increased by verapamil and Hoechst-33342. In conclusion, WIF-B9/R cells are a useful model of polarized cells to study, in the absence of Bsep/BSEP, hepatic phase III of the detoxification process of several compounds whose canalicular transport is mediated by ABC proteins.

Introduction Nous avons montre que la consommation d’un repas de sucres simples recrute de facon transitoire la proteine de transport du glucose et du fructose, GLUT2, dans la membrane en bordure en brosse des enterocytes de souris. Il en resulte une augmentation de la capacite d’absorption intestinale de sucre. Nous avons donc montre que les sucres alimentaires favorisent l’insertion de GLUT2 dans la bordure en brosse, alors que l’insuline la previent par son internalisation. Cette internalisation de GLUT2 par l’insuline est perdue chez les souris rendues resistantes a l’insuline par la consommation d’un regime riche en fructose (65 %) pendant un mois. Materiels et methodes Le but de ce travail etait de connaitre les contributions respectives de la resistance a l’insuline et du fructose sur le defaut d’internalisation de GLUT2. Nous avons nourri des souris avec un regime, riche en graisse saturees (72 %) et pauvre en sucre ( Resultats Les souris developpent une intolerance au glucose et une resistance a l’insuline a partir de 2 mois de ce regime hyperlipidique. Le taux de proteine GLUT2 est diminue de 70 % par rapport a celui de souris nourries avec un regime riche en fructose. Comme chez les souris rendues resistantes a l’insuline par le regime riche en fructose, GLUT2 est majoritairement localise dans la bordure en brosse des enterocytes des souris soumises a un regime hyperlipidique en condition de clamp euglycemique et hyperinsulinemique. La localisation de GLUT2 est insensible a l’insuline. De plus, nous avons observe que la proportion de GLUT2 dans les membranes basolaterales etait reduite chez les souris resistantes a l’insuline par rapport aux souris nourries avec un regime standard (standard > fructose > graisse saturees). Conclusion Ces observations indiquent que l’etat de resistance a l’insuline est associe a une localisation apicale permanente de GLUT2 dans les membranes en bordure en brosse independamment de la presence de sucre dans le regime des souris. Cette distribution pathologique de GLUT2 dans les membranes plasmiques des enterocytes est un facteur aggravant de la resistance a l’insuline parce qu’il augmente l’absorption intestinale des sucres.