Hepatocellular carcinoma (HCC) is the fifth most frequent cancer worldwide. Sorafenib is the only drug available that improves the overall survival of HCC patients. P-glycoprotein (P-gp), Multidrug resistance-associated proteins 2 and 3 (MRP2 and 3) and Breast cancer resistance protein (BCRP) are efflux pumps that play a key role in cancer chemoresistance. Their modulation by dietary compounds may affect the intracellular accumulation and therapeutic efficacy of drugs that are substrates of these transporters. Genistein (GNT) is a phytoestrogen abundant in soybean that exerts its genomic effects through Estrogen-Receptors and Pregnane-X-Receptor (PXR), which are involved in the regulation of the above-mentioned transporters. We evaluated the effect of GNT on the expression and activity of P-gp, MRP2, MRP3 and BCRP in HCC-derived HepG2 cells. GNT (at 1.0 and 10 μM) increased P-gp and MRP2 protein expression and activity, correlating well with an increased resistance to sorafenib cytotoxicity as detected by the methylthiazole tetrazolium (MTT) assay. GNT induced P-gp and MRP2 mRNA expression at 10 but not at 1.0 μM concentration suggesting a different pattern of regulation depending on the concentration. Induction of both transporters by 1.0 μM GNT was prevented by cycloheximide, suggesting translational regulation. Downregulation of expression of the miR-379 by GNT could be associated with translational regulation of MRP2. Silencing of PXR abolished P-gp induction by GNT (at 1.0 and 10 μM) and MRP2 induction by GNT (only at 10 μM), suggesting partial mediation of GNT effects by PXR. Taken together, the data suggest the possibility of nutrient-drug interactions leading to enhanced chemoresistance in HCC when GNT is ingested with soy rich diets or dietary supplements.

Oxidative stress is a frequent cause underlying drug-induced hepatotoxicity. Benznidazole (BZL) is the only antitripanocide agent available for treatment of Chagas disease in endemic areas. Its use is associated with side effects, including increases in biomarkers of hepatotoxicity. However, BZL potential to cause oxidative stress has been poorly investigated. Here, we evaluated the effect of a pharmacologically relevant BZL concentration (200 μM) at different time points on redox status and the counteracting mechanisms in the human hepatic cell line HepG2. BZL increased reactive oxygen species (ROS) after 1 and 3 h of exposure, returning to normality at 24 h. Additionally, BZL increased glutathione peroxidase activity at 12 h and the oxidized glutathione/total glutathione (GSSG/GSSG+GSH) ratio that reached a peak at 24 h. Thus, an enhanced detoxification of peroxide and GSSG formation could account for ROS normalization. GSSG/GSSG+GSH returned to control values at 48 h. Expression of the multidrug resistance-associated protein 2 (MRP2) and GSSG efflux via MRP2 were induced by BZL at 24 and 48 h, explaining normalization of GSSG/GSSG+GSH. BZL activated the nuclear erythroid 2-related factor 2 (Nrf2), already shown to modulate MRP2 expression in response to oxidative stress. Nrf2 participation was confirmed using Nrf2-knockout mice in which MRP2 mRNA expression was not affected by BZL. In summary, we demonstrated a ROS increase by BZL in HepG2 cells and a glutathione peroxidase- and MRP2 driven counteracting mechanism, being Nrf2 a key modulator of this response. Our results could explain hepatic alterations associated with BZL therapy.

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Lipopolysaccharide (LPS) from Gram (-) bacteria induces inflammatory cholestasis by impairing the expression/localization of transporters involved in bile formation (e.g., Bsep, Mrp2). Therapeutic options for this disease are lacking. Ursodeoxycholic acid (UDCA) is the first choice therapy in cholestasis, but its anticholestatic efficacy in this hepatopathy remains to be evaluated. To asses it, male Wistar rats received UDCA for 5 days (25 mg/Kg/day, i.p.) with or without LPS, administered at 8 a.m. of the last 2 days (4 mg/Kg/day, i.p.), plus half of this dose at 8 p.m. of the last day. Then, plasma alkaline phosphatase (ALP), bile flow, basal and taurocholate-stimulated bile acid output, total glutathione output, and total/plasma membrane liver protein expression of Bsep and Mrp2 by confocal microscopy were assessed. mRNA levels of both transporters were assessed by Real-time PCR. Plasma pro-inflammatory cytokines (IL-6 and TNF-α) were measured by ELISA. Our results showed that, UDCA attenuated LPS-induced ALP plasma release and the impairment in the excretion of the Bsep substrate, taurocholate. This was associated with an improved Bsep expression at both mRNA and protein levels, and by an improved localization of Bsep in plasma membrane. UDCA failed to reduce the increase in plasma pro-inflammatory cytokines induced by LPS and Mrp2 expression/function. In conclusion, UDCA protects the hepatocyte against the damaging effect of bile acids accumulated by the LPS-induced secretory failure. This involved an enhanced synthesis of Bsep and an improved membrane stability of the newly synthesized transporters.

Many drugs can induce liver injury, characterized by hepatocellular, cholestatic or mixed hepatocellular-cholestatic lesions. While an inflammatory stress is known to aggravate hepatocellular injury caused by some drugs much less evidence exists for cholestatic features. In this study, the influence of pro-inflammatory cytokines (IL-6, IL-1β and TNF-α), either individually or combined, on cytotoxic and cholestatic properties of antibiotics was evaluated using differentiated HepaRG cells. Six antibiotics of various chemical structures and known to cause cholestasis and/or hepatocellular injury in clinic were investigated. Caspase-3 activity was increased with all these tested hepatotoxic drugs and except with erythromycin, was further augmented in presence of cytokines mainly when these were co-added as a mixture. TNF-α and IL-1β aggravated cytotoxicity of TVX more than IL-6. Bile canaliculi (BC) dilatation induced by cholestatic drugs was increased by co-treatment with IL-6 and IL-1β but not with TNF-α. Reduced accumulation of carboxy-dichlorofluorescein, a substrate of the multi-drug resistance-associated protein 2, in antibiotic-induced dilatated BC, was further extended in presence of individual or mixed cytokines. In conclusion, our data demonstrate that pro-inflammatory cytokines either individually or in mixture, can modulate cholestatic and/or cytotoxic responses to antibiotics and that the extent of these effects is dependent on the cytokine and the cholestatic antibiotic.

TNFα is a cytokine whose levels are increased in inflammatory pathologies that are associated with cholestasis. Endocytic internalization of Abcc2 (multidrug resistance-associated protein 2), a canalicular transporter of organic anions that is implicated in the clearance of clinically important drugs, is a phenomenon that occurs in inflammatory liver diseases, and it has been established that cytokines act as mediators. However, the intracellular mechanism involved in this effect remains unknown. The aim of the present work was to characterize the internalization of Abcc2 induced by TNFα and to study the role of ERK1/2 and reactive oxygen species as signaling mediators of transporter internalization. Using rat hepatocyte couplets, we found that TNFα (6.25 pg/ml) induced a decrease in Abcc2 activity estimated by the accumulation of the Abcc2 substrate glutathione methylfluorescein in the canalicular vacuole that was accompanied by internalization of Abcc2 from the canalicular membrane. Inhibition of MEK1/2 (upstream of ERK1/2) partially prevented TNFα effects on Abcc2 internalization and activity impairment. Reactive oxygen species (ROS) scavengers such as vitamin C and mannitol partially prevented both TNFα-induced decrease in Abcc2 activity and ERK1/2 phosphorylation. Apocynin, a NADPH oxidase inhibitor, prevented the increase in ROS and the phosphorylation of ERK1/2 produced by TNFα. Taken together, these results indicate that TNFα activates a pathway involving NADPH oxidase, ROS and MEK1/2-ERK1/2 that is partially responsible for the internalization of Abcc2. This internalization leads to an altered transport activity of Abcc2 that could impair drug disposal, enhancing drug toxicity in patients suffering from inflammatory liver diseases.

Estradiol-17β-D-glucuronide (E17G), through the activation of different signaling proteins, induces acute endocytic internalization of canalicular transporters in rat, including multidrug resistance-associated protein 2 (Abcc2) and bile salt export pump (Abcb11), generating cholestasis. Insulin-like growth factor 1 receptor (IGF-1R) is a membrane-bound tyrosine kinase receptor that can potentially interact with proteins activated by E17G. The aim of this study was to analyze the potential role of IGF-1R in the effects of E17G in isolated perfused rat liver (IPRL) and isolated rat hepatocyte couplets. In vitro, IGF-1R inhibition by tyrphostin AG1024 (TYR, 100 nM), or its knock-down with siRNA, strongly prevented E17G-induced impairment of Abcc2 and Abcb11 function and localization. The protection by TYR was not additive to that produced by wortmannin (PI3K inhibitor, 100 nM), and both protections share the same dependency on microtubule integrity, suggesting that IGF-1R shared the signaling pathway of PI3K/Akt. Further analysis of the activation of Akt and IGF-1R induced by E17G indicated a sequence of activation GPR30-IGF-1R-PI3K/Akt. In IPRL, an intraportal injection of E17G triggered endocytosis of Abcc2 and Abcb11, and this was accompanied by a sustained decrease in the bile flow and the biliary excretion of Abcc2 and Abcb11 substrates. TYR did not prevent the initial decay, but it greatly accelerated the recovery to normality of these parameters and the reinsertion of transporters into the canalicular membrane. In conclusion, the activation of IGF-1R is a key factor in the alteration of canalicular transporter function and localization induced by E17G, and its activation follows that of GPR30 and precedes that of PI3K/Akt.

ATP binding cassette (ABC) transporters are involved in drug absorption, distribution and elimination. They also mediate multidrug resistance in cancer cells. Isoflavones, such as genistein (GNT), belong to a class of naturally-occurring compounds found at high concentrations in commonly consumed soya based-foods and dietary supplements. GNT and its metabolites interact with ABC transporters as substrates, inhibitors and/or modulators of their expression. This review compiles information about regulation of ABC transporters by GNT with special emphasis on the three major groups of ABC transporters involved in excretion of endo- and xenobiotics as follows: Pglycoprotein (MDR1, ABCB1), a group of multidrug resistance associated proteins (MRPs, ABCC subfamily) and ABCG2 (BCRP), an ABC half-transporter. The impact of these regulations on potential GNT-drug interactions is further considered.