Aldo D. Mottino

Differential effects of silymarin and its active component silibinin on plasma membrane stability and hepatocellular lysis

Silymarin (SIL) is a natural extract with hepatoprotective properties composed mainly of flavonolignans, with silibinin (SB) being its principal constituent. SB is thought to be the main responsible for SIL hepatoprotective properties, although this has not been corroborated systematically. We analysed comparatively the effects of SIL and SB on hepatocellular plasma membrane stability. SIL (500 microM concentration in SB) protected significantly the plasma membrane disruption induced by Triton X-100 (TX-100) and taurochenodeoxycholate (TCDC), both in isolated plasma membrane (assessed by recording the plasma membrane transition from bilayer to micelle using the R18 self-quenching assay) and in isolated rat hepatocytes (assessed by the release into the incubation medium of the cytosolic enzymes lactate dehydrogenase and alanine aminotransferase). Contrarily, SB (500 microM) exacerbated plasma membrane disruption induced by TX-100 in both systems at detergent concentrations relevant to induce hepatocellular lysis, although it displayed some stabilizing properties at higher concentrations. SB showed a lower stabilizing effect against TCDC-induced plasma membrane disruption when assayed in both models. In addition, SB exposure made the plasma membrane more labile to disruption induced by osmotic lysis. These results show that SIL and SB have differential effects on membrane stability; whereas SIL shows consistently stabilizing effects, SB exacerbates hepatocellular lysis or exerts only minimal stabilizing effects. This differential behaviour should be taken into account when considering the pros and cons of using purified SB vs. the whole SIL extract in medicinal formulations for liver diseases.

Pregnane X receptor mediates the induction of P-glycoprotein by spironolactone in HepG2 cells

We evaluated the effect of spironolactone (SL), a well-known inducer of biotransformation and elimination pathways, on the expression and activity of P-glycoprotein (P-gp/ABCB1/MDR1), a major xenobiotic transporter, in HepG2 cells, as well as the potential mediation of pregnane X nuclear receptor (PXR). Cells were exposed to SL (1, 5, 10, 20 or 50 μM) for 48 h. Expression of P-gp and its mRNA levels were estimated by Western blotting and real time PCR, respectively. P-gp activity was inversely correlated with the ability of the cells to accumulate the model substrate rhodamine 123 (Rh123, 5 μM), in the presence or absence of verapamil (50 μM), a P-gp inhibitor. At the highest dose of SL tested, P-gp and MDR1 mRNA levels were significantly increased (73 and 108%) with respect to control cells. Rh123 accumulation was concomitantly reduced and verapamil was able to abolish this effect, confirming P-gp participation. Additionally, we tested the cytotoxicity of doxorubicin, a model substrate of P-gp, under inducing conditions. HepG2 cells treated with SL exhibited higher viability, i.e. less doxorubicin toxicity, than control cells, consistent with P-gp up-regulation. When HepG2 cells were treated with SL in the presence of ketoconazole (KTZ), a non-specific nuclear receptor inhibitor, the up-regulation of P-gp was suppressed. To further identify the nuclear receptor involved, cells were transfected with a siRNA directed against human PXR, leading to a 74% decrease in PXR protein levels, which totally abolished SL induction of P-gp. We conclude that SL up-regulates P-gp expression, likely at transcriptional level, and its efflux activity in HepG2 cells. This effect is mediated by PXR. Thus, ligands of PXR such as SL may alter the disposition and toxicity of other xenobiotics, including drugs of therapeutic use, that are P-gp substrates.

Regulation of Multidrug Resistance Proteins by Genistein in a Hepatocarcinoma Cell Line: Impact on Sorafenib Cytotoxicity

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.

Phosphoinositide 3‐kinase/protein kinase B signaling pathway is involved in estradiol 17β‐d‐glucuronide–induced cholestasis: Complementarity with classical protein kinase c

Estradiol 17β‐D‐glucuronide (E217G) is an endogenous, cholestatic metabolite that induces endocytic internalization of the canalicular transporters relevant to bile secretion: bile salt export pump (Bsep) and multidrug resistance–associated protein 2 (Mrp2). We assessed whether phosphoinositide 3‐kinase (PI3K) is involved in E217G‐induced cholestasis. E217G activated PI3K according to an assessment of the phosphorylation of the final PI3K effector, protein kinase B (Akt). When the PI3K inhibitor wortmannin (WM) was preadministered to isolated rat hepatocyte couplets (IRHCs), it partially prevented the reduction induced by E217G in the proportion of IRHCs secreting fluorescent Bsep and Mrp2 substrates (cholyl lysyl fluorescein and glutathione methylfluorescein, respectively). 2‐Morpholin‐4‐yl‐8‐phenylchromen‐4‐one, another PI3K inhibitor, and an Akt inhibitor (Calbiochem 124005) showed similar protective effects. IRHC immunostaining and confocal microscopy analysis revealed that endocytic internalization of Bsep and Mrp2 induced by E217G was extensively prevented by WM; this effect was fully blocked by the microtubule‐disrupting agent colchicine. The protection of WM was additive to that afforded by the classical protein kinase C (cPKC) inhibitor 5,6,7,13‐tetrahydro‐13‐methyl‐5‐oxo‐12H‐indolo[2,3‐a]pyrrolo[3,4‐c]carbazole‐12‐propanenitrile (Gö6976); this suggested differential and complementary involvement of the PI3K and cPKC signaling pathways in E217G‐induced cholestasis. In isolated perfused rat liver, an intraportal injection of E217G triggered endocytosis of Bsep and Mrp2, and this was accompanied by a sustained decrease in the bile flow and the biliary excretion of the Bsep and Mrp2 substrates [3H]taurocholate and glutathione until the end of the perfusion period. Unlike Gö6976, WM did not prevent the initial decay, but it greatly accelerated the recovery to normality of these parameters and the reinsertion of Bsep and Mrp2 into the canalicular membrane in a microtubule‐dependent manner. Conclusion: The PI3K/Akt signaling pathway is involved in the biliary secretory failure induced by E217G through sustained internalization of canalicular transporters endocytosed via cPKC. (HEPATOLOGY 2010)

Gender-related differences in the amount and functional state of rat liver UDP-glucuronosyltransferase

The basis for gender-dependent differences in rates of glucuronidation of xenobiotics is uncertain. To clarify this issue, the glucuronidation of p-nitrophenol was compared in liver microsomes from adult male and female rats. The activity of native UDP-glucuronosyltransferase was 47% higher in microsomes from male than from female rats. Immunoblotting of microsomal protein with anti-UDP-glucuronosyltransferase antiserum revealed 66% more immunoreactive protein in male microsomes. A kinetic method for measuring glucuronidating enzyme content confirmed the result of the immunoblot. Responses of UDP-glucuronosyltransferase to activation by palmitoyllysophosphatidylcholine or high pressure indicated that the activity of the enzyme was more latent in male than in female microsomes. Differences in enzyme latency could be due to differences in membrane structure. A comparison of microsomal fatty acid composition revealed significantly higher levels of oleic and linoleic acids and lower levels of stearic and docosahexaenoic acids in male than in female microsomes. The phospholipid composition, ratio of cholesterol:phospholipid, and membrane fluidity were similar in male and female microsomes. These results indicate that gender-dependent differences in UDP-glucuronosyltransferase activity are due to differences in both the amount and functional state of the enzyme.

Effect of repeated administration with subtoxic doses of acetaminophen to rats on enterohepatic recirculation of a subsequent toxic dose

Development of resistance to toxic effects of acetaminophen (APAP) was reported in rodents and humans, though the mechanism is only partially understood. We examined in rats the effect of administration with subtoxic daily doses (0.2, 0.3, and 0.6g/kg, i.p.) of APAP on enterohepatic recirculation and liver toxicity of a subsequent i.p. toxic dose of 1g/kg, given 24h after APAP pre-treatment. APAP and its major metabolite APAP-glucuronide (APAP-Glu) were determined in bile, urine, serum and liver homogenate. APAP pre-treatment was not toxic, as determined by serum markers of liver damage and neither induced oxidative stress as demonstrated by assessment of ROS generation in liver or glutathione species in liver and bile. APAP pre-treatment induced a partial shift from biliary to urinary elimination of APAP-Glu after administration with the toxic dose, and decreased hepatic content and increased serum content of this conjugate, consistent with a marked up-regulation of its basolateral transporter Mrp3 relative to apical Mrp2. Preferential secretion of APAP-glu into blood decreased enterohepatic recirculation of APAP, thus attenuating liver exposition to the intact drug, as demonstrated 6h after administration with the toxic dose. The beneficial effect of interfering the enterohepatic recirculation was alternatively tested in animals receiving activated charcoal by gavage to adsorb APAP of biliary origin. The data indicated decreased liver APAP content and glutathione consumption. We conclude that selective up-regulation of Mrp3 expression by APAP pre-treatment may contribute to development of resistance to APAP hepatotoxicity, at least in part by decreasing its enterohepatic recirculation.

Beneficial Effect of Spironolactone Administration on Ethynylestradiol-Induced Cholestasis in the Rat: Involvement of Up-Regulation of Multidrug Resistance-Associated Protein 2

The effect of spironolactone (SL) administration on 17α-ethynylestradiol (EE)-induced cholestasis was studied, with emphasis on expression and activity of Mrps. Adult male Wistar rats were divided into the following groups: EE (5 mg/kg daily for 5 days, s.c.), SL (200 μmol/kg daily for 3 days, i.p.), EE+SL (same doses, SL administered the last 3 days of EE treatment), and controls. SL prevented the decrease in bile salt-independent fraction of bile flow induced by EE, in association with normalization of biliary excretion of glutathione. Western blot studies indicate that EE decreased the expression of multidrug resistance-associated protein 2 (Mrp2) by 41% and increased that of Mrp3 by 200%, whereas SL only affected Mrp2 expression (+60%) with respect to controls. The EE+SL group showed increased levels of Mrp2 and Mrp3 to the same extent as that registered for the individual treatments. Real-time polymerase chain reaction studies indicated that up-regulation of Mrp2 and Mrp3 by SL and EE, respectively, was at the transcriptional level. To estimate Mrp2 and Mrp3 activities, apical and basolateral excretion of acetaminophen glucuronide (APAP-glu), a common substrate for both transporters, was measured in the recirculating isolated perfused liver model. Biliary/perfusate excretion ratio was decreased in EE (-88%) and increased in SL (+36%) with respect to controls. Coadministration of rats with SL partially prevented (-53%) impairment induced by EE in this ratio. In conclusion, SL administration to EE-induced cholestatic rats counteracted the decrease in bile flow and biliary excretion of glutathione and APAP-glu, a model Mrp substrate, findings associated with up-regulation of Mrp2 expression.

Regulation of Synthesis and Trafficking of Canalicular Transporters and its Alteration in Acquired Hepatocellular Cholestasis. Experimental Therapeutic Strategies for its Prevention

Bile formation is an osmotic process driven by the vectorial transport of actively transferred biliary components across the basolateral (sinusoidal) and apical (canalicular) hepatocyte membranes, the latter being the rate-limiting step of the overall blood-to-bile transfer. The ATP-binding cassette (ABC) superfamily of membrane transporters comprises novel ATP-dependent carriers that mediate canalicular transfer of several endogenous and exogenous substrates, and therefore play a key role in bile formation. Gene expression, as well as the balance between vesicular targeting and internalization of these transporters to/from the canalicular membrane are highly regulated processes. This balance is affected in several models of hepatocellular cholestasis, and these alterations may either initiate or perpetuate the cholestatic manifestations. This review describes the regulation of the normal activity of hepatocellular ABC transporters, focusing on the involvement of transcription factors and signaling pathways in the regulation of carrier synthesis, dynamic localization and phosphorylation status. Its alteration in different experimental models of cholestasis, such as those induced by estrogens, lipopolysaccharide (endotoxin), monohydroxylated bile salts and oxidative stress, is also reviewed. Finally, several experimental therapeutic approaches based upon the administration of compounds known/thought to induce carrier synthesis (e.g., protein synthesis inducers), to counteract etiological factors responsible for the cholestatic disease (e.g., corticoids in lipopolysaccharide-induced cholestasis) or to stimulate exocytic insertion of canalicular transporters (e.g., cAMP, silymarin or tauroursodeoxycholate) are described with respect to their ability to prevent cholestatic alterations; the role of signaling molecules as putative downstream mediators of their effects are also discussed.

Regulation of expression and activity of multidrug resistance proteins MRP2 and MDR1 by estrogenic compounds in Caco-2 cells. Role in prevention of xenobiotic-induced cytotoxicity

ABC transporters including MRP2, MDR1 and BCRP play a major role in tissue defense. Epidemiological and experimental studies suggest a cytoprotective role of estrogens in intestine, though the mechanism remains poorly understood. We evaluated whether pharmacologic concentrations of ethynylestradiol (EE, 0.05pM to 5nM), or concentrations of genistein (GNT) associated with soy ingestion (0.1-10μM), affect the expression and activity of multidrug resistance proteins MRP2, MDR1 and BCRP using Caco-2 cells, an in vitro model of intestinal epithelium. We found that incubation with 5pM EE and 1μM GNT for 48h increased expression and activity of both MRP2 and MDR1. Estrogens did not affect expression of BCRP protein at any concentration studied. Irrespective of the estrogen tested, up-regulation of MDR1 and MRP2 protein was accompanied by increased levels of MDR1 mRNA, whereas MRP2 mRNA remained unchanged. Cytotoxicity assays demonstrated association of MRP2 and MDR1 up-regulation with increased resistance to cell death induced by 1-chloro-2,4-dinitrobenzene, an MRP2 substrate precursor, and by paraquat, an MDR1 substrate. Experiments using an estrogen receptor (ER) antagonist implicate ER participation in MRP2 and MDR1 regulation. GNT but not EE increased the expression of ERβ, the most abundant form in human intestine and in Caco-2 cells, which could lead in turn to increased sensitivity to estrogens. We conclude that specific concentrations of estrogens can confer resistance against cytotoxicity in Caco-2 cells, due in part to positive modulation of ABC transporters involved in extrusion of their toxic substrates. Although extrapolation of these results to the in vivo situation must be cautiously done, the data could explain tentatively the cytoprotective role of estrogens against chemical injury in intestine.

Inhibitory effect of bilirubin on complement-mediated hemolysis

We investigated the in vitro action of the bile pigments, unconjugated bilirubin (UB) and bilirubin monoglucuronide (BMG) on complement (C) cascade reaction. Both UB and BMG inhibited hemolysis in the classical pathway (CP) in a dose-dependent manner at low micromolar concentrations, UB showing a stronger effect than BMG. The analysis of the action of UB on the hemolytic activity of the C1, C4, C2 and C-EDTA components of the C cascade revealed that the C1 step was the most inhibited. An enzyme immunoassay was developed to evaluate the effect of UB on the binding of C1q, one of the subcomponents of C1, to human IgM and IgG. The study demonstrated that the unconjugated pigment interferes both the C1q-IgM and -IgG interactions, thus tentatively explaining the inhibitory action of UB on hemolytic activity of C1. We conclude that the anti-complement effect of UB is mainly exerted on the C1 component, the recognition unit of CP. The potential clinical implication of the reported effects in hyperbilirubinemia is discussed.