Enrique J. Sánchez Pozzi

Ursodeoxycholic acid in cholestasis: linking action mechanisms to therapeutic applications

UDCA (ursodeoxycholic acid) is the therapeutic agent most widely used for the treatment of cholestatic hepatopathies. Its use has expanded to other kinds of hepatic diseases, and even to extrahepatic ones. Such versatility is the result of its multiple mechanisms of action. UDCA stabilizes plasma membranes against cytolysis by tensioactive bile acids accumulated in cholestasis. UDCA also halts apoptosis by preventing the formation of mitochondrial pores, membrane recruitment of death receptors and endoplasmic-reticulum stress. In addition, UDCA induces changes in the expression of metabolizing enzymes and transporters that reduce bile acid cytotoxicity and improve renal excretion. Its capability to positively modulate ductular bile flow helps to preserve the integrity of bile ducts. UDCA also prevents the endocytic internalization of canalicular transporters, a common feature in cholestasis. Finally, UDCA has immunomodulatory properties that limit the exacerbated immunological response occurring in autoimmune cholestatic diseases by counteracting the overexpression of MHC antigens and perhaps by limiting the production of cytokines by immunocompetent cells. Owing to this multi-functionality, it is difficult to envisage a substitute for UDCA that combines as many hepatoprotective effects with such efficacy. We predict a long-lasting use of UDCA as the therapeutic agent of choice in cholestasis.

Ca2+-dependent protein kinase C isoforms are critical to estradiol 17β-D-glucuronide–induced cholestasis in the rat†

The endogenous estradiol metabolite estradiol 17β‐D‐glucuronide (E217G) induces an acute cholestasis in rat liver coincident with retrieval of the canalicular transporters bile salt export pump (Bsep, Abcc11) and multidrug resistance‐associated protein 2 (Mrp2, Abcc2) and their associated loss of function. We assessed the participation of Ca2+‐dependent protein kinase C isoforms (cPKC) in the cholestatic manifestations of E217G in perfused rat liver (PRL) and in isolated rat hepatocyte couplets (IRHCs). In PRL, E217G (2 μmol/liver; intraportal, single injection) maximally decreased bile flow, total glutathione, and [3H] taurocholate excretion by 61%, 62%, and 79%, respectively; incorporation of the specific cPKC inhibitor Gö6976 (500 nM) in the perfusate almost totally prevented these decreases. In dose‐response studies using IRHC, E217G (3.75–800 μM) decreased the canalicular vacuolar accumulation of the Bsep substrate cholyl‐lysylfluorescein with an IC50 of 54.9 ± 7.9 μM. Gö6976 (1 μM) increased the IC50 to 178.4 ± 23.1 μM, and similarly prevented the decrease in the canalicular vacuolar accumulation of the Mrp2 substrate, glutathione methylfluorescein. Prevention of these changes by Gö6976 coincided with complete protection against E217G‐induced retrieval of Bsep and Mrp2 from the canalicular membrane, as detected both in the PRL and IRHC. E217G also increased paracellular permeability in IRHC, which was only partially prevented by Gö6976. The cPKC isoform PKCα, but not the Ca2+‐independent PKC isoform, PKCϵ, translocated to the plasma membrane after E217G administration in primary cultured rat hepatocytes; Gö6976 completely prevented this translocation, thus indicating specific activation of cPKC. This is consistent with increased autophosphorylation of cPKC by E217G, as detected via western blotting. Conclusion: Our findings support a central role for cPKC isoforms in E217G‐induced cholestasis, by inducing both transporter retrieval from the canalicular membrane and opening of the paracellular route. (HEPATOLOGY 2008;48:1885‐1895.)

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.

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.

G‐protein‐coupled receptor 30/adenylyl cyclase/protein kinase A pathway is involved in estradiol 17ß‐d‐glucuronide‐induced cholestasis

Estradiol‐17ß‐d‐glucuronide (E17G) activates different signaling pathways (e.g., Ca2+‐dependent protein kinase C, phosphoinositide 3‐kinase/protein kinase B, mitogen‐activated protein kinases [MAPKs] p38 and extracellular signal‐related kinase 1/2, and estrogen receptor alpha) that lead to acute cholestasis in rat liver with retrieval of the canalicular transporters, bile salt export pump (Abcb11) and multidrug resistance‐associated protein 2 (Abcc2). E17G shares with nonconjugated estradiol the capacity to activate these pathways. G‐protein‐coupled receptor 30 (GPR30) is a receptor implicated in nongenomic effects of estradiol, and the aim of this study was to analyze the potential role of this receptor and its downstream effectors in E17G‐induced cholestasis. In vitro, GPR30 inhibition by G15 or its knockdown with small interfering RNA strongly prevented E17G‐induced impairment of canalicular transporter function and localization. E17G increased cyclic adenosine monophosphate (cAMP) levels, and this increase was blocked by G15, linking GPR30 to adenylyl cyclase (AC). Moreover, AC inhibition totally prevented E17G insult. E17G also increased protein kinase A (PKA) activity, which was blocked by G15 and AC inhibitors, connecting the links of the pathway, GPR30‐AC‐PKA. PKA inhibition prevented E17G‐induced cholestasis, whereas exchange protein activated directly by cyclic nucleotide/MAPK kinase, another cAMP downstream effector, was not implicated in cAMP cholestatic action. In the perfused rat liver model, inhibition of the GPR30‐AC‐PKA pathway totally prevented E17G‐induced alteration in Abcb11 and Abcc2 function and localization. Conclusion: Activation of GPR30‐AC‐PKA is a key factor in the alteration of canalicular transporter function and localization induced by E17G. Interaction of E17G with GPR30 may be the first event in the cascade of signaling activation. (Hepatology 2014;59:1016–1029)

Analysis of p-nitrophenol glucuronidation in hepatic microsomes from lactating rats

In the present study, hepatic p-nitrophenol glucuronidation was analyzed comparatively in virgin female, lactating mother and nonlactating mother rats (the last two groups 19-21 days post-partum). Enzyme assays were performed in native and activated microsomal suspensions. Activation was achieved either by including UDP-N-acetylglucosamine in the incubation mixtures or by preincubating native microsomes with optimal concentrations of Triton X-100 or palmitoyl-lysophosphatidylcholine. When UDP-N-acetylglucosamine was used as activator, enzyme activity increased in both lactating (about 80% increment) and nonlactating mothers (about 30% increment) as compared with virgin females. From an analysis of the degree of activation by Triton X-100 and palmitoyl-lysophosphatidylcholine, it can be inferred that the pregnancy-delivery event decreased the latency of UDP-glucuronosyltransferase activity that was detectable even 3 weeks post-partum, irrespective of whether suckling newborns were or were not kept with their mothers (lactating and nonlactating mothers, respectively). The estimation of apparent Vmax toward UDP-glucuronic acid in palmitoyl-lysophosphatidylcholine-activated microsomes, which allows an estimation of the amount of the enzyme, showed that lactation increased the number of catalytic units (about 40%). Hepatic UDP-glucuronic acid content was 70% higher in lactating rats than in other groups. The lipid composition and membrane fluidity (using 1,6-diphenyl-1,3,5-hexatriene as probe) were also analyzed in microsomes from all groups. A significant decrease in the unsaturation index that correlated with the rigidization of microsomal membranes was consistent with the changes in the degree of enzyme latency observed in lactating and nonlactating mothers. In conclusion, lactating rats exhibited enhanced p-nitrophenol UDP-glucuronosyltransferase activity as well as an increase in the hepatic content of UDP-glucuronic acid. These findings and the fact that lactation increased the liver to body weight ratio emphasize the role of the liver in the metabolism of planar phenolic derivatives in these circumstances.

Preventive effect of silymarin against taurolithocholate-induced cholestasis in the rat

Increased amounts of monohydroxylated bile salts (BS) have been found in neonatal cholestasis, parenteral nutrition-induced cholestasis and Bylers disease, among others. We analyzed whether the hepatoprotector silymarin (SIL), administered i.p. at the dose of 100mg/kg/day for 5 days, prevents the cholestatic effect induced by a single injection of the model monohydroxylated BS taurolithocholate (TLC, 30 micromol/kg, i.v.) in male Wistar rats. TLC, administered alone, reduced bile flow, total BS output, and biliary output of glutathione and HCO(3)(-) during the peak of cholestasis (-75, -67, -81, and -80%, respectively, P<0.05). SIL prevented partially these alterations, so that the drops of these parameters induced by TLC were of only -41, -25, -60, and -64%, respectively (P<0.05 vs. TLC alone); these differences between control and SIL-treated animals were maintained throughout the whole (120 min) experimental period. Pharmacokinetic studies showed that TLC decreased the intrinsic fractional constant rate for the canalicular transport of both sulfobromophthalein and the radioactive BS [14C]taurocholate by 60 and 68%, respectively (P<0.05), and these decreases were fully and partially prevented by SIL, respectively. SIL increased the hepatic capability to clear out exogenously administered TLC by improving its own biliary excretion (+104%, P<0.01), and by accelerating the formation of its non-cholestatic metabolite, tauromurideoxycholate (+70%, P<0.05). We conclude that SIL counteracts TLC-induced cholestasis by preventing the impairment in both the BS-dependent and -independent fractions of the bile flow. The possible mechanism/s involved in this beneficial effect will be discussed.

Enhancement of intestinal UDP-glucuronosyltranferase activity in partially hepatectomized rats.

To evaluate whether a temporary hepatic insufficiency may affect intestinal glucuronidation, we determined UDP-glucuronosyltransferase activity towards bilirubin and p-nitrophenol in rat jejunum and liver after partial hepatectomy. Enzyme assays were performed in native, and in UDP-N-acetylglucosamine- or palmitoyl lysophosphatidylcholine-activated microsomes at different times post-hepatectomy. Content of enzyme was analyzed by Western blot. Microsomal cholesterol/phospholipid ratio, phospholipid and total fatty acid classes were also determined to evaluate the possible influence on enzyme activity. The results show that while hepatic microsomes exhibited no change in UDP-glucuronosyltransferase activity (for both substrates) with respect to shams at any time of study, intestinal activities increased significantly 48 h after surgery, returning to sham values 96-h post-hepatectomy. Western blotting confirmed the increase (about 50% for both substrates 48-h post-hepatectomy) in intestinal UDP-glucuronosyltransferase activity. No variations were observed in hepatic and intestinal microsomal lipid composition in agreement with the absence of modification in the percent of activation by palmitoyl lysophosphatidylcholine. In conclusion, jejunum but not liver, was able to produce a compensatory increase in conjugation capacity during a transitory loss of hepatic mass. The phenomenon is associated to a modification in the amount of UDP-glucuronosyltransferase, rather than to changes in the characteristics of the enzyme environment.

GALACTOSAMINE PREVENTS ETHINYLESTRADIOL-INDUCED CHOLESTASIS

Ethinylestradiol (EE) induces intrahepatic cholestasis in experimental animals being its derivative, ethinylestradiol 17β-glucuronide, a presumed mediator of this effect. To test whether glucuronidation is a relevant step in the pathogenesis of cholestasis induced by EE (5 mg/kg b.wt. s.c. for 5 consecutive days), the effect of simultaneous administration of galactosamine (200 mg/kg b.wt. i.p.) on biliary secretory function was studied. A single injection of this same dose of galactosamine was able to decrease hepatic UDP-glucuronic acid (UDP-GA) levels by 85% and excretion of EE-17β-glucuronide after administration of a tracer dose of [3H]EE by 40%. Uridine (0.9 g/kg b.wt. i.p.) coadministration reverted the effect of galactosamine on hepatic UDP-GA levels and restored the excretion of [3H]EE-17β-glucuronide. When administered for 5 days, galactosamine itself did not alter any of the serum markers of liver injury studied (aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase) or biliary secretory function. When coadministered with EE, galactosamine partially prevented the impairment induced by this estrogen in total bile flow, the bile-salt-independent fraction of bile flow, basal bile salt secretion, and the secretory rate maximum of tauroursodeoxycholate. Uridine coadministration partially prevented galactosamine from exerting its anticholestatic effects. In conclusion, galactosamine administration partially prevented EE-induced cholestasis by a mechanism involving decreased UDP-GA availability for subsequent formation of EE 17β-glucuronide. The evidence thus supports the hypothesis that EE 17β-glucuronide is involved in the pathogenesis of EE cholestasis.