Regulation of hepatic P-gp expression and activity by genistein in rats.

P-glycoprotein (P-gp) is an ABC transporter exhibiting high pharmacotoxicological relevance by extruding a wide range of cytotoxic compounds out of the cells. Previously, we demonstrated that the phytoestrogen genistein (GNT) modulates P-gp expression in hepatocellular carcinoma in vitro. Although several beneficial effects (e.g., antioxidant, antimutagenic, anticancer) have been attributed to GNT, the molecular mechanisms have not been totally elucidated. In the present work, we evaluated the effect of GNT on P-gp expression in rat liver, kidney and ileum. We found that GNT (5 mg/kg daily s.c. 3 days) increased hepatic P-gp expression and also Mdr1a (one of the genes encoding P-gp) mRNA levels. Renal and intestinal P-gp remained unchanged after GNT treatment. Hepatic P-gp activity measured with rhodamine-123 and digoxin, both well-known P-gp substrates, was also increased. In vitro experiments using hepatocyte primary cell culture demonstrated that inhibition of ER-α with ICI182/780 did not prevent Mdr1a mRNA up-regulation by GNT (10 µM). In contrast, Mdr1a induction was suppressed after pregnane X receptor (PXR) inhibition by sulforaphane and knockdown of this nuclear receptor. These findings were confirmed in vivo by using the PXR antagonist ketoconazole. In conclusion, we demonstrated the induction of hepatic P-gp expression and activity by GNT in vivo, with PXR being a likely mediator. This suggests that GNT, at concentrations observed in plasma of individuals consuming the phytoestrogen in the diet or through supplements, could affect the clearance of relevant P-gp substrates of therapeutic use as well as toxicity of environmental and food toxicants.

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.

Impaired localisation and transport function of canalicular Bsep in taurolithocholate induced cholestasis in the rat.

BACKGROUND: Taurolithocholate induced cholestasis is a well established model of drug induced cholestasis with potential clinical relevance. This compound impairs bile salt secretion by an as yet unclear mechanism. AIMS: To evaluate which step/s of the hepatocellular bile salt transport are impaired by taurolithocholate, focusing on changes in localisation of the canalicular bile salt transporter, Bsep, as a potential pathomechanism. METHODS: The steps in bile salt hepatic transport were evaluated in rats in vivo by performing pharmacokinetic analysis of (14)C taurocholate plasma disappearance. Bsep transport activity was determined by assessing secretion of (14)C taurocholate and cholyl-lysylfluorescein in vivo and in isolated rat hepatocyte couplets (IRHC), respectively. Localisation of Bsep and F-actin were assessed both in vivo and in IRHC by specific fluorescent staining. RESULTS: In vivo pharmacokinetic studies revealed that taurolithocholate (3 micro mol/100 g body weight) diminished by 58% canalicular excretion and increased by 96% plasma reflux of (14)C taurocholate. Analysis of confocal images showed that taurolithocholate induced internalisation of Bsep into a cytosolic vesicular compartment, without affecting F-actin cytoskeletal organisation. These effects were reproduced in IRHC exposed to taurolithocholate (2.5 micro M). Preadministration of dibutyryl-cAMP, which counteracts taurolithocholate induced impairment in bile salt secretory function in IRHC, restored Bsep localisation in this model. Furthermore, when preadministered in vivo, dibutyryl-cAMP accelerated recovery of both bile flow and bile salt output, and improved by 106% the cumulative output of (14)C taurocholate. CONCLUSIONS: Taurolithocholate impairs bile salt secretion at the canalicular level. Bsep internalisation may be a causal factor which can be prevented by dibutyryl-cAMP.

Beneficial effects of silymarin on estrogen-induced cholestasis in the rat: a study in vivo and in isolated hepatocyte couplets.

The effect of silymarin (SIL) on 17alpha-ethynylestradiol (EE)-induced cholestasis was evaluated in rats. EE (5 mg/kg, subcutaneously, daily, for 5 days) decreased both the bile-salt-dependent and the bile-salt-independent fractions of the bile flow. The decrease in the former was associated to a reduction in the bile salt pool size (-58%), and this effect was completely prevented by SIL. This compound also counteracted the inhibitory effect induced by EE on HCO(3)(-) but not glutathione output, 2 major determinants of the bile-salt-independent bile flow. EE decreased the secretory rate maximum (SRM) of tauroursodeoxycholate, (-71%) and bromosulfophthalein (BSP; -60%), as well as the expression of the BSP canalicular carrier, mrp2; SIL failed to increase mrp2 expression, and had only a marginal beneficial effect on both tauroursodeoxycholate and BSP SRM values. However, the two-compartment model-based kinetic constant for BSP canalicular transfer was significantly improved by SIL (+262%). SIL decreased rather than increased CYP3A4, the cytochrome P450 isoenzyme involved in the oxidative metabolism of EE, and had no inhibitory effect on the UDP-glucuronosyltrasferase isoforms involved in the formation of its 17beta-glucuronidated, more cholestatic metabolite. Pretreatment of isolated rat hepatocyte couplets with silibinin, the major, active component of SIL, counteracted the estradiol 17beta-glucuronide-induced decrease in the percentage of couplets secreting apically the fluorescent bile acid analogue, cholyl-lysyl-fluorescein. These results show that SIL protects against EE-induced cholestasis by normalizing mainly the decrease in the bile salt pool size and HCO(3)(-) output, and probably by counteracting the cholestatic effect of its cholestatic, glucuronidated metabolite.

Molecular basis of perinatal changes in UDP-glucuronosyltransferase activity in maternal rat liver.

The molecular basis of perinatal changes occurring in major UDP-glucuronosyltransferase (UGT) family 1 isoforms and in UGT2B1, a relevant isoform belonging to family 2, was analyzed in rat liver. Nonpregnant, pregnant (19-20 days of pregnancy), and two groups of postpartum animals corresponding to early and middle stages of lactation (2-4 and 10-12 days after delivery, respectively) were studied. UGT activity determined in UDP-N-acetylglucosamine-activated microsomes revealed that bilirubin, p-nitrophenol, and ethynylestradiol (17beta-OH and 3-OH) but not androsterone and estrone glucuronidation rates, were decreased in pregnant rats. Decreased enzyme activities returned to control values after delivery. p-Nitrophenol, androsterone, and estrone conjugation rate increased in postpartum rats. Western blot analysis performed with anti-peptide-specific (anti-1A1, 1A5, 1A6, and 2B1) antibodies revealed decreased levels of all family 1 isoforms and UGT2B1 during pregnancy. In postpartum animals, protein level recovered (1A5 and 2B1) or even increased (1A1 and 1A6) with respect to control rats. Northern blot analysis suggested that expression of UGT proteins is down-regulated at a post-translational level during pregnancy and that increased levels of 1A1 and 1A6 observed in postpartum rats were associated to increased mRNA. To establish whether prolactin is involved in up-regulation of UGT1A1 and 1A6 postpartum, ovariectomized rats were treated with 300 microg of ovine prolactin per day for 7 days. The data indicated that prolactin was able to increase expression of UGT1A6 (protein and mRNA) but not 1A1. Thus, prolactin is the likely mediator of the increased expression of UGT1A6 observed in maternal liver postpartum.

Expression of multidrug resistance-associated protein 2 in small intestine from pregnant and postpartum rats.

We analyzed the expression of multidrug resistance-associated protein 2 (mrp2) in the small intestine of control female rats and in rats during late pregnancy (19-20 days of pregnancy) and lactation (2-4, 10-14, and 21 days after delivery). Western blot analysis was performed on brush-border membranes prepared from different regions of the small intestine. Expression of mrp2 was maximal in the proximal segments for all experimental groups, was preserved in pregnant rats, and increased by 100% in postpartum rats by late lactation with respect to control animals. Northern blot analysis of mrp2 mRNA revealed a positive correlation with protein levels. Transport of S-glutathione-dinitrophenol (DNP-SG) from the intestinal cell to the lumen was analyzed in the everted intestinal sac model. Secretion of DNP-SG was not altered in pregnant rats but increased in lactating animals by late lactation. Intestinal mrp2 mRNA, protein, and transport activity are increased in lactating rats, suggesting that this may represent an adaptive mechanism to minimize the toxicity of dietary xenobiotics in response to increased postpartum food consumption.

Expression and localization of multidrug resistant protein mrp2 in rat small intestine.

The expression of multidrug resistance-associated protein isoform 2 (mrp2), the ATP-dependent export pump that mediates the transport of glucuronic acid-, glutathione-, and sulfate-conjugated derivatives, was studied in rat small intestine. The small intestine was divided into nine equal segments, and mrp2 content was analyzed in homogenate and brush border membrane preparations by Western analysis. mrp2 protein was present mainly in brush border membrane of the proximal segments and gradually decreased from jejunum to the distal ileum. We also analyzed the content of mrp2 in three different populations of proximal enterocytes obtained from the upper and lower villus and the crypt regions. The export pump was mainly expressed in the villus cells and to a lesser degree in the crypt cells of the epithelium. Immunohistochemical analysis performed in duodenum, jejunum, and ileum confirmed in situ the Western blot findings. Analysis of mRNA encoding mrp2 in proximal and distal segments revealed a similar content in both regions, whereas distribution along the villus-crypt axis was similar to the protein gradient. Because conjugating enzymes are distributed similarly to mrp2, we conclude that they may act coordinately to contribute to first-pass metabolism of drugs and other xenobiotics in the proximal small intestine.

Effect of silymarin on biliary bile salt secretion in the rat.

The effect of the hepatoprotector silymarin on bile secretion, with particular regard to bile salt secretion, was studied in Wistar rats. Silymarin (25, 50, 100, and 150 mg/kg/day, i.p., for 5 days) induced a dose-dependent increase in bile flow and bile salt secretion, the maximal effect being reached at a dose of 100 mg/kg/day (+17 and +49%, for bile flow and bile salt output, respectively; P < 0.05). Assessment of bile salt composition in bile revealed that stimulation of the bile salt secretion was accounted for mainly by an increase in the biliary secretion of beta-muricholate and, to a lesser extent, of alpha-muricholate, chenodeoxycholate, ursodeoxycholate, and deoxycholate. The maximum secretory rate (T(m)) of bile salts, as assessed by infusing the non-hepatotoxic bile salt tauroursodeoxycholate i.v. at stepwise-increasing rates, was not influenced by silymarin. The flavonolignan also increased the endogenous bile salt pool size (+53%, P < 0.05) and biliary bile acid excretion after bile acid pool depletion (+54%, P < 0.05), a measure of de novo bile salt synthesis. These results suggest that silymarin increases the biliary excretion and the endogenous pool of bile salts by stimulating the synthesis, among others, of hepatoprotective bile salts, such as beta-muricholate and ursodeoxycholate.

Quantitative and qualitative gender-related differences in jejunal glutathione S-transferase in the rat effect of testosterone administration.

Gender-related differences and the regulation by testosterone of glutathione S-transferase were studied in rat jejunum. We analyzed enzyme activity and the relative content of GST subunits. Four experimental groups of adult rats were studied: normal males, castrated males, castrated males injected with testosterone and normal females. Glutathione S-transferase activity was assayed using 1-chloro-2,4-dinitrobenzene and 1,2-dichloro-4-nitrobenzene as substrates. Differences in subunit composition among groups were evaluated by western blot analysis. The results demonstrated that 1-chloro-2,4-dinitrobenzene conjugation rate is higher in normal males than in normal females and castrated males. Testosterone administration to castrated males raised the activity up to the level observed in normal males. No significant difference in glutathione S-transferase activity towards 1,2-dichloro-4-nitrobenzene was observed among groups. Western blot analysis revealed that males and females differ in all subunits tested that is, rGSTA2, rGSTM1, rGSTM2 and rGSTP1, and that testosterone regulates the content of rGSTM1, rGSTM2 and rGSTP1. In conclusion, jejunal GST shows a gender-dependent regulation affecting both enzyme activity and subunit composition, and testosterone appears to be one of the factors involved.

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.

Induction of phase II biotransformation reactions in rat jejunum during lactation. Possible involvement of prolactin.

The effect of lactation on UDP-glucuronosyltransferase (UGT) and Glutathione S-transferase (GST) activities was studied in jejunum from mother rats, 14 (LM14) and 21 (LM21) days after delivery. p-Nitrophenol glucuronidation rate was increased in LM14 and LM21 rats while conjugation of bilirubin and estrone was not affected and androsterone glucuronidation was decreased. Additional studies, including Western blotting and microsomal lipid analysis, revealed that the enhancement in p-nitrophenol UGT activity is most likely associated with an inductive process rather than with a modification in enzyme constraint. GST activity towards 1-chloro-2,4-dinitrobenzene (CDNB) was also increased in LM14 and LM21 while activity towards 1,2-dichloro-4-nitrobenzene (DCNB) was not affected. Western blotting revealed a significant increase in the cytosolic content of mu (rGSTM2) and pi (rGSTP1) class subunits in LM14 and LM21 groups, while the alpha class subunit rGSTA2 remained unchanged. To evaluate the potential modulatory role of prolactin on the same enzyme systems, ovariectomized rats were treated with ovine prolactin (oPRL) at doses of 100, 200 and 300 microg/100 g body wt. per day for 4 days. Hormone administration affected UGT activities towards p-nitrophenol and androsterone and GST activity towards CDNB in a way and magnitude consistent with those produced in lactating rats, while conjugation of estrone, bilirubin and DCNB were unchanged. Western blotting data were also consistent with those of lactating rats. These results indicate that UGT and GST activities are increased in rat jejunum during lactation, due to induction of some specific isoforms, and that prolactin is the likely mediator of these effects.

Prolactin increases the hepatic content of mu-class subunits of glutathione S-transferase in the rat.

Hepatic glutathione S-transferase (GST) activity is increased in postpartum female rats, a phenomenon that depends on the lactation stimulus. Here we evaluated the effect of prolactin (PRL) administration on hepatic enzyme activity and on the expression of the major subunits of the alpha- (rGSTA1, rGSTA2, rGSTA3) and mu-classes (rGSTM1, rGSTM2). A similar study was conducted in lactating (LM) and in nonlactating (NLM) mother rats 14 days after delivery and in virgin female rats (V). Ovine PRL (oPRL) was administered to ovariectomized rats at daily doses of 75, 150, 200, and 300 microg/100 g b.wt. (PRL1, PRL2, PRL3, and PRL4, respectively) for 4 consecutive days. GST activity was measured using 1-chloro-2,4-dinitrobenzene as substrate. The relative content of the different subunits was determined by Western blot. oPRL produced a dose-dependent increase in GST activity (60% at the highest dose). Subunit analysis performed in PRL2 and PRL4 revealed a substantial enhancement in rGSTM2 and to a lesser extent in rGSTM1, in response to oPRL. The effect was also dose-dependent. alpha-Class subunits were increased only slightly after hormone treatment. A 60% increase in GST activity was observed for LM relative to NLM and V. As was observed for PRL treatment, the increase was associated with changes in the expression of mu-class subunits whereas alpha-class subunits were not affected by lactation. Taken together these data would indicate a role of PRL in regulating GST activity postpartum via an increase in the content of mu-class subunits, particularly rGSTM2.

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.

Differential induction of glutathione S-transferase subunits by spironolactone in rat liver, jejunum and colon.

The effect of spironolactone pretreatment on glutathione S-transferase activity and on the relative content of the principal subunits (Ya, Yc, Yb1, Yb2 and Yp or 1, 2, 3, 4 and 7 respectively) was studied in rat liver, jejunum and colon. Male Wistar rats were injected with spironolactone i.p. at daily doses of 50, 100 and 200 micromol/kg body wt for 3 consecutive days. Glutathione S-transferase activities were assayed using 1-chloro-2,4-dinitrobenzene as substrate. Changes in subunit composition were evaluated by Western blot analysis in rats treated with the highest dose of spironolactone. The results demonstrated a dose-dependent increase in enzyme activity in liver, while in jejunum the three tested doses exhibited the same magnitude of induction. No significant difference in glutathione S-transferase activity was observed between control and treated rats for the colon. Immunoblot analysis revealed more Ya and Yp protein in liver (140 and 118% increase respectively) and jejunum (45 and 145% increase respectively) from treated rats. While Ya and Yp relative contents were similar in jejunum, the latter subunit slightly contributed to total GST in liver, even in SL-treated animals. The inducer produced no change in subunit composition in colon. In conclusion, spironolactone was able to increase glutathione S-transferase activity mainly by induction of Ya subunit in liver and Yp subunit in jejunal mucosa, without affecting colonic enzyme.

Transepithelial fate of bilirubin in the isolated rat kidney.

The renal handling of bilirubin in the rat was studied using an isolated kidney preparation by means of the determination of total pigment concentration decay in the perfusion medium and its renal clearance. Unconjugated bilirubin was incorporated in the perfusate at a concentration of about 4 microg/ml. In order to establish the potential role of secretion in renal handling of the pigment, experiments were also performed incorporating in the perfusate different doses of nicotinic acid (NA) (0.1 and 1.0 mM final concentration), which is considered an alternative substrate for the organic anion transport system, or probenecid (Prob) (0.1 and 1.0 mM final concentration), the classical inhibitor of organic anion transport process. The magnitude of pigment uptake from the perfusion medium, estimated by a first order exponential decay constant, was decreased in a dose-dependent way by NA (40 and 76% decrease for 0.1 and 1.0 mM of NA, respectively) and Prob (57 and 88% decrease for 0.1 and 1.0 mM of Prob, respectively). NA and Prob also induced a diminution in the ratio of pigment renal clearance to glomerular filtration rate (24 and 48% decrease for 0.1 and 1.0 mM of NA and 52 and 55% decrease for 0.1 and 1.0 mM of Prob). Based on these findings, it can be proposed that tubular secretion through the proximal cells contributes significantly to renal pigment depuration. In order to establish the possible contribution of cellular metabolism to the secretory process, a different set of experiments was conducted. The content of bilirubin mono and diconjugates (BMC and BDC) were determined in urine, in arterial and venous samples and in renal cortex. Studies performed using either an open or a closed circulating system, revealed that after conjugation in the renal cell, pigment derivatives can be secreted into both the tubule and the venous compartments. Total bilirubin concentration as well as the relative content of BMC and BDC in urine increased over time, representing the sum of both conjugates about 50% of the total pigment excreted by the end of experiments. Consequently, our results support the existence of a tubular transepithelial transport of bilirubin, playing the metabolism of the pigment an important role in this process.

Ovine prolactin increases hepatic UDP-glucuronosyltransferase activity in ovariectomized rats.

The potential role of prolactin in modulating hepatic UDP- glucuronosyltransferase (UGT) activity was studied. Ovariectomized adult female rats were treated with ovine prolactin (oPRL) at doses of 150, 200, 260, and 310 micrograms/100 g b.wt. per day, for 4 days. Enzyme assays were performed in native and activated microsomes with p-nitrophenol as substrate. Activation was achieved either by including UDP-N-acetylglucosamine in the incubation mixtures or by preincubating native microsomes with palmitoyl-lysophosphatidylcholine. Data obtained with UDP-N-acetylglucosamine as activator showed that increasing doses of oPRL produced a progressive increase in enzyme activity up to a maximum of about 35% over basal values. Immunoblotting of microsomal protein with anti-UGT antiserum revealed also a dose-dependent increase in the immunoreactive protein. A kinetic method for measuring glucuronidating enzyme content confirmed the result of the immunoblot. oPRL induced minor changes in the physicochemical properties of the microsomal membrane. Consistent with this observation, studies performed with palmitoyl-lysophosphatidylcholine as activator showed no change in UGT latency, suggesting that the functional characteristics of the enzyme were not substantially affected by oPRL. The current data support the conclusion that prolactin may act as a modulator of UGT activity by increasing the amount of enzyme.

Enhancement of intestinal bilirubin UDP-glucuronosyltransferase activity by modification of microsomal lipid composition.

Microsomal membranes from rat small intestine exhibit a higher cholesterol/phospholipid ratio and a lower phosphatidyl-choline/sphingomyelin ratio than those of the liver, which could negatively influence membrane-bound enzymes like bilirubin UDP-glucuronosyltransferase. To study the effect of in vitro modifications in the lipid composition of intestinal microsomes on bilirubin glucuronidating activity, two strategies were employed. On one hand, microsomal lipids were modified in order to mimic those of the liver tissue; on the other hand, cholesterol content of microsomal membranes was increased or decreased with respect to the normal value. Lipid changes were carried out by both an enzyme-mediated and a detergent-mediated procedure. Irrespective of the methodology employed, when a depletion in the cholesterol content was produced, enzyme activity increased about 40%, and when lipid composition approached that of the liver tissue, which not only decreased cholesterol but also modified phospholipid classes, enzyme activity increased about 80%. Both lipid modifications produced a 'fluidification' of microsomal membranes measured by fluorescence anisotropy of 1,6-diphenylhexatriene, being the effect of the approach to the liver higher than that of the decrease of cholesterol. In turn, the enrichment in cholesterol of microsomal membranes led to a decrease of enzyme activity of about 20% and to a 'rigidization' of the membranes. The present findings suggest that in rat intestine, bilirubin glucuronidation is strongly influenced by microsomal lipids. In particular, there seems to be an inverse association between enzyme activity and the cholesterol content of membrane.

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.

Differential effect of ursodeoxycholate and its taurine conjugate on biliary transport maximum of bilirubin in the rat.

The effects of ursodeoxycholate and its taurine conjugate on biliary Tm of bilirubin were evaluated in rats. Ursodeoxycholate was administered at four different doses (4, 8, 12 or 16 mumol per 100 g body wt i.v., followed by an i.v. infusion of 0.3, 0.6, 0.9 or 1.2 mumol/min per 100 g body wt, respectively), whereas tauroursodeoxycholate was administered only at the maximal dose. A dose-dependent diminution of bilirubin Tm was observed during ursodeoxycholate administration, which ranged from no effect at the lowest dose to a virtual excretory blockage at the highest dose. This was associated with an increase in bilirubin concentrations in both plasma and liver as well as in the fractional amount of conjugated pigment in both sites, suggesting an impairment of bilirubin transfer at the canalicular level. Incomplete taurine conjugation of ursodeoxycholate well correlated with these effects. Unlike ursodeoxycholate, tauroursodeoxycholate had no inhibitory effect on bilirubin Tm, although a slight inhibition of bilirubin uptake and bilirubin conjugation became apparent. Taken together, these results suggest that ursodeoxycholate interferes with the hepatobiliary transport of bilirubin by impairing its transfer at the canalicular level and that incomplete taurine conjugation appears to be a key factor determining this effect.