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Objective We aimed to examine the dynamics of serum Wisteria floribunda agglutinin-positive human Mac-2-binding protein glycosylation isomer (M2BPGi) in patients with acute liver injury. Methods Serum M2BPGi levels at the time of the diagnosis (n=77) and normalization of the serum alanine aminotransferase (ALT) level (n=26) were examined retrospectively. The difference in the serum M2BPGi level according to the etiology, and the correlations with other laboratory parameters were evaluated. Results The serum M2BPGi level at the time of the diagnosis was increased in 59 of 77 patients [2.3 cutoff index (COI); range, 0.31-11.1 COI] and was significantly decreased at the time of serum ALT normalization (0.68 COI; range, 0.15-1.87 COI; p<0.0001). The serum M2BPGi level was positively correlated with the duration for which serum ALT normalization was achieved (n=46, Spearman rho=0.53, p<0.0001). A multivariate analysis identified total bilirubin (T-bil), albumin, ALT, alkaline phosphatase, and etiology (e.g., drug-induced liver injury or etiology unknown) as independent factors for increased serum M2BPGi. In patients with infectious mononucleosis, the serum M2BPGi level was higher relative to the degree of increase of serum ALT or T-bil levels in comparison to other etiologies. Conclusion The serum M2BPGi level in patients with acute liver injury reflects the magnitude and duration of liver injury. However, it should be noted that the degree of increase of serum M2BPGi in patients with acute liver injury may differ according to the etiology.

We investigated the effects of orange juice (OJ) or hesperidin, a component of OJ, on the pharmacokinetics of pravastatin (PRV) and the expression of both protein and mRNA of multidrug resistance-associated protein 2 (Mrp2) in the rat small intestine and liver. Eight-week-old male Sprague-Dawley rats were used in this study. OJ or a 0.079% hesperidin suspension was administered orally for 2 days. Tap water was given as a control. A single dose of PRV at 100  mg/kg p.o. was administered after 2 days of OJ, hesperidin, or tap water ingestion. The AUC, C(max), and t(1/2) values of PRV were significantly increased in OJ group. Mrp2 protein and mRNA levels in the small intestine and liver, respectively, were significantly decreased after the ingestion of OJ. The same results were obtained with hesperidin. These results suggest that the changes in PRV pharmacokinetic parameters and the decrease in Mrp2 expression caused by OJ are due to hesperidin in the juice.

Nitric oxide (NO) and the partial pressure of oxygen (pO(2)) in the liver were simultaneously quantified in rats with partial hepatic ischemia/reperfusion injury (PHIRI). Real-time NO/pO(2) monitoring and immunohistochemical analysis for superoxide dismutase and inducible nitric oxide synthase (iNOS) and endothelial NOS (eNOS) were performed to evaluate the protective effects of a dihydropyridine-type calcium-channel blocker--CV159--on PHIRI. Serum high-mobility-group box-1 (HMGB-1) was measured to assess cellular necrosis. Moreover, we used in vitro/ex vivo electron paramagnetic resonance spin trapping to assess the hydroxyl radical (*OH)-scavenging activity (OHSA) of CV159 and the liver tissue. The NO levels were significantly higher in CV159-treated rats than in control rats throughout the ischemic phase. Immediately after reperfusion, the levels temporarily increased in waves and then gradually decreased in the treated rats but remained constant in the control rats. pO(2) was continually higher in the treated rats. In these rats, hepatic eNOS expression increased, whereas iNOS expression decreased. The treated rats exhibited significantly higher cytosolic and mitochondrial concentrations NOx (NO(2)+NO(3)). The serum HMGB-1 levels significantly decreased in the treated rats. Moreover, CV159 directly scavenged *OH and both mitochondrial and cytosolic OHSA were preserved in the treated rats. Thus, CV159-mediated inhibition of intracellular Ca(2+) overloading may effectively minimize organ damage and also have *OH-scavenging activity and the cytoprotective effects of eNOS-derived NO.

A relatively large amount of human liver tissue was required to determine the exact activity of human hepatic CYP3A. Although, the quantity of available human liver tissue samples is limited. We measured levels of CYP3A4 mRNA by RT-PCR with a radiolabeled primer specific for CYP3A4 and compared mRNA expression with CYP3A4 protein level and metabolic activity in liver. The level of CYP3A4 mRNA was correlated with the levels of CYP3A4 protein and activity. Our results suggest that CYP3A4 protein and activity levels can be predicted from CYP3A4 mRNA levels determined by RT-PCR and using a very small amount of liver tissue.

We examined the influence of five phenothiazine derivatives on the activity and the expression of hepatic cytochrome P450 (CYP) 1A, 2B, 2C6, 2C11, 2E1 and 3A2 in male Sprague-Dawley rats. Twenty mg/kg of phenothiazine, chlorpromazine, and thioridazine, or 1 mg/kg of trifluoperazine and perphenazine was administered intraperitoneally for 4 consecutive days. Phenothiazine increased the total CYP content and induced CYP1A, CYP2B, and CYP3A. Western blot analysis showed marked induction of CYP1A1 by phenothiazine. Chlorpromazine induced CYP2B, and CYP3A without a significant increase in the CYP content. Thioridazine decreased the total CYP content and reduced CYP2C11, CYP2E1, and CYP3A. Neither trifluoperazine nor perphenazine significantly altered the catalytic activity or the protein level of any enzyme examined. These results suggest that the antipsychotic phenothiazine derivatives examined here hardly affect CYP1A at the pharmacological doses, while phenothiazine does induce it. Although a relatively short period and small dose of administration might counteract the influence, trifluoperazine and perphenazine have less effect on CYP subfamilies than chlorpromazine or thioridazine.

Background/aims: Hydrophobic bile acids, such as deoxycholic acid produce cholestatic liver injury. Ursodeoxycholic acid has been shown to be useful in the treatment of cholestatic liver disease., Methods: In this study, we investigated the effects of deoxycholic acid or ursodeoxycholic acid (1% of diet, for 14 days) and their combination (1% each) on expression of hepatic cytochrome P450 isozymes, their related enzyme activities and mRNA level in rats., Results: Adding 1% deoxycholic acid to chow caused a marked increase in serum total bilirubin (47-fold) and total bile acid (8-fold) concentrations and in alkaline phosphatase (2.5-fold, p<0.01) and alanine aminotransferase activities (23.5-fold, p<0.01). Adding the same dose of ursodeoxycholic acid along with the deoxycholic acid mitigated both the rise in serum total bilirubin and bile acid concentrations and that in alkaline phosphatase and alanine aminotransferase activities, although the use of ursodeoxycholic acid alone did not affect any of the above. Feeding 1% deoxycholic acid caused a decrease (48% of control) in total cytochrome P450 content in hepatic microsomes. Addition of 1% ursodeoxycholic acid along with the 1% deoxycholic acid completely prevented the decrease in total cytochrome P450 content. Feeding ursodeoxycholic acid alone did not affect the total cytochrome P450 content. The expression of cytochrome P450 2B1, 2E1, 3A2, 2C6, 2C11 and 4A1 proteins in hepatic microsomes was decreased by deoxycholic acid (44, 51, 23, 59, 30 and 74% of control, respectively). Likewise, the activities of cytochrome P450 2B1 (pentoxyresorufin O-depentylation), 2E1 (aniline p-hydroxylation) and 3A2 (testosterone 6beta-hydroxylation) isozymes and the 3A2 mRNA levels in liver were decreased by deoxycholic acid. Addition of 1% ursodeoxycholic acid to 1% deoxycholic acid also prevented the decrease in these cytochrome P450 proteins, related enzyme activities and mRNA levels in liver., Conclusions: These results indicate that, in rats with deoxycholic acid-induced liver injury, ursodeoxycholic acid prevents the decrease in hepatic cytochrome P450 isozymes and suggest that ursodeoxycholic acid is useful for the treatment of liver injury in terms of aiding the normalization of the hepatic drug-metabolizing system.

We examined hepatic cytochrome P450 (CYP) induction in rat foetuses and neonates by phenobarbital administered through placenta or breast feeding. In an intraperitoneal study, phenobarbital was administered intraperitoneally to mother rats once a day for 7 consecutive days before delivery. The livers were removed from foetuses, neonates, and mothers just before and 5 and 10 days after delivery. In oral administration study, water containing phenobarbital was given orally ad libitum from day 13 of pregnancy to 3 weeks after delivery (end of lactation). The livers were removed from neonates and mothers just before and one week after delactation. Phenobarbital administered intraperitoneally increased both the activity and the protein expression of CYP2B in 5-day-old neonates, even though the administration ended before delivery. This increase had disappeared in 10-day-old neonates. In mother rats, phenobarbital increased CYP2B just before and 5 days after delivery, while no increase was detected 10 days after delivery. Phenobarbital administered orally also increased both the activity and the protein expression of CYP2B of neonates and mothers during lactation and this increase also disappeared 1 week after delactation. Neither activity nor protein expression of CYP3A were induced in perinates at any age examined in either administration route. In mother rats, increase in CYP3A was found only just before delivery in the peritoneal administration study. Our results suggest that phenobarbital administered through placenta or breast milk transiently induces hepatic CYP2B in newborn rats but that the influence of phenobarbital does not last long.

The aim of the present study was to investigate the effects of the estrous cycle and physiological dose of estradiol (E2) and the gender difference on several hepatic drug-metabolising enzyme activities. Eight-week old female Sprague-Dawley rats at different stages of the estrous cycle [proestrous (P), estrous (E), and diestrous (D)] and 8-week old male rats were used in this study (n = 5, respectively). Serum E2 level at D was higher than that at E and lower than that at P. The hepatic cytochrome P450 content, aniline hydroxylase, ethoxycoumarin O-deethylase and aminopyrine N-demethylase activities in male were significantly higher than those in female at any stage of the estrous cycle and these activities in female remained unchanged during the estrous cycle. Neither the estrous cycle nor the gender difference affected the cytochrome b5 content. NADPH-cytochrome c reductase (fPT) activity at P was similar to that in male, and was significantly higher than that in E and D. fPT activity was increased by the administration of physiological dose of E2 in ovariectomized rats. Uridine diphosphate glucuronyltransferase (UDP-GT) activity at E was significantly lower than that at P or D and that activity in female was significantly lower than that in male. These results indicate that the estrous cycle, especially serum E2 level, may affect both fPT and UDP-GT activities.

We examined the influence of ageing and gender on the activity of dihydropyrimidine dehydrogenase (DPD) in male and female Sprague-Dawley rats at 3, 8, 40 and older than 60 weeks of age. The body and liver weights of male rats at 8 weeks and older of age were heavier than those of female rats at the same age and the ratio of liver to body weight was significantly reduced with ageing. The amount of produced 5FU metabolites, dihydrofluorouracil and fluoroureidopropionic acid, remained unchanged in the reaction mixture with the cytosol from any group of the rats. The formation rates of 5FU metabolite were 1.03+/-0.08/1.12+/-0.06, 0.98+/-0.11/1.16+/-0.10, 1.13+/-0.09/1.13+/-0.11 and 1.17+/-0.16/1.24+/-0.08 nmol min(-1) mg protein(-1) (mean+/-SD, male/female) in rats at 3, 8, 40 and older than 60 weeks of age, respectively. The results suggest that the DPD activity in the rat liver may not be affected by ageing or gender.

We immunochemically measured the contents of 9 different cytochrome P450 (CYP) isoenzymes expressed in the liver and compared them between two groups: one group of 6 infant and 4 perinatal patients and one group of 10 patients after infancy (over 1 year old). CYP protein expressed in human liver can be divided into three groups on the basis of expression pattern: (a) CYP2A6, 2C9, 2D6, 2E1, and 3A were present in all samples and no difference was observed between the two groups; (b) CYP1A2, 2B6, and 2C8 were expressed more after infancy than during infancy; and (c) CYP3A7, which has been considered a major CYP enzyme in fetal liver microsomes, was expressed in all infants as well as the four perinatal patients, whereas it was detected in only 2 patients after infancy. These results implied that CYP2A6, 2C9, 2D6, 2E1, and 3A are already expressed during perinatal and infant period, while CYP1A2, 2B6, and 2C8 are expressed highly in subjects over 1 year old, and CYP3A7 disappeared after infancy.

We estimate the influence of five 5-hydroxytryptamine receptor (5-HT3) antagonists on the activity of dihydropyrimidine dehydrogenase (DPDase), the rate-limiting enzyme in 5-fluorouracil (5FU) metabolism. The activity of DPDase from the rat liver was compared in the cytosol mixture of 5FU incubated with or without each of five 5-HT3 antagonists. DPDase activity was not altered in the presence of any 5-HT3 antagonist studied here. It may be inferred from these results that the any 5-HT3 receptor antagonist examined in this study has little or no effect on fluorouracil catabolism.

We investigated bile flow and biliary excretion of bile acids in the Eisai hyperbilirubinemic rat, a Sprague-Dawley mutant rat with conjugated hyperbilirubinemia, using both in vivo and in vitro models. In vivo bile flow was lower in Eisai hyperbilirubinemic rats than in the control rats before and after taurocholate was infused. After taurocholate was infused, bile acid output was similar in the Eisai hyperbilirubinemic rats and control rats. In the isolated perfused rat liver, biliary excretion of bile acids was higher in the Eisai hyperbilirubinemic rats than in the control rats after a high-dose infusion of taurocholate (0.33 mumol/min/gm liver). Infusion of taurochenodeoxycholate (0.22 mumol/min/gm liver) did not produce cholestasis and did not reduce the biliary excretion of bile acids in the Eisai hyperbilirubinemic rats. Taurochenodeoxycholate significantly increased the phospholipid/bile acid molar ratio and slightly reduced bile acid-induced alkaline phosphatase output into bile. The release of lactate dehydrogenase from the perfused liver 30 min after the start of the taurochenodeoxycholate infusion was 10 times lower in the Eisai hyperbilirubinemic rats than in the control rats (2.0 +/- 0.8 vs. 28.7 +/- 6.8 mU/min/gm liver). When the isolated perfused rat liver was infused with a 1-min pulse of horseradish peroxidase (25 mg), we observed an early and late peak of biliary excretion of horseradish peroxidase. The Eisai hyperbilirubinemic rats showed a significant increase in the late peak.(ABSTRACT TRUNCATED AT 250 WORDS)

We investigated the effects of papaverine on bile acid excretion into bile in the presence of infusions of taurocholic acid, tauroursodeoxycholic acid, taurochenodeoxycholic acid and taurodehydrocholic acid in a single-pass, isolated perfused rat liver model. Although continuous infusion of papaverine (1.6 mumol/min) did not reduce bile acid excretion in the presence of low-dose (1.0 mumol/min) infusions of taurocholic acid or tauroursodeoxycholic acid, papaverine significantly inhibited biliary excretion of bile acids in the presence of low-dose taurochenodeoxycholic acid (-50%) and high-dose (3.0 mumol/min) taurocholic acid (-54%), tauroursodeoxycholic acid (-37%) and taurodehydrocholic acid (-31%). During continuous infusion of taurocholic acid (3 mumol/min), a 15-min infusion of papaverine (3.2 mumol/min) reduced bile acid excretion significantly; however, total uptake of bile acid was slightly decreased by the papaverine infusion. Bile acid excretion increased over the baseline value after the papaverine infusion was stopped and then returned to baseline. These results suggest that papaverine does not affect the uptake phase of bile acids at the sinusoidal membrane but may affect the intracellular transport phase or the excretory phase at the bile canalicular membrane. When taurocholic acid was infused at a constant rate of 3 mumol/min for 20 min without papaverine and then stopped, bile acid excretion decreased gradually and was nearly zero by 52 min. Cumulative bile acid excretion in the 52 min after the end of the infusion reached 3.3 +/- 0.2 mumol/gm liver and represented the storage capacity of the liver.(ABSTRACT TRUNCATED AT 250 WORDS)

The secondary messenger cyclic AMP plays an important role in regulating biliary excretory function by stimulating the transcytotic vesicle transport system, whereas papaverine exerts an inhibitory effect on this system. We therefore investigated their effects on bile acid-induced cytotoxicity and intrahepatocytic content of bile acid in primary cultured rat hepatocytes. Simultaneous addition of 1 mM dibutyryl cyclic AMP (DBcAMP), an analogue of cAMP, with 1 mM taurochenodeoxycholic acid (TCDCA) significantly decreased the release of lactate dehydrogenase (LDH) as compared with the case with 1 mM TCDCA alone (7.1 +/- 0.13% of total versus 10.7 +/- 0.3%). In contrast, 0.1 mM papaverine approximately doubled the amount of LDH (22.0 +/- 0.6% of total versus 10.7 +/- 0.3%; P < 0.01). The intracellular content of TCDCA 180 min after the administration of 1 mM TCDCA alone was 20.8 +/- 0.7 nmol/mg protein, that after simultaneous administration of 1 mM DBcAMP, 16.2 +/- 1.0 nmol/mg protein, and that after the simultaneous administration of 0.1 mM papaverine, 38.5 +/- 1.9 nmol/mg protein. A clear correlation between the release of LDH from hepatocytes and the intracellular content of TCDCA was thus observed. When given together with 1 mM taurocholic acid (TCA) or 1 mM tauroursodeoxycholic acid (TUDCA), papaverine exerted little effect on cytotoxicity or intrahepatocytic bile acid content. When cells were bathed in a medium free of bile acid after pretreatment with 1 mM TCDCA and 1 mM DBcAMP, additional exposure to DBcAMP for 30 min significantly stimulated reduction of intracellular TCDCA content (30.2 +/- 0.4% of total versus 44.0 +/- 1.4%).(ABSTRACT TRUNCATED AT 250 WORDS)

Papaverine is a nonspecific smooth muscle relaxant and a phosphodiesterase inhibitor. Its effects on biliary excretion of lipids and horseradish peroxidase were investigated in a single-pass isolated perfused rat liver model. A constant infusion of papaverine (1.6 mumol/min; 40 mumol/L) significantly increased bile flow (microliters per minute per gram of liver) before (2.03 +/- 0.09 vs. 1.0 +/- 0.06) and after sodium taurocholate infusion (2.77 +/- 0.10 vs. 1.88 +/- 0.11). However, papaverine significantly and reversibly reduced biliary excretion of phospholipids and cholesterol (nanomoles per minute per gram of liver) after a 1.0 mumol/min sodium taurocholate infusion, from 7.45 +/- 0.83 and 1.42 +/- 0.15 to 1.75 +/- 0.18 and 0.39 +/- 0.06, respectively (p less than 0.01), whereas secretion of bile acids was unaffected. When a 1-min pulse of horseradish peroxidase (25 mg) was infused in isolated perfused rat liver after a continuous infusion of N6,O-2'-dibutyryladenosine 3',5'-cyclic monophosphate (0.25 mumol/min; 6.25 mumol/L), horseradish peroxidase appeared in bile in an early (4 to 6 min) and late (20 to 25 min) peak. Papaverine significantly reduced the late peak, from 1.211 +/- 0.264 to 0.498 +/- 0.107 (p less than 0.01). Papaverine had no significant effects on either cyclic AMP or cyclic GMP in the liver and bile, although it has been reported that papaverine is a phosphodiesterase inhibitor. These findings indicate that papaverine inhibits biliary excretion of lipids but not bile acids, and they suggest that papaverine has an inhibitory effect on transcytotic vesicle transport independent of an increase of cyclic nucleotides in hepatocytes.