Changes in the pattern of bile acids in the nuclei of rat liver cells during hepatocarcinogenesis.

Bile acids reach the nuclei of hepatocytes, where they may play an important role in controlling gene expression by binding to nuclear receptors. In previous studies, changes in the amounts of the different molecular species of bile acids in the hepatocyte nucleus during rat liver regeneration have been reported. The aim of the present work was to investigate whether this also occurs during rat hepatocarcinogenesis. Liver cell nuclei were isolated after homogenization of livers from healthy adult rats (controls) and from rats at different time points during chemically induced hepatocarcinogenesis, corresponding to the stages of foci (12 weeks), hepatoma (20 weeks) and carcinoma (32 weeks). Bile samples from the cannulated common bile duct were collected for 1h from different sets of animals undergoing hepatocarcinogenesis. Bile acids in bile, liver homogenates and isolated nuclei were measured by GC-MS. Because the yield of nuclei isolated changed during the course of hepatocarcinogenesis (control, 20.1%; 12 weeks, 23.6%; 20 weeks, 7.8%; 32 weeks, 5.1%), amounts of bile acids in nuclei were corrected for the amount of DNA in each preparation. During hepatocarcinogenesis, bile acid concentrations in liver homogenates were reduced to approximately half the values obtained in control livers, while the levels of bile acids in both isolated nuclei and bile were not decreased. Hepatocarcinogenesis induced changes in the composition of bile acid pools. These were manifest as an increase in the proportion of cholic acid and a decrease in that of ursodeoxycholic acid in both bile and liver. These modifications differed from the changes seen in the nuclear bile acid pool, where a decrease in the proportion of cholic acid together with an increase in that of ursodeoxycholic acid were the major changes observed during hepatocarcinogenesis. With regard to the 'flat' bile acids (allo-cholic acid plus Delta(5)- or Delta(4)-unsaturated bile acids), a marked hepatocarcinogenesis-induced increase in the output of these species in bile was found. However, these bile acids were only found in liver homogenates at the hepatoma stage, whereas they were not detected in isolated nuclei at any stage of hepatocarcinogenesis. In summary, these results support the existence of a bile acid pool in hepatocyte nuclei whose composition differs from that of the extranuclear bile acid pool. Moreover, they indicate that, during hepatocarcinogenesis, the composition of the nuclear pool undergoes important alterations.

Cytokine-mediated inhibition of ketogenesis is unrelated to nitric oxide or protein synthesis.

Cytokines play an important role in the lipid disturbances commonly associated with sepsis. Ketogenesis is inhibited during sepsis, and tumor necrosis factor alpha (TNF alpha) and interleukin-6 (IL-6) have been suggested to mediate this impairment, irrespective of the ketogenic substrate (fatty acid or branched chain ketoacid). However, the underlying mechanism of cytokine action is still unknown. First we investigated the possible role of the induction of nitric oxide (NO) synthesis, using rat hepatocyte monolayers. Hepatocytes were incubated for 6 h, with either alpha -ketoisocaproate (KIC) (1 mM) or oleic acid (0.5 mM) in the presence or absence of TNF alpha (25 microg/L) and IL-6 (15 microg/L). In some experiments, cells were incubated with NO synthase (NOS) inhibitors. The ketone body (beta -hydroxybutyrate and acetoacetate) production and nitrite production were measured in the incubation medium. Our results indicated no involvement of nitric oxide in the inhibitory action of cytokines on ketogenesis. Secondly, we showed that cycloheximide (10(-4)M) did not counteract the cytokine-mediated ketogenesis decrease; hence, the effects of cytokines on ketogenesis are not protein synthesis-dependent. The cytokine-mediated inhibition of ketogenesis is therefore unrelated to either NO production or protein synthesis.

Increased levels of typically fetal bile acid species in patients with hepatocellular carcinoma.

The aim of this work was to investigate the reappearance during liver neoplasia of bile acids (BAs) species, which are unusual in healthy adults, but common in fetuses. Serum and urine samples were collected from patients with hepatocellular carcinoma (HCC; n=27), and for comparative purposes, with liver cirrhosis (n=49), liver metastasis (n=19), chronic viral hepatitis (n=11) and healthy volunteer (control group; n=26) groups. BAs were identified and measured by GC--MS. Hypercholanaemia was found in all groups of patients. In HCC, this was characterized by a marked increase in the chenodeoxycholate/cholate ratio in both serum and urine. Although increased levels of BAs, with hydroxylations at unusual positions, and oxo-BAs were found in HCC, these were not significantly different from those observed in other groups. However, BAs with a flat structure, i.e. Delta(4)-unsaturated- and 5 alpha- or allo-BAs, which were almost absent in healthy subjects, were markedly increased in the serum and urine of HCC patients. They were also detected, although in much lower amounts, in liver metastasis and liver cirrhosis, but not in viral hepatitis. Flat-BAs were better detected in urine than in serum. Urinary Delta(4)-unsaturated-BA output was significantly lower in patients with small tumours (<3 cm) compared with those with higher size tumours. No correlation between flat-BA output into urine and serum alpha-fetoprotein or total BAs was found. These results suggest that Delta(4)- and/or allo-BAs are particularly elevated in patients with HCC, which may be a potentially useful complementary, rather than alternative, marker for early detection of liver neoplasia.

Exogenous Mg-ATP induces a large inhibition of pyruvate kinase in intact rat hepatocytes.

Mg-ATP infusion in vivo has been reported to be beneficial both to organ function and survival rate in various models of shock. Moreover, a large variety of metabolic effects has been shown to occur in several tissues due to purinergic receptor activation. In the present work we studied the effects of exogenous Mg-ATP in rat liver cells perifused with dihydroxyacetone to investigate simultaneously gluconeogenetic and glycolytic pathways. We found a significant effect on oxidative phosphorylation as characterized by a decrease in oxygen consumption rate and in the cellular ATP-to-ADP ratio associated with an increase in lactate-to-pyruvate ratio. In addition, exogenous Mg-ATP induced rapid and reversible inhibition of both gluconeogenesis and glycolysis. The main effect on gluconeogenesis was located at the level of the fructose cycle, whereas the decrease in glycolysis was due to a strong inhibition of pyruvate kinase. Although pyruvate kinase inhibition induced by exogenous Mg-ATP was allosteric when assessed in vitro after enzyme extraction, we found a large decrease in the apparent maximal velocity when kinetics were assessed in vivo in intact perifused hepatocytes. This newly described short-term regulation of pyruvate kinase occurs only in the intact cell and may open new potentials for the pharmacological regulation of pyruvate kinase in vivo.

Cholephilic characteristics of a new cytostatic complex of cisplatin with glycocholate (Bamet-R2).

The aim of this work was to investigate both the existence of enterohepatic circulation of cisplatin-cholylglycinate complex, Bamet-R2, and the relevance of biliary versus urinary excretion of this compound. Two experimental models were used: (i) intraluminal perfusion of 'in situ' ileum in anaesthetized rats bearing a biliary catheter that permitted bile sample collection and (ii) conscious rats in which a permanent intraarterial catheter had been implanted to carry out sequential blood sampling after intravenous (i.v.) or intragastric (i.g.) drug administration. Total platinum in serum, bile, ileum, liver, urine and feces was measured by flameless atomic absorption spectroscopy. Serum concentration versus time curves obtained after i.v. administration of 1 micromol Bamet-R2 or cisplatin revealed that the area under the curve was significantly higher for Bamet-R2 than for cisplatin (+48%). Non-ultrafiltrable platinum accounted for 54.8 and 48.4% of serum platinum 168 h after cisplatin and Bamet-R2 i.v. administration, respectively. When the animals received i.g. 1 micromol cisplatin or Bamet-R2, serum concentrations of total platinum were markedly higher (three-fold) after Bamet-R2 than after cisplatin administration. The area under the curve was, also in this case, significantly higher for Bamet-R2 than for cisplatin (+28%). This was in part due to the enhanced intestinal absorption of Bamet-R2, as confirmed in experiments on perfused rat ileum, where a markedly higher amount of the drug was found in ileum tissue and bile after perfusion with media containing Bamet-R2 as compared with experiments where cisplatin instead of Bamet-R2 was added to perfusion media. Moreover, after i.v. administration to conscious rats, excretion of Bamet-R2 by the kidney was three-fold lower than that of cisplatin, while elimination of the former compound into feces was four-fold higher than that of the latter. In summary, these results indicate that in addition to the previously reported cytostatic activity of Bamet-R2, this complex has interesting cholephilic characteristics typical of bile acids, such as low urinary excretion together with enhanced intestinal absorption and biliary secretion, probably endowed by the cholylglycyl moiety included in the Bamet-R2 molecule.

Transport and biotransformation of the new cytostatic complex cis-diammineplatinum(II)-chlorocholylglycinate (Bamet-R2) by the rat liver.

Rat liver uptake and bile output of the cytostatic complex cis-diammineplatinum(II)-chlorocholylglycinate (Bamet-R2) were studied. Up to 100 microM, Bamet-R2 uptake by rat hepatocytes in primary culture followed saturation kinetics (Vmax = 0.65 +/- 0.12 nmol/5 min per mg protein; K(M) = 45.2 +/- 10.7 microM). Bamet-R2 uptake was lower than that of cholylglycinate (CG) but higher than that of cisplatin. Replacement of 116 mM NaCl by 116 mM choline chloride did not significantly reduce Bamet-R2 uptake. Addition of 500 microM CG, cholic acid, estrone sulfate, or ouabain to 50 microM Bamet-R2-containing incubation media inhibited Bamet-R2 uptake. No liver biotransformation of Bamet-R2 occurred, as indicated by HPLC analysis of bile collected from anesthetized rats after intravenous administration of the drug. Bamet-R2 uptake and secretion into bile by isolated rat livers exceeded those of cisplatin but were lower than those of CG. Differences between Bamet-R2 and CG were more marked for bile output than for liver uptake. Thus, higher Bamet-R2 than CG or cisplatin liver content was found. Co-administration of Bamet-R2 and CG revealed that CG induced a slight reduction in Bamet-R2 uptake and a marked inhibition in Bamet-R2 bile output. By contrast, Bamet-R2 had no effect on CG on either liver uptake or bile output. In sum, the present data indicate that Bamet-R2 is efficiently taken up and secreted into bile by the rat liver by mechanisms shared in part by natural bile acids.

In vitro test to determine the effect of cytostatic drugs on co-cultured rat hepatocytes and hepatoma cells.

Using uptake of the fluorescent bile acid derivative cholylglycylamido-fluorescein (FITC-GC) as a measurement of liver cell population size and function, the antiproliferative and toxic effects of the well known cytostatic drug, cisplatin was evaluated on rapidly growing rat hepatoma McA-RH7777 cells and rat hepatocytes in primary culture under non-proliferating conditions. Co-culture set up to mimic the in vivo situation of tumour and extratumoural liver tissue exposed to cytostatic chemotherapy does not markedly affect the survival or the growth dynamics of both cell types. FITC-GC uptake as corrected for DNA and protein contents in the dish was significantly lower in hepatoma cells than in rat hepatocytes throughout the experimental period (96 h). Effect of 0.1-100 microM cisplatin exposure from 24 to 96 h of culture on cell population size, as measured by protein and DNA contents in the culture dishes, were consistent with changes observed in total FITC-GC uptake. Cisplatin concentrations lower than 50 microM did not affect FITC-GC uptake by rat hepatocytes. By contrast, a progressively increasing effect on hepatoma cells as from 2 microM cisplatin was observed. Two phases in the decay of FITC-GC uptake versus cisplatin concentrations were found in co-cultures exposed to this drug. The first segment, between 2 microM and 50 microM, was characterized by a slow decay that matched the response of hepatoma cells to cisplatin exposure. This was considered to be due to the antiproliferative effect of cisplatin. The second segment, with a steeper decay, matched the effect of cisplatin on hepatocytes. This was interpreted as being due to non-specific toxicity. These results suggest that FITC-GC uptake by co-culture of hepatocytes and tumour cells provides a useful experimental model to explore the mechanism of action and the size of beneficial effect window for new drugs in vitro.

Rat liver transport and biotransformation of a cytostatic complex of bis-cholylglycinate and platinum (II).

BACKGROUND/AIMS: Bile acids have previously been used as shuttles for directing organic drugs to the liver. The aim of this study was to investigate liver transport and biotransformation of a new cytostatic bioinorganic complex (Bamet-H2), that was obtained by binding platinum(II) to two cholylglycinate moieties. METHODS: Using rat hepatocytes in primary culture, the kinetics of cholylglycinate, cisplatin and Bamet-H2 uptake were studied. Sodium-dependency of Bamet-H2 uptake was investigated by replacement of 116 mM NaCl by 116 mM choline chloride. Liver biotransformation was investigated by HPLC analysis of bile samples collected from anesthetized rats following intravenous Bamet-H2 administration. Using isolated rat liver preparations, which were perfused with erythrocyte- and albumin-free Krebs-Henseleit solutions for 40 min, measurement of cholylglycinate, cisplatin and Bamet-H2 uptake and bile output was carried out. Interaction between Bamet-H2 and cholylglycinate for liver transport was studied by co-administration of 1 microM Bamet-H2 plus 500 microM cholylglycinate and 1 microM [14C]-cholylglycinate plus 500 microM Bamet-H2. RESULTS: Both cholylglycinate and Bamet-H2 uptake by rat hepatocytes followed saturation kinetics. Comparison between the two compounds indicated that the Vmax (22.2 versus 8.5 nmol.5 min(-1).mg protein(-1)), and Kt (365 versus 171 microM) were higher for Bamet-H2 uptake. The efficiency of Bamet-H2 uptake (Vmax/Kt) was significantly reduced (-35%) in the absence of sodium. Cisplatin uptake by rat hepatocytes was approximately 10-fold lower than that for Bamet-H2 at any dose used. Moreover, this was not saturable up to 400 microM cisplatin. Bamet-H2 was not biotransformed during its intrahepatic residence in anesthetized rats. Bamet-H2 uptake and secretion into bile by isolated rat livers exceeded cisplatin but were less than cholylglycinate. Differences between Bamet-H2 and cholylglycinate were more marked for bile output than for liver uptake. Thus, higher drug liver content was found after perfusion with Bamet-H2 than with cholylglycinate or cisplatin. Co-administration of Bamet-H2 and cholylglycinate revealed the existence of partial cross-inhibition in both liver uptake and bile output. Bamet-H2 induced a more profound alteration on cholylglycinate uptake and bile secretion than cholylglycinate on both process for Bamet-H2. CONCLUSION: These results suggest that in the transfer of Bamet-H2 from the sinusoids to the canaliculi both bile acid and non-bile acid transport systems are involved.

Effect of bile acids on hepatobiliary transport of cisplatin by perfused rat liver.

The liver and kidney collaborate in the excretion of the cytostatic drug, cis-diamminedichloroplatinum(II) (cisplatin) from the body. Enhancement of this process is envisaged as a way of reducing cisplatin toxicity, thus allowing increases in the doses administered. In this sense, using different compounds, several attempts have been made to enhance cisplatin biliary excretion. In this study, the ability of endogenous compounds belonging to the bile acid family to improve cisplatin excretion by the isolated perfused rat liver was investigated. A highly choleretic bile acid (ursodexoycholic acid) and two others bile acids with marked micelle-forming properties (glycocholic acid and chenodeoxycholic acid) were chosen for study. When these drugs were given at concentrations (1 microM) that did not affect the viability of liver preparations, a correlation between the biliary excretion of platinum and bile acid output was found. This was not due to the incorporation of cisplatin into mixed micelles because no correlation between the biliary output of lecithin or cholesterol and platinum was observed. Moreover, a wash-out effect of bile acids was probably not the cause of bile acid-induced platinum output into bile because no correlation between this and bile flow was found. An enhancement in cisplatin transport processes by the hepatocyte or by direct binding of cisplatin to bile acid monomers or aggregates cannot be ruled out. In spite of the biliary induction of cisplatin output, the net excretion of platinum was reduced under bile acid administration. This was related to lower platinum contents in the liver tissue, probably due to an inhibition of the ability of the hepatocyte to take up and/or retain cisplatin while subject to bile acid infusion. In summary, our results indicate that bile acids reduce the net excretion of cisplatin by the liver even though they induce an enhancement in the transport of this compound from the hepatocyte into bile.

ATP-dependent bile acid transport across microvillous membrane of human term trophoblast.

The main fate for fetal bile acids is to be transferred to the mother by the trophoblast. In this study, ATP-dependent bile acid transport across the maternal- and the fetal-facing plasma membranes (mTPM and fTPM, respectively) of the human trophoblast was investigated. With the use of [14C]glycocholate (GC) and a rapid-filtration technique, GC transport by mTPM and fTPM was measured in the absence or the presence of 3 mM ATP plus an ATP-regenerating system. GC efflux from preloaded mTPM or fTPM vesicles was found to be insensitive to ATP. By contrast, GC uptake by mTPM, but not by fTPM, was significantly increased (approximately threefold) by ATP. This was temperature sensitive and occurred into an osmotically reactive space. Kinetic analysis revealed that GC uptake by mTPM was saturable and fit the Michaelis-Menten equation both in the absence and in the presence of ATP. ATP-dependent transport was not abolished by a protonophore (carbonyl cyanide p-trifluormethoxyphenyl hydrazone) together with 100 mM K+ (in = out) plus a K+ ionophore (valinomycin). It specifically required hydrolyzable ATP, although CTP had a slight stimulatory effect. Neither Na+ nor Cl- (100 mM, in = out) was mandatory. Moreover, 100 mM gradients of either Na+ (in << out) or Cl- (in >> out) had no effect on ATP-dependent GC uptake. This was inhibited by vanadate and bile acid analogues but not by several cholephilic organic anions and a variety of adenosine triphosphatase inhibitors. These results provide strong evidence for the existence of an ATP-dependent transport system for bile acids across the apical membrane of human trophoblast, which may play an important role in the control of the overall fetal-maternal bile acid traffic.