Influence of agents affecting monooxygenase activity on taurolithocholic acid-induced cholestasis.

In rats, pretreatment with certain ketones results in enhanced taurolithocholic acid (TLCA)-induced reduction in bile flow, whereas pretreatment with inhibitors of protein synthesis diminishes the effect on bile flow of cholestatic regimens. In the present study, the possible role of cytochrome P-450 in the ketone potentiation phenomenon was investigated. Male rats were pretreated with inducers or inhibitors of hepatic cytochrome P-450 and the impact of these pretreatments on TLCA-induced cholestasis assessed. Phenobarbital, 3-methylcholanthrene, chlordecone or mirex were used as inducers, and SKF 525-A, piperonyl butoxide, or cobaltous chloride as inhibitors of monooxygenase activity. Phenobarbital and 3-methylcholanthrene pretreatment enhanced TLCA-induced reduction of bile flow, while mirex and chlordecone were without effect. The three inhibitors of monooxygenase activity did not diminish TLCA-induced cholestasis. Instead, piperonyl butoxide and cobaltous chloride appeared to enhance the action of TLCA. Consequently, an increase in cytochrome P-450 (or specific isozymes) as a common denominator in the potentiation phenomenon appears unlikely. While hepatic proteins may play an important role in the potentiation of TLCA-induced cholestasis following pretreatment with ketones, the pattern of potentiation after pretreatment of rats with different inducers or inhibitors of cytochrome P-450 does not appear to implicate this family of proteins.

Functional changes of the biliary tree associated with experimentally induced cholestasis: sulfobromophthalein on manganese-bilirubin combinations.

Administration of combinations of manganese (Mn) and bilirubin (BR) to rats results in a severe, but reversible diminution of bile flow, an effect that can be abolished if sulfobromophthalein (BSP) is given at a specific time prior to BR. Some studies suggest that changes in the bile canalicular membrane (BCM) are critical to the response. One aim of the present work was to determine if functional changes in BCM also become more marked with increasing doses of BR. A second aim was to investigate the protective effects of BSP on MnBR-altered biliary function. The permeability of the biliary tree was evaluated by the segmented retrograde intrabiliary injection (SRII) procedure in male Sprague-Dawley rats treated with varying combinations and dosages of Mn, BR, and BSP. [3H]Mannitol and [3H]inulin were used as marker substances of the biliary tree (canalicular membrane and tight junctions, respectively). Administration of Mn, followed 15 min later by BR, led to a reduction in bile flow that was dose-dependent on BR. The percentage recovery of both inulin and mannitol in bile after SRII also decreased significantly with increasing dosages of BR. When BSP was given 10 min before BR, MnBR-induced reduction in bile flow was abolished. BSP treatment also prevented MnBR-induced reduction in biliary recovery of both inulin and mannitol after SRII; this was more evident with mannitol than with inulin. BSP protection against MnBR cholestasis depends upon when it is administered relative to BR injection. The relationship of BSP relative to BR injection was comparable for both reduced bile flow and the recoveries of marker substances in bile after SRII. The data are consistent with the conclusion that changes in biliary tree permeability, particularly at the canalicular membrane, likely lead to MnBR-induced cholestasis.

The influence of severity of bile flow reduction, cycloheximide, and methyl isobutyl ketone pretreatment on the kinetics of taurolithocholic acid disposition in the rat.

Pretreatment of rats with methyl isobutyl ketone (MIBK) potentiates the effect of taurolithocholic acid (TLCA) on bile flow, while cycloheximide pretreatment diminishes the cholestatic response. Experiments were performed to determine if the effects of the pretreatments were related to changes in the kinetic disposition of TLCA. Groups of rats were pretreated daily with either 7.5 mmol MIBK/kg po for 3 days or 3.55 mumol cycloheximide/kg ip for 2 days prior to an iv challenge of TLCA. Bile and blood samples were collected for 3 hr and the blood concentrations and biliary excretion of TLCA monitored. The severity of the bile flow reduction had a marked effect on the kinetic pattern of TLCA. The volume of distribution and bile disposition constant of TLCA decreased inversely with the severity of bile flow reduction, while the blood disposition constant increased. The total clearance of TLCA was not affected, but increasing the severity of the cholestasis altered the contribution of biliary and extrabiliary clearance to total clearance. The changes in the kinetics of TLCA observed in MIBK- and cycloheximide-pretreated rats were consistent with the effects the pretreatments exerted on TLCA-induced reduction in bile flow. They were interpreted to be the result of the effects of the pretreatments rather than their cause. Thus, pretreatment with MIBK and cycloheximide appears to exert a modulating effect on TLCA-induced cholestasis by mechanisms unrelated to an alteration of TLCA kinetic profile.

Dose-dependent cytotoxicity of chlorinated hydrocarbons in isolated rat hepatocytes.

The aim was to determine if isolated suspended hepatocytes could differentiate between the effects of four chlorinated hydrocarbons that are hepatotoxic in vivo and four that are not. Membrane integrity was assessed by measuring alanine aminotransferase (ALT) release after 30- to 180-min incubations in vitro. From the results, the chlorinated hydrocarbons fell into three groups: tetrachloroethylene and 1,1,2,2-tetrachloroethane were the most potent cytotoxicants; CCl4, 1,1,2-trichloroethane, and trichloroethylene exhibited intermediate cytotoxicity; and low cytotoxicity was observed with CHCl3, 1,1,1-trichloroethane, and 1,1-dichloroethylene. Cytotoxicity ranking correlated poorly with the reported in vivo hepatotoxicity of these agents. The effect of adding SKF-525A on the cytotoxicity of tetrachloroethylene and CCl4 was also assessed. In addition, hepatocytes from rats pretreated with 2,5-hexanedione were used to determine if they were more susceptible to the effects of CHCl3, CCl4, or tetrachloroethylene. SKF-525A decreased the cytotoxicity of both CCl4 and tetrachloroethylene, whereas pretreatment with 2,5-hexanedione enhanced their effect. The effects of both SKF-525A and 2,5-hexanedione on CCl4 in vitro are consistent with in vivo findings. However, tetrachloroethylene is not hepatotoxic in vivo, suggesting that SKF-525A might act by stabilizing plasma membranes rendering the hepatocyte more resistant to lysis. Overall, the results cast doubts on the use of ALT release from isolated hepatocytes as an appropriate in vitro model for assessing hepatotoxic properties of chlorinated hydrocarbons.

Effect of inhibition of protein synthesis on cholestasis induced by taurolithocholate, lithocholate, and a manganese-bilirubin combination in the rat.

Taurolithocholate, lithocholate, and a manganese-bilirubin combination produced a rapid reduction in bile flow after i.v. injection in the rat. The effect was diminished or blocked completely by pretreating the animals with cycloheximide or ethionine, known inhibitors of protein synthesis. The injection sequence and time period between administration of the inhibitor of protein synthesis and the cholestatic agent influenced the degree to which they modulated the cholestatic effect. The results indicate that uninterrupted protein synthesis is required for the expression of maximal reduction of bile flow by taurolithocholate, lithocholate, and a manganese-bilirubin combination.

Tissue residues of clopidol (3,5-dichloro-2,6-dimethyl-4-pyridinol) in chickens in relation to withdrawal times.

Broiler chickens were fed on a commercial diet containing 0.0125% clopidol for 34 days. They were killed 0-10 days after withdrawal of the premedicated feed and clopidol was determined in liver and muscle samples by a sensitive gas-liquid chromatography (g.l.c.) method. During the first two post-withdrawal days the clopidol concentration in the liver decreased rapidly from 7 to 0.5 mg/kg and the level in muscle declined from 3 to 0.1 mg/kg. There was little decline in the clopidol concentrations from days 2 to 10, the levels during this period being 0.2-0.8 mg/kg in liver and 0.05-0.2 mg/kg in muscle. In addition to the above experimental study, liver and muscle samples collected at a Swedish slaughterhouse from broiler chickens raised on clopidol-containing feed were analysed for residues of this drug. Large variations were found in the clopidol levels in broilers from different producers. The levels in the liver ranged from 0.05 to 8.0 mg/kg and those in muscle from 0.03 to 3.5 mg/kg. The present results emphasize the need to carry out field studies to check the levels of feed additive residues in edible tissues from chickens.