Pathogenesis of lithocholate-induced intrahepatic cholestasis: role of glucuronidation and hydroxylation of lithocholate.

It has been shown that lithocholic glucuronide is more cholestatic than lithocholic acid (LCA), as well as its taurine and glycine conjugates. Furthermore, LCA hydroxylation is thought to be a major detoxifying mechanism. Therefore, the role of LCA glucuronidation and hydroxylation was investigated during the development of LCA-induced cholestasis and recovery from it. Male rats received a bolus intravenous injection of [14C]LCA (12 mumol/100 g body weight) and bile samples were collected every 30 min for 5 h. Bile flow (BF) was reduced immediately after LCA injection, dropping to 40% of basal BF at 60 min. It then started to increase, reaching normal bile flow values at 3.5 h. Morphologically, canalicular lesions were dominant at 60 min and virtually absent at 2 h. At 60 min (maximal cholestasis), 30% of the LCA injected was secreted in bile, 20% was found in plasma while the other 50% was recovered in the liver and distributed mainly in plasma membranes, microsomes and cytosol. At the end of the experiment (normal BF), 20% of the LCA injected was still in the liver but was present mainly in the cytosol. In bile, within 30 min after injection, 46% of the LCA secreted was lithocholic glucuronide, 24% was conjugated with taurine and glycine, and 21% was in the form of hydroxylated bile acids. During the recovery period, lithocholic glucuronide secretion decreased to 18-25%. Taurine and glycine conjugate secretion increased to a maximum of 43% at 60 min, after which it was reduced to 21-28%. In contrast, hydroxylated metabolites were elevated during the recovery periods, reaching a maximum (45%) at 120 min and remaining constant thereafter. These results suggest that: (i) LCA binding to plasma membranes and microsomes appeared to correlate with the development of cholestasis; (ii) LCA glucuronidation may initiate and/or contribute to LCA-induced cholestasis; and (iii) hydroxylation predominates during recovery from cholestasis.

Hepatic subcellular distribution of manganese in manganese and manganese-bilirubin induced cholestasis.

Administration of non-cholestatic doses of manganese (Mn2+) followed by injection of bilirubin (BR) results in a severe reduction in rat bile flow. Male Sprague-Dawley rats were given various doses of Mn2+ (2, 4.5, 8, and 18 mg/kg, i.v.) and killed 0.25, 1, 3, or 5 hr later. 54Mn2+ was used to evaluate Mn2+ content (micrograms/g protein) in different liver fractions: homogenate, mitochondria, microsomes, cytoplasm, nuclei-membrane fraction and liver cell plasma membrane fractions, one containing bile canalicular complexes (LCPM-BCM), the other containing sinusoidal membranes (LCPM-PM). In LCPM-BCM and LCPM-PM, two time-related patterns of Mn2+ content were observed. With non-cholestatic doses (2, 4.5, and 8 mg/kg), Mn2+ content decreased with time and rarely exceeded 50 micrograms/g protein. With 18 mg/kg (a cholestatic dose), Mn2+ content increased with time and reached values over 100 micrograms/g protein (3-5 hr), reflecting possible modification in membrane structure. BR caused a marked increase in Mn2+ content (at a dose of 4.5 mg Mn2+/kg) in LCPM-BCM (240%), approaching values seen with 18 mg Mn2+/kg, whereas in LCPM-PM it was less striking (50%). These and other results obtained with various treatments (cholestatic and non-cholestatic) suggest than Mn2+ concentration in bile canalicular membranes is a critical factor in both forms of cholestasis, and that BR can facilitate Mn2+ incorporation in the bile canalicular membrane.

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.

Effects of acetone and methyl n-butyl ketone on hepatic mixed-function oxidase.

The administration of ketones potentiates CCl4 hepatotoxicity; however, the potencies of the ketones differ. The aim of the present study was to assess potential differences between acetone and methyl n-butyl ketone (MnBK) on cytochrome P-450. The effects of single and repetitive doses of acetone and MnBK were determined in male rats by estimating the rate of metabolite formation of three substrates and the hepatic content of cytochrome P-450. A single treatment with acetone (13.5 mmol/kg or greater) enhanced the oxidation of aniline and 7-ethoxycoumarin, whereas repetitive treatments also increased aminopyrine demethylation and cytochrome P-450 content. Single and repetitive treatments of MnBK (15 mmol/kg) augmented the oxidation of all three substrates and increased cytochrome P-450 content. The effects of the ketones on cytochrome P-450 isozymes were characterized using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Acetone and MnBK increased the 52.1 and 54.1 kD forms and, in addition, MnBK tended to increase the 50.6 kD species. The data indicate that ketones differ in the type of isozymes induced and in the degree of induction. The higher potency of MnBK, compared to acetone, is probably associated with the fact that MnBK affects a greater number of isozymes than acetone.

Phase I and phase II metabolism of lithocholic acid in hepatic acinar zone 3 necrosis. Evaluation in rats by combined radiochromatography and gas-liquid chromatography-mass spectrometry.

In the present study, lithocholic acid (LCA) metabolism was assessed by radiochromatography and gas-liquid chromatography-mass spectrometry, and its relationship to cholestasis was investigated. In addition, the role of the perivenous zone in LCA-induced cholestasis and LCA biotransformation was examined by using bromobenzene (BZ), a chemical that causes selective necrosis of hepatocytes in this zone. LCA injection induced cholestasis of comparable amplitude in both control and BZ-treated rats. The biliary recovery of bile salts (BS) was 65-70% 2 hr after LCA injection. Excretion of LCA and its cholestatic metabolite, LCA glucuronide, was similar in both groups, although LCA excretion was delayed in BZ-treated animals. The appearance of LCA and LCA glucuronide in bile occurred early, and their proportion decreased with time. Concentrations of choleretic hydroxylated metabolites were low immediately after LCA injection but increased with time. 3 alpha,6 beta-Dihydroxy-5 beta-cholanoic and 3 alpha,6 beta,7 beta-trihydroxy-5 beta-cholanoic acids were the major species arising from LCA, indicating the importance of 6 beta hydroxylation in LCA detoxification in rats. Other metabolites were found, but their contribution was either minor or negligible. Overall amounts of hydroxylated metabolites were comparable in both groups, but trihydroxylated metabolites predominated over their dihydroxylated counterparts in control rats, whereas the production of dihydroxylated forms was more pronounced in BZ-treated animals. These results suggest that the destruction of perivenous hepatocytes does not exacerbate LCA-induced cholestasis, and that there may be an acinar zonation of LCA biotransformation to trihydroxylated metabolites in the rat liver.

Quantitative aspects in the assessment of liver injury.

Liver function data are usually difficult to use in their original form when one wishes to compare the hepatotoxic properties of several chemical substances. However, procedures are available for the conversion of liver function data into quantal responses. These permit the elaboration of dose-response lines for the substances in question, the calculation of median effective doses and the statistical analysis of differences in liver-damaging potency. These same procedures can be utilized for estimating the relative hazard involved if one compares the liver-damaging potency to the median effective dose for some other pharmacologie parameter. Alterations in hepatic triglycerides, lipid peroxidation, and the activities of various hepatic enzymes can also be quantitiated in a dose-related manner. This permits the selection of equitoxic doses required for certain comparative studies and the selection of doses in chemical interaction studies. The quantitative problems involved in low-frequency adverse reactions and the difficulty these present in the detection of liver injury in laboratory animals are discussed.

Animal models.

A review of the major categories of animal test procedures used in the toxicological assessment of drugs is presented. Problems that persist include low incidence responses, the need for innovation in toxicological methods and current societal attitudes about drugs and chemicals. A better understanding of the biological phenomena involved in toxicological responses is needed to properly interpret animal data and eventually extrapolate these findings to humans.

Manganese-bilirubin effect on cholesterol accumulation in rat bile canalicular membranes.

Manganese-bilirubin (Mn-BR)-induced cholestasis in rats is associated with altered lipid composition of various hepatic subcellular fractions. Increased bile canalicular (BCM) cholesterol content in Mn-BR cholestasis and the intracellular source of the accumulating cholesterol were investigated. To label the total hepatic cholesterol pool, male Sprague-Dawley rats were given ip 3H-cholesterol, followed 18 h later by 2-14C-mevalonic acid (a precursor of cholesterol synthesis). To induce cholestasis, manganese (Mn, 4.5 mg/kg) and bilirubin (BR, 25 mg/kg) were injected iv; animals were killed 30 min after BR injection; canalicular and sinusoidal membranes, microsomes, mitochondria, and cytosol were isolated. Total cholesterol content of each fraction was determined by spectrophotometric techniques as well as radiolabeled techniques. In Mn-BR cholestasis, the total cholesterol concentrations of BCM and cytosol were significantly increased. Also, the contribution of 14C-labeled cholesterol (newly synthesized cholesterol) was enhanced in all isolated cellular fractions. The results are consistent with the hypothesis that accumulation of newly synthesized cholesterol in BCM is involved in Mn-BR cholestasis. An enhanced rate of synthesis of cholesterol, however, does not appear to be the causal event, as the activity of HMG-CoA reductase (rate-limiting enzyme in cholesterol synthesis), assessed in vitro, was decreased following Mn-BR treatment. Treatment with the Mn-BR combination may affect other aspects of intracellular cholesterol dynamics.

The effect of ethanol on the absorption, accumulation and biotransformation of xenobiotics by the isolated perfused rabbit lung.

The effects of acutely administered ethanol on absorption, accumulation and biotransformation of several model compounds were examined in the isolated perfused lung (IPL). To determine pulmonary accumulation imipramine and paraquat were studied. To determine the effect of ethanol on the absorption of substances from the airways into the blood sulfanilic acid was used as an example of a relatively lipid soluble compound while mannitol was chosen as a non-lipid soluble compound. Aldrin was chosen to demonstrate the effects of circulating ethanol on pulmonary biotransformation. The results obtained with imipramine and paraquat indicate that after a 60-min perfusion period 93% and 20% of the original amount added are respectively lost from the blood perfusate. Ethanol at an initial concentration of 300 mg/100 ml has no apparent effect either on the rate or the quantities accumulated by the lungs. When sulfanilic or mannitol were administered into the airways both compounds appeared in the perfusate and ethanol had no effect on this absorption. Finally we obtained evidence that aldrin is converted to dieldrin by the IPL but that the quantities metabolized are not modified by the presence of ethanol.

Chloral hydrate enhances ethanol-induced inhibition of methanol oxidation in mice.

Several studies indicated that chloral hydrate can prolong the disappearance time of ethanol from blood in mice. This seems to result from inhibition of the enzyme alcohol dehydrogenase by chloral hydrate and trichloroethanol, its main metabolite. We examined the effect of both these compounds on the disappearance time of methanol in mice. Also the effect of a combination of ethanol and chloral hydrate on the disappearance time of methanol was examined. Several groups of six mice each received methanol (1 g/kg i.p.) followed immediately by one of the following treatments: saline (10 ml/kg); chloral hydrate (0.4 g/kg); trichloroethanol (0.36 g/kg); ethanol (4 g/kg); or a combination of chloral hydrate (0.2 g/kg) and ethanol (4 g/kg). The concentrations of methanol in blood were measured at 1, 2, 4, and 8 h after its administration and were used to calculate some approximate indicators of methanol elimination in each group. The results show that all the above treatments do prolong the disappearance time of methanol in the blood of mice to varying extents. The ethanol-chloral hydrate combination produced the most pronounced effect.

1,3-Butanediol-induced increases in ketone bodies and potentiation of CCl4 hepatotoxicity.

Evidence previously reported suggest that 1,3-butanediol (BD) enhances the hepatotoxic effect of a single small dose of carbon tetrachloride (CCl4) in a dose-related manner. The present study provides additional information concerning the quantitative relationship between the severity of the ketotic state produced by BD and the magnitude of the potentiation observed and emphasizes the use of ketone bodies (KB) to predict the potential hazard of the BD-CCl4 interaction. Liver damage was modulated in male Sprague-Dawley rats by varying the concentration of the BD solutions ingested prior to a CCl4 challenge (0.1 ml/kg, i.p.). These data were compared to ketone bodies in plasma, hepatic tissue and urine. BD produced a dose-dependent metabolic ketosis observable at dosages between 1.1 and 9.9 g/kg per day given for 7 days. Plasma and liver data correlated well together. Concomitantly, potentiation of the CCl4-induced liver injury was also dose-related for the same dosage range; the minimum effective dosage of BD for potentiation was estimated as 1.1 g/kg per day. The linear correlations between hepatic or plasma KB values and the indices of hepatic dysfunction (ALT, OCT) were highly significant. Using a semiquantitative method, a correlation was also found for the urinary KB data. These results suggest that plasma KB concentrations might be useful for predicting possible potentiation of the hepatonecrotic effect of CCl4 by BD.

Temporal analysis of rat liver injury following potentiation of carbon tetrachloride hepatotoxicity with ketonic or ketogenic compounds.

The administration of some ketonic or ketogenic compounds prior to a challenging dose of CCl4 potentiates the hepatic damage induced by this haloalkane. However, nothing is known about the recovery from the liver injury in these cases of chemically induced potentiation. To investigate this problem, we performed a temporal analysis of the hepatotoxic response of male Sprague-Dawley rats to CCl4 following a single pretreatment (p.o.) with: n-hexane, 2-hexanone, 2,5-hexanedione (15 mmol/kg in corn oil), isopropanol, acetone (33 and 34 mmol/kg in water, respectively); or the vehicle alone (10 ml/kg). They received, 18 h later, an i.p. injection of CCl4 (0.1, 0.75 or 1.0 ml/kg) and were killed 24-120 h later. Liver damage was assessed biochemically (ALT, OCT) and morphologically. A good correlation between biochemical and morphological results was observed. The ketonic or ketogenic compounds studied potentiated the liver injury produced by 0.1 ml/kg CCl4. Relative ranking orders regarding severity of maximal hepatic damage induced and time needed for complete recovery of liver injury were established; time of recovery was dependent on the maximal severity of the lesion, regardless of the potentiation. The results show that the temporal evolution of CCl4-induced liver injury is not markedly influenced by the administration of ketonic or ketogenic compounds as pretreatments, but rather depends on the severity of the maximal damage induced by the overall treatment.

Pharmacodynamic and metabolic interactions between ethanol and two industrial solvents (methyl n-butyl ketone and methyl isobutyl ketone) and their principal metabolites in mice.

MnBK and MiBK prolong the duration of ketamine-, pentobarbital-, thiopental- and ethanol-induced loss of righting reflex (LRR) in mice. In equimolar doses, (5 mmol/kg i.p.), both isomers were equipotent with respect to the enhancement of ketamine-, pentobarbital-, and thiopental-induced LRR. However, MnBK was significantly more effective (twice as effective) than its isomer with respect to enhancing ethanol-induced LRR. An attempt to explain the difference in effectiveness between the two isomers was carried out. The effects of both ketones and their principal metabolites, (2-hexanol (2-HOL), 2,5-hexanedione (2,5-HD), 4-methyl-2-pentanol (4-MPOL) and 4-hydroxy 4-methyl-2-pentanone (HMP)) on ethanol-induced LRR and ethanol elimination were studied in mice. The ketones and their metabolites were dissolved in corn oil and injected intraperitoneally 30 min before 4 g/kg ethanol for LRR and 2 g/kg for ethanol elimination. Ethanol-induced LRR was significantly prolonged by the following dosages (mmol/kg), MnBK, 5; MiBK, 5; 2-HOL, 2.5; 4-MPOL, 2.5; and HMP, 2.5; 2,5-HD, 2.5, however exerted no effect. Concentrations of ethanol in blood or brain upon return of the righting reflex were similar in solvent-treated and control animals. The mean elimination rate of ethanol was slower in groups pretreated with MnBK or 2-HOL as compared to control animals. Ethanol elimination in animals pretreated with MiBK, HMP, 4-MPOL, or 2,5-HD was similar to that in control animals. These ketones are known to have some central depressant action on their own. This by itself could lead to prolongation of ethanol-induced LRR. However, MnBK, as well as one of its principal metabolites, (2-HOL), markedly reduced ethanol elimination. This could explain the observation that MnBK has a greater potentiating effect on ethanol-induced LRR that its isomer, MiBK, which does not affect ethanol elimination.

Alteration of lipid composition of hepatic membranes associated with manganese-bilirubin induced cholestasis.

One hypothesis concerning the pathogenesis of manganese-bilirubin (Mn-BR)-induced cholestasis is that the molecular organization of the bile canalicular membrane is altered. The purpose of the present study was to evaluate lipid composition and fluidity of hepatic membranes during cholestasis in male Sprague-Dawley rats. To induce cholestasis, manganese (Mn, 4.5 mg/kg, intravenously [i.v.]) was given 15 min before bilirubin (BR, 25 mg/kg, i.v.). The rats were killed 30 min after BR injection, at which time bile flow was decreased by approximately 40% compared to control values. Liver cell plasma membranes enriched in canalicular fractions (BCM) and plasma membranes enriched in sinusoidal and lateral fractions (PM), microsomes, mitochondria and cytosol were isolated by differential centrifugation. Total lipids were extracted and measured colorimetrically. To assess fluidity, membranes were incubated in vitro with fluorescent probes [1,6-diphenyl-1,3,5-hexatriene and 1-(4'-trimethyl-ammonium-phenyl)-6-phenyl-1,3,5-hexatriene]. After Mn-BR treatment, BCM cholesterol incorporation increased markedly (about 3-fold) accompanied by a decrease in fluidity. BCM phospholipid content was unaltered by the cholestatic challenge. In PM-enriched fractions, the changes in cholesterol and phospholipid content after Mn-BR treatment were not statistically significant (P > 0.05) compared to controls. Furthermore, the biochemical alterations in PM were not accompanied by changes in membrane fluidity. These results support the hypothesis that altered lipid composition and fluidity of BCM are involved in the pathogenesis of Mn-BR cholestasis.

Dose-dependent modification of 1,1-dichloroethylene toxicity by acetone.

The ability of acetone to potentiate the toxicity of 1,1-dichloroethylene (DCE) in male rats was investigated. A biphasic potentiation of DCE-induced hepatotoxicity was observed; low doses (5 and 10 mmol/kg, p.o.) of acetone were active, whereas higher doses (15 and 30 mmol/kg) were not. Nephrotoxicity was not affected.

Tissue concentrations of methyl isobutyl ketone, methyl n-butyl ketone and their metabolites after oral or inhalation exposure.

Quantitative relationships between plasma, liver and lung methyl isobutyl ketone (MiBK) and methyl n-butyl ketone (MnBK) concentrations after oral or inhalation exposure were established. Their respective metabolites (4-methyl-2-pentanol, 4-hydroxy-methyl isobutyl ketone, 2-hexanol, and 2,5-hexanedione) were also quantified. Male Sprague-Dawley rats were exposed for 3 days to MiBK or MnBK vapors (4 h/day) or treated orally for 3 days with a MiBK- or MnBK-corn oil solution. Both ketones and their respective metabolites in plasma or tissue concentrations were determined by gas chromatography. MiBK and MnBK plasma and tissue concentrations increased in a dose-related manner with the administered dose irrespective of the route of administration. Metabolite concentrations, however, were influenced by the route of administration.

Inhalation versus oral administration of acetone: effect of the vehicle on the potentiation of CCl4-induced liver injury.

Acetone potentiation of liver injury is greater when corn oil is given with acetone 18 h prior to a challenge with CCl4. This study aimed to further characterize the effects of the vehicle used to administer acetone on the severity of acetone-potentiated CCl4-induced liver injury. The more severe acetone-potentiated liver injury observed when corn oil was the vehicle does not seem to be due to greater liver acetone concentrations. When corn oil was used as the vehicle to administer acetone, liver and blood CCl4 concentrations were not significantly different from those where water was the vehicle. Therefore the relationship between blood or liver acetone concentration and plasma ALT activity for orally-administered acetone was modified by corn oil. Liver triglyceride concentration measured 18 h after a gavage of corn oil was significantly higher than that for the water-treated group. A direct effect of corn oil on liver, in particular a promotion of the propagation phase in the lipid peroxidation process induced by CCl4, is proposed to explain the increase in acetone-potentiated CCl4-induced liver injury.

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.

Metabolic fate of methyl n-butyl ketone, methyl isobutyl ketone and their metabolites in mice.

The metabolic fate of methyl n-butyl ketone (MnBK) and its isomer methyl isobutyl ketone (MiBK) was studied in mice. The concentrations of both ketones and their metabolites in blood and brain were measured at different time intervals after their administration. The principal metabolites of MnBK were 2-hexanol (2-HOL) and 2,5-hexanedione (2,5-HD), while those of MiBK were 4-methyl-2-pentanol (4-MPOL) and 4-hydroxy-4 methyl-2-pentanone (HMP). The administration of 2-hexanol by itself led to the appearance of both MnBK and 2,5-hexanedione which, when administered by itself, did not lead to the appearance of either MnBK or 2-hexanol. The administration of 4-methyl-2-pentanol resulted in the appearance of MiBK and HMP. The administration of HMP did not result in the appearance of MiBK or 4-MPOL. These results indicate that the metabolic fate of MnBK and MiBK is similar to that reported in other species.

The effect of alpha-naphthylisothiocyanate on bile secretion prior to and during the onset of cholestasis in the rat.

The effect of alpha-naphthylisothiocyanate (ANIT) on bile flow, erythritol clearance, bile acid excretion and bilirubin excretion was studied in rats. The median time-to-effect (Et50) for the appearance of cholestasis was about 15 h. ANIT failed to exert a gradual effect on bile flow or erythritol clearance before the onset of cholestasis. However, 3 h before complete cessation of bile flow, a rapid decline in bile flow and bile acid excretion was observed. Bile acid-independent flow was markedly reduced. However, the bile acid-dependent component was also affected.