Role of perivenous hepatocytes in taurolithocholate-induced cholestasis in vivo.

The magnitude of cholestasis induced by taurolithocholic acid (TLCA) and its relationship with phase I metabolism were analyzed in rats treated with bromobenzene (BZ), a chemical that causes selective necrosis of perivenous (zone 3) hepatocytes. Forty-eight hours after BZ administration (600 mg/Kg bw), a single dose of 20 micromol/Kg bw of TLCA was injected. Bile was collected during 180 min and bile flow and total bile acid excretion rate were determined. Biliary bile acid composition was analyzed by gas-liquid chromatography-mass spectrometry. BZ administration did not affect the development of TLCA-induced cholestasis, but exacerbated the bile acid-induced decrease in bile flow during the period of recovery from cholestasis. Biliary excretion of total bile acids after TLCA injection relative to basal value was not effected by BZ. The analysis of bile acid composition in bile revealed that TLCA was partially converted to hyodeoxycholic and muricholic acids. The cumulative excretion of all exogenous bile acids and their contribution to the composition of the biliary bile acid pool were not substantially affected by zone 3 necrosis, suggesting that synthesis and secretion of hydroxylated derivatives of TLCA were maintained by zone 1 and 2 hepatocytes. The relative content of endogenous bile acids was not affected by BZ during TLCA-induced cholestasis. Thus, it seems unlikely that the exacerbation of the cholestasis in BZ-treated rats is due to different choleretic properties and/or toxicity of the bile acid pool.

Chlorinated methanes and liver injury: highlights of the past 50 years.

The chlorinated methanes, particularly carbon tetrachloride and chloroform, are classic models of liver injury and have developed into important experimental hepatoxicants over the past 50 years. Hepatocellular steatosis and necrosis are features of the acute lesion. Mitochondria and the endoplasmic reticulum as target sites are discussed. The sympathetic nervous system, hepatic hemodynamic alterations, and role of free radicals and biotransformation are considered. With carbon tetrachloride, lipid peroxidation and covalent binding to hepatic constituents have been dominant themes over the years. Potentiation of chlorinated methane-induced liver injury by alcohols, aliphatic ketones, ketogenic compounds, and the pesticide chlordecone is discussed. A search for explanations for the potentiation phenomenon has led to the discovery of the role of tissue repair in the overall outcome of liver injury. Some final thoughts about future research are also presented.

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.

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.

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.

Effect of dosing vehicle on the hepatotoxicity of CCl4 and nephrotoxicity of CHCl3 in rats.

There are conflicting results in the literature concerning the effect of gavage vehicle, corn oil (CO) versus aqueous suspension, on the toxicity of haloalkanes. The purpose of our study was to assess the influence of oral dosing vehicle on the acute hepatotoxicity of CCl4 and nephrotoxicity of CHCl3. Male Sprague-Dawley rats, fed ad libitum, were treated (po) with single doses of CCl4 or CHCl3 using corn oil (CO), or an aqueous preparation (5%) of Emulphor (EL620) or Tween-85 (Tw-85) as vehicle (10 ml/kg). Rats were killed 48 h after treatment. Blood was collected for plasma alanine aminotransferase (ALT) determination and renal cortical slices were prepared for p-aminohippuric acid (PAH) incorporation. The comparison, between gavage vehicles, of the slopes and ED50 of the dose-response curves, although not significantly different, indicated clear trends for enhanced potency with CO for CHCl3 nephrotoxicity but not for CCl4 hepatotoxicity. However, ALT values, a measure of the severity of effect for CCl4, also indicated that CO, when compared to EL620 and Tw-85, tended to enhance CCl4 hepatotoxicity at low toxicity incidence. Furthermore, CO clearly enhanced the severity of effect for CHCl3 nephrotoxicity, as measured by the slice-to-medium PAH ratios, at high dosage. The greater severity of the lesion produced by exposure to these chemicals, when administered in CO, is consistent with the trends observed for their potency (dose-response curves). Our results agree with an increased toxicity of haloalkanes by the gavage vehicle CO reported in the literature. Thus, CO should be considered a potential confounder in hepato- and nephrotoxicity assays.

Ketone potentiation of intrahepatic cholestasis: effect of two aliphatic isomers.

Occupational exposure to methyl isobutyl ketone (MiBK) or methyl n-butyl ketone (MnBK) normally occurs by inhalation. The present study reports that exposure to both ketones can potentiate cholestasis experimentally induced by taurolithocholic acid (TLC, 30 mumol/kg) or by a combination of manganese (Mn, 4.5 mg/kg) and bilirubin (BR, 25 mg/kg). Male Sprague-Dawley rats were exposed for 3 d, 4 h/d, to MiBK or MnBK vapors using 0.5, 1, 1.5, or 2 times the minimal effective concentration (MEC). The estimated MiBK or MnBK MEC for potentiating TLC- or Mn-BR-induced cholestasis were 400 and 150 ppm, respectively. Eighteen hours after ketone exposure, rats were injected i.v. with TLC or Mn-BR. Bile flow was measured from 15 to 150 min after the cholestatic regimen. Rats exposed to MiBK or MnBK exhibited an enhanced diminution in bile flow compared to controls that was dose-dependent with the inhaled ketone dose. The dose-effect characteristics of the potentiation phenomenon were established. Results indicate that MiBK or MnBK inhalation potentiated both TLC and Mn-BR cholestasis in a dose-related fashion. Quantitative differences, however, exist between both ketones with respect to their ability to potentiate both models. Comparison between the two isomers was established, and MnBK was found to be more potent than MiBK.

Altered cholesterol synthesis as a mechanism involved in methyl isobutyl ketone-potentiated experimental cholestasis.

Mechanisms by which ketones potentiate manganese-bilirubin (Mn-BR)-induced cholestasis are unknown. The purpose of the present study was to investigate the effect of methyl isobutyl ketone (MiBK), a widely used ketonic solvent, at the level of the bile canalicular membrane (BCM) and to verify if altered membrane lipid dynamics could be involved in MiBK-potentiated Mn-BR cholestasis. Male Sprague-Dawley rats were exposed 4 hr/day for 3 days to MiBK vapors (200 or 600 ppm). Eighteen hours after the last exposure, manganese (Mn, 4.5 mg/kg) was given i.v. followed 15 min later by bilirubin (BR, 25 mg/kg). Rats were killed 30 min after BR; liver cell plasma membranes (bile canalicular and sinusoidal), microsomes, mitochondria, and cytosol were isolated by differential centrifugation. Lipids were extracted and cholesterol was measured in each fraction. After Mn-BR and MiBK exposure (600 ppm), results indicated a marked increase in BCM cholesterol content compared to rats exposed to air only. This increase was greater than that due to Mn-BR or MiBK given alone. Also, results indicated that cholesterol increased in a dose-related fashion in BCM after MiBK exposure, whereas PM cholesterol remained unaltered. To identify the source of the increased BCM cholesterol and to permit distinction between de novo cholesterol synthesis and subcellular shifts, the hepatic lipid pool was labeled in vivo with [3H]-cholesterol and [2-14C]-mevalonic acid, a cholesterol synthesis precursor. Results showed that after 600 ppm MiBK exposure, 14C-labeled cholesterol was greater than 3H-labeled cholesterol, indicating that the contribution of de novo cholesterol synthesis to the total cholesterol content of the various isolated hepatocellular fractions was more important than the contribution of intracellular pools. Therefore, increased BCM cholesterol content and enhanced accumulation of newly synthesized cholesterol appear to be involved in MiBK potentiation of Mn-BR-induced cholestasis.

Ketone potentiation of haloalkane-induced hepatotoxicity: CCl4 and ketone treatment on hepatic membrane integrity.

Previous results in male Sprague-Dawley rats indicate that acetone (A), methyl ethyl ketone (MEK), and methyl isobutyl ketone (MiBK) pretreatments (3 d, p.o.) at a dosage of 6.8 mmol/kg potentiate CCl4 hepatotoxicity. The potentiation potency profile observed was MiBK > A > MEK. In the present study, male Sprague-Dawley rats were treated for 3 d with 6.8 mmol/kg (p.o.) of A, MEK, or MiBK using Emulphor as vehicle (10 ml/kg). Rats were either killed 18 h after the last pretreatment or treated with CCl4 (prepared in corn oil) and then killed 48 h later. Livers were perfused; purified plasma membrane (PM), sinusoidal (SM) and basal canalicular membrane (BCM) fractions were prepared. Membrane fluidity was monitored by fluorescence polarization using 1,6-diphenyl-1,3,5-hexatriene (DPH) or 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH). The following membrane enzymes were measured to monitor membrane purity and treatment effects: 5'-nucleotidase (5N), leucine aminopeptidase (LAP), and alkaline phosphatase (AP). Our results suggest that CCl4 modifies membrane integrity as indicated by a decrease in liver membrane 5N, LAP, and AP activity. CCl4 also increased the fluidity of the lipid and protein portions of the liver membranes as measured by the DPH and TMA-DPH fluorescence probes, respectively. Of the three ketones, only A altered CCl4 effects on plasma membrane enzymes and decreased BCM fluidity. The data only partially support increased susceptibility of liver membranes by ketone pretreatment as a factor implicated in the mechanism of potentiation of CCl4-induced hepatotoxicity.

Rat hepatocytes with elevated metallothionein expression are resistant to N-methyl-N'-nitro-N-nitrosoguanidine cytotoxicity.

Metallothionein (MT) is a small cysteine-rich metal-binding protein involved in Zn and Cu homeostasis as well as in heavy metal detoxication. It is also believed that when MT is overexpressed, it can confer resistance against alkylating agents. However, the mechanisms involved are still poorly understood. The purpose of the present work was to investigate whether metal treatment, which induces MT synthesis, could protect isolated rat hepatocytes against the cytotoxic effects of the alkylating agents methyl methanesulfonate (MMS) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Exposure to 12.5 microM ZnSO4 for 18 hr raised MT levels approximately 15-fold (as measured by the 109Cd-heme assay). When these cells were exposed to increasing concentrations of MNNG, a significant reduction in cell death (as measured by lactate dehydrogenase leakage into extracellular medium) was observed (LC50 = 468 +/- 20 microM vs 362 +/- 13 microM for control cells). On the other hand, Zn pretreatment was not accompanied by resistance against MMS toxicity. In addition, the synthesis of graded amounts of MT, achieved by incubation with various concentrations of Zn or Cu, led to a high correlation between MT levels and the extent of hepatocyte survival. Cd (another MT inducer) failed to protect hepatocytes from MNNG cytotoxicity. Time-course studies also revealed a good correlation between the onset of MT induction by Zn (> 3 hr) and that of protection against MNNG (> 3 hr). The stability of MT in the presence of MNNG was studied by incubating 109Cd-labeled MT with MNNG and by analyzing the mixture using Sephadex G-75 Chromatography. Direct interaction of MNNG with rabbit liver (Cd,Zn)-MT was demonstrated by the release of 109Cd bound to MT. Similar results were obtained with 109Cd-exposed hepatocytes, 109Cd being redistributed from MT to high-molecular-weight proteins after incubation with MNNG. None of the metals used to induce MT modulated glutathione (GSH) because it remained at control levels after 18 hr. However, within 15 min of incubation, MNNG had completely depleted GSH in both control and Zn-pretreated hepatocytes equally. This was followed by a marked decline in MT levels. Taken together, these results suggest that Zn- and Cu-induced tolerance against killing by MNNG appears to be related to the accumulation of MT. The mechanism of protection might reside in the antioxidant properties of MT and on its ability to scavenge electrophilic species.

Ketone potentiation of haloalkane-induced hepato- and nephrotoxicity. II. Implication of monooxygenases.

Previous results in Sprague-Dawley rats indicate that acetone (A), methyl ethyl ketone (MEK), and methyl isobutyl ketone (MiBK) pretreatment (3 d, po) at dosages of 6.8 and 13.6 mmol/kg potentiate CCl4 hepatotoxicity and CHCl3 nephrotoxicity, respectively. The potentiation potency profile observed was MiBK > A > MEK for liver and A > MEK > or = MiBK for kidney toxicity (Raymond & Plaa, 1995). In the present study, hepatic and renal microsomes from A-, MEK-, and MiBK-pretreated rats (6.8 or 13.6 mmol/kg) were examined for cytochrome P-450 content, substrate-specific monooxygenase activity (aminopyrine and benzphetamine N-demethylase, aniline hydroxylase) and in vitro covalent binding of 14CHCl3 and 14CCl4. Of the three ketones, only MiBK significantly increased P-450 content of liver and renal cortical microsomes. Similarly, 14CCl4 covalent binding under aerobic and anaerobic conditions was significantly increased by MiBK pretreatment only. 14CHCl3 covalent binding by renal cortical microsomes was significantly increased only under aerobic conditions by MiBK pretreatment. MiBK (13.6 mmol/kg) increased (threefold) aminopyrine N-demethylation in both liver and kidney, but only benzphetamine N-demethylation (two-fold, at 6.8 and 13.6 mmol/kg) in liver; A and MEK had no effect on either monooxygenase. All ketones at dosages of 6.8 and 13.6 mmol/kg increased aniline hydroxylation in liver (two-fold) and kidney (fivefold). Comparable profiles for P-450 induction, haloalkane covalent binding, and aminopyrine or benzphetamine N-demethylase activity were observed in liver and kidney microsomes. This profile was consistent with the ketone potentiation potency ranking profile observed in vivo for liver but not kidney injury. These findings affirm the importance of ketone-enhanced bioactivation for potentiation of CCl4 hepatotoxicity but suggest an alternative mechanism for CHCl3 nephrotoxicity.

Dose- and route-dependent alteration of metabolism and toxicity of chloroform in fed and fasting rats.

Effects of overnight food deprivation on the metabolism and toxicity of chloroform (CHCl3) administered to rats per os (po), intraperitoneally (ip), or by inhalation (inh) at different doses were investigated. Rats that had been either deprived of food overnight or normally fed were challenged with CHCl3 either po (0, 100, 200, or 400 mg/kg), ip (0, 100, 200, or 400 mg/kg), or inh (0, 50, 100, or 500 ppm for 6 hr). Overnight fasting increased CHCl3 metabolism in vitro about threefold with a decrease of liver glutathione content to 67%. The fasting caused route- and dose-dependent alteration in the metabolism and toxicity of CHCl3. The area-under-the-curve (AUC) of blood CHCl3 concentration was invariably smaller following po than ip administration, and CHCl3 administered po caused more severe hepatic damage than did the same amount of CHCl3 administered ip. With po administration, the AUC (toxicity) of CHCl3 in fasting rats was significantly smaller (higher) than that of fed rats at a dose as small as 100 mg/kg, whereas, with ip administration at such a small dose, fasting caused no significant alteration in the AUC (toxicity). When rats were exposed by inhalation to CHCl3 vapor, food deprivation had little or no effect on either the blood concentration or the toxicity until the exposure concentration was raised to 500 ppm. The present study indicates that po administration is different from both ip and inh administration with regard to the effect of enzyme induction on the toxicokinetics of CHCl3, mainly due to the first-pass metabolism unique to po administration.

1-Naphthyl isocyanate and 1-naphthylamine as metabolites of 1-naphthylisothiocyanate.

The importance of the bioactivation of 1-naphthylisothiocyanate was studied. Forty minutes after 1-naphthylisothiocyanate administration to rats, bile was collected over a 2.5-h period; the liver was then excised and homogenized. 1-naphthylisothiocyanate and its metabolites in bile and liver of rats were identified and quantified using coupled gas chromatography-mass spectrometry. Three main compounds were found in all 1-naphthylisothiocyanate-treated animals. They were identified as 1-naphthyl isocyanate, 1-naphthylamine and the parent compound, 1-naphthylisothiocyanate. When rats were given cycloheximide, which attenuates 1-naphthylisothiocyanate toxicity, 30 min before 1-naphthylisothiocyanate (300 mg/kg), 1-naphthyl isocyanate concentration was significantly lower than in rats receiving only 1-naphthylisothiocyanate. The appearance of 1-naphthylamine was also inhibited by cycloheximide, although not to the same extent as 1-naphthyl isocyanate. On the other hand, phenobarbital, which potentiates 1-naphthylisothiocyanate hepatotoxicity, enhanced 1-naphthyl isocyanate and 1-naphthylamine formation. It is suggested that 1-naphthyl isocyanate, 1-naphthylamine and the highly reactive sulfur released from 1-naphthylisothiocyanate might be involved in the hepatotoxic effect of 1-naphthylisothiocyanate.

Ketone potentiation of haloalkane-induced hepato- and nephrotoxicity. I. Dose-response relationships.

Carbon tetrachloride (CCl4) induced hepatotoxicity and chloroform (CHCl3) induced nephrotoxicity were evaluated in male Sprague-Dawley rats pretreated with acetone (A), methyl ethyl ketone (MEK), and methyl isobutyl ketone (MiBK). Dose-response relationships for A, MEK, and MiBK potentiation of CCl4-induced hepatotoxicity and CHCl3-induced nephrotoxicity were compared. A, MEK, and MiBK pretreatment at a dosage of 6.8 mmol/kg, given daily for 3 d, markedly potentiated CCl4-induced liver toxicity as indicated by a decrease in the CCl4 ED50 to 3.4, 4.6, and 1.8 mmol/kg, respectively, compared to vehicle-pretreated rats (17.1 mmol/kg). Similarly, pretreatment with these ketones (13.6 mmol/kg) potentiated CHCl3 kidney toxicity but to a lesser degree; CHCl3 ED50 values for vehicle-, A-, MEK-, and MiBK-pretreated rats were 3.4, 1.6, 2.1, and 2.2 mmol/kg, respectively. Our results indicate a potency ranking profile for the potentiation of CCl4 hepatotoxicity of MiBK > A > MEK and of A > MEK > or = MiBK for CHCl3 nephrotoxicity. These dissimilar ranking profiles could be due to differences in mechanisms of action for the two target sites.

Induction of metallothionein gene expression by epidermal growth factor and its inhibition by transforming growth factor-beta and dexamethasone in rat hepatocytes.

Metallothionein (MT) is a small cysteine-rich protein thought to be mainly involved in metal regulation and detoxification. The implication of MT in cell growth and differentiation has also been suggested. This latter hypothesis was further investigated in adult rat hepatocytes induced to proliferate by epidermal growth factor (EGF). Exposure of hepatocytes to EGF resulted in significant increases (approximately twofold) in MT protein and MT-1 messenger RNA (mRNA) levels, which were maximal after 48 hours. As revealed by nuclear run-on analysis, these changes were the result of transcriptional activation. Increases of MT occurred concomitantly with stimulation of DNA synthesis (48 hours). Addition of ZnSO4 or dexamethasone (Dex) was also effective at inducing MT protein (approximately 3.6 to 3.3 times) and mRNA. Combined addition of Zn and EGF produced an additive increase in MT protein and MT-1 mRNA levels. When both Dex and EGF were present together, the EGF-induced MT protein and mRNA expression was lost, whereas it had only minor inhibitory effects on DNA synthesis. Transforming growth factor beta (TGF-beta), a known antagonist of EGF on hepatocytes, blocked the EGF-induced MT accumulation and stimulation of DNA synthesis. In addition, under the same conditions, the EGF-induced c-fos mRNA accumulation was blocked by Dex whereas TGF-beta had no effect. These results show that growth factors believed to play a role in liver regeneration can also modulate MT gene expression in vitro. This modulation does not strictly parallel that of DNA synthesis. The possibility that c-fos stimulation may play a role in MT induction by EGF cannot be ruled out.

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.