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Background & Aims: A single dose of alcohol causes transient hepatic mitochondrial DNA (mtDNA) depletion in mice followed by increased mtDNA synthesis and an overshoot of mtDNA levels. We determined the effect of repeated alcohol binges on hepatic mtDNA in mice., Methods: Ethanol (5 g/kg) was administered by gastric intubation daily for 4 days, and mtDNA levels, synthesis, and integrity were assessed by slot blot hybridization, in organello [3H]deoxythymidine triphosphate incorporation, and long polymerase chain reaction analysis, respectively., Results: mtDNA levels were decreased for 48 hours after the last dose, with no overshoot phenomenon later on. Two and 24 hours after the fourth dose, long polymerase chain reaction experiments showed DNA lesions that blocked the progress of the polymerases and in organello mtDNA synthesis was decreased, although DNA polymerase gamma activity was unchanged with synthetic templates. Mitochondria exhibited ultrastructural abnormalities, and respiration was impaired 2 and 24 hours after the fourth binge. Cytochrome P450 2E1, mitochondrial generation of peroxides, thiobarbituric acid reactants, and ethane exhalation were increased., Conclusions: After repeated doses of ethanol, the accumulation of unrepaired mtDNA lesions (possibly involving lipid peroxidation-induced adducts) blocks the progress of polymerase gamma on mtDNA and prevents adaptive mtDNA resynthesis, causing prolonged hepatic mtDNA depletion.

Background & Aims: For similar ethanol consumption, some subjects only develop macrovacuolar steatosis whereas others develop severe liver lesions. A genetic dimorphism encodes for either alanine or valine in the mitochondrial targeting sequence of manganese superoxide dismutase and could modulate its mitochondrial import., Methods: The DNA of 71 white patients with alcoholic liver disease and 79 white blood donors was amplified and genotyped., Results: The frequency of the alanine-encoding allele and the percentage of alanine homozygotes were higher in all patients than in controls and increased with the severity of liver lesions. The percentage of alanine homozygotes was 19% in controls, 17% in alcoholic patients with macrovacuolar steatosis, 43% in patients with microvesicular steatosis, 58% in patients with alcoholic hepatitis, and 69% in patients with cirrhosis. Alcohol consumption in alcoholics was similar whatever the genotype. Alanine homozygosity did not change the risk of developing macrovacuolar steatosis in alcoholics, but increased by 3-fold that of microvesicular steatosis, and 6- and 10-fold that of alcoholic hepatitis and cirrhosis., Conclusions: Homozygosity for alanine in the mitochondrial targeting sequence of manganese superoxide does not modify alcohol consumption and the risk of macrovacuolar steatosis in alcoholics but is a major risk factor for severe alcoholic liver disease.

Background & Aims: Ethanol causes oxidative stress in the hepatic mitochondria of experimental animals and mitochondrial DNA deletions in alcoholics. We postulated that ethanol intoxication may cause mitochondrial DNA strand breaks., Methods: Effects of an intragastric dose of ethanol (5 g/kg) on hepatic mitochondrial DNA levels, structure, and synthesis were determined by slot blot hybridization, Southern blot hybridization, and in vivo [3H]thymidine incorporation, respectively., Results: Two hours after ethanol administration, ethane exhalation (an index of lipid peroxidation) increased by 133%, although hepatic lipids were unchanged. Mitochondrial DNA was depleted by 51%. Its supercoiled form disappeared, whereas linearized forms increased. Long polymerase chain reaction evidenced lesions blocking polymerase progress on the mitochondrial genome. Mitochondrial transcripts decreased. Subsequently, [3H]thymidine incorporation into mitochondrial DNA increased, and mitochondrial DNA levels were restored. In contrast, nuclear DNA was not fragmented and its [3H]thymidine incorporation was unchanged. Liver ultrastructure only showed inconstant mitochondrial lesions. Ethanol-induced mitochondrial DNA depletion was prevented by 4-methylpyrazole, an inhibitor of ethanol metabolism, and attenuated by melatonin, an antioxidant., Conclusions: After an alcoholic binge, ethanol metabolism causes oxidative stress and hepatic mitochondrial DNA degradation in mice. DNA strand breaks may be involved in the development of mitochondrial DNA deletions in alcoholics.

Background & Aims: 4,4'-Diethylaminoethoxyhexestrol (DEAEH), amiodarone, and perhexiline cause steatohepatitis in humans. The mechanisms of these effects are unknown for DEAEH and have not been completely elucidated for amiodarone and perhexiline. The aim of this study was to determine these mechanisms., Methods: Rat liver mitochondria, cultured rat hepatocytes, or rats were treated with these drugs, and the effects on mitochondrial respiration, beta-oxidation, reactive oxygen species formation, and lipid peroxidation were determined., Results: DEAEH accumulated in mitochondria and inhibited carnitine palmitoyl transferase I and acyl-coenzyme A dehydrogenases; it decreased beta-oxidation and caused lipid deposits in hepatocytes. DEAEH also inhibited mitochondrial respiration and decreased adenosine triphosphate (ATP) levels in hepatocytes. DEAEH, amiodarone, and perhexiline augmented the mitochondrial formation of reactive oxygen species and caused lipid peroxidation in rats., Conclusions: Like amiodarone and perhexiline, DEAEH accumulates in mitochondria, where it inhibits both beta-oxidation (causing steatosis) and respiration. Inhibition of respiration decreases ATP and also increases the mitochondrial formation of reactive oxygen species. The latter oxidize fat deposits, causing lipid peroxidation. We suggest that ATP depletion and lipid peroxidation may cause cell death and that lipid peroxidation products may account, in part, for other steatohepatitis lesions.

Background & Aims: Germander was withdrawn from the market after its use for weight control caused an epidemic of hepatitis. Its toxicity was shown to be caused by diterpenoids and their cytochrome P4503A-mediated metabolic activation into electrophilic metabolites that deplete cellular thiols. The aim of the present study was to determine the mechanisms of cell death., Methods: Isolated rat hepatocytes were incubated for 2 hours with germander diterpenoids (100 micrograms/mL)., Results: Diterpenoids decreased cell glutathione, increased cytosolic [Ca2+], activated Ca(2+)-dependent tissue transglutaminase forming a cross-linked protein scaffold, and caused internucleosomal DNA fragmentation and the ultrastructural features of apoptosis. Cell death was prevented by decreasing metabolic activation (with troleandomycin), preventing depletion of glutathione (with cystine), blocking activation of Ca(2+)-modulated enzymes (with calmidazolium), or inhibiting internucleosomal DNA fragmentation (with aurintricarboxylic acid). Apoptosis was increased and diterpenoids caused overexpression of p53 and interleukin 1 beta-converting enzyme in rats treated with dexamethasone (cytochrome P4503A inducer). Apoptosis was also increased by a diet deficient in sulfur amino acids., Conclusions: The germander furano diterpenoids cause apoptosis within 2 hours in isolated rat hepatocytes. Electrophilic metabolites may stimulate apoptosis by decreasing thiols, increasing [Ca2+], and activating Ca(2+)-dependent transglutaminase and endonucleases.

Background & Aims: Aging is associated with and may be caused by acquired somatic mutations of the mitochondrial genome. In Wilson's disease, inherited mutations of a nuclear gene encoding a copper transporter cause accumulation of copper in the liver, particularly within mitochondria. Because copper has prooxidant properties and the mitochondrial genome is particularly susceptible to oxidative damage, we hypothesized that Wilson's disease may cause premature oxidative aging of mitochondrial DNA., Methods: Hepatic DNA was screened for large mitochondrial DNA deletion(s) in 16 patients with Wilson's disease and 67 control subjects. Deleted mitochondrial DNA copies were amplified by polymerase chain reaction and were sequenced., Results: Although 15 of the 16 patients with Wilson's disease were 30 years old or younger, 8 of them (50%), including the 6 patients with cirrhosis (100%), had diverse mitochondrial DNA deletions, whereas only 2 controls (3%), aged 39 and 45 years, showed a mitochondrial DNA deletion., Conclusions: Wilson's disease is associated with frequent, diverse, and early deletions of mitochondrial DNA. Accumulation of prooxidant copper within hepatic mitochondria may cause this premature oxidative aging of mitochondrial DNA. Thus, inherited mutations of a nuclear gene may cause somatic mutations of the mitochondrial genome in this condition.

Background & Aims: Tacrine administration (1-3 mg/kg) may lead to sinusoidal concentrations in the micromolar range and produce liver dysfunction in 50% of recipients. The aim of this study was to determine the cellular effects of tacrine that account for liver dysfunction., Methods: The effects of tacrine on mitochondrial function were determined in isolated rat liver mitochondria, cultured rat hepatocytes, and isolated human lymphocytes., Results: In vitro, tacrine was taken up by rat liver mitochondria, decreased their membrane potential, and stimulated their respiration. Ex vivo, respiration was increased in rat mitochondria isolated 30 minutes after the administration of 2 mg of tacrine per kilogram. After 7 days of culture, tacrine (2.5 mumol/L) decreased rat hepatocyte adenosine triphosphate levels. Ten micromolar decreased 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium reduction and neutral red uptake without modifying cell glutathione, the morphology of the endoplasmic reticulum, or protein synthesis. Tacrine (1.25 mumol/L) decreased levels of adenosine triphosphate in human lymphocytes., Conclusions: The weak base tacrine exerts a protonophoric effect in mitochondria that wastes energy and decreases levels of adenosine triphosphate in rat and human cells. These effects are visible after clinically relevant doses of tacrine and might be involved in tacrine-induced liver dysfunction.

Background & Aims: Nitric oxide forms inactive iron-nitrosyl complexes within hepatic mitochondria in vitro. However, when formed in vivo, NO might react instead with hemoglobin. The aim of this study was to compare the effects of cell-derived NO on rat hepatocyte mitochondria in vitro and in vivo., Methods: First, hepatocytes were cultured in vitro for 24 hours under a porous membrane supporting macrophages that were stimulated by endotoxin. Second, hepatic macrophage hyperplasia was induced in vivo by preadministration of killed Corynebacterium parvum; 7 days later, rats received endotoxin and were killed after 6 hours. Third, mitochondria were exposed to sodium nitroprusside in vitro, washed, mixed with blood, and recovered., Results: Iron-nitrosyl complexes and hepatocyte mitochondrial dysfunction were observed in the in vitro model and prevented by an NO synthase inhibitor. In the in vivo model, however, despite a 130-fold increase in plasma nitrate levels and formation of hemoglobin-NO complexes in blood, no iron-nitrosyl complex was detected in hepatic mitochondria, and hepatic mitochondrial function was not impaired. In the third model, mitochondria lost preformed iron-nitrosyl complexes when exposed to blood., Conclusions: Although NO reacts with hepatocyte mitochondria in vitro, in vivo it reacts with sinusoidal hemoglobin without detectable impairment of hepatic mitochondrial function.

Background/aims: Autoantibodies against cytochrome P450 are found in some forms of autoimmune hepatitis. Cytochrome P450 is synthesized and mainly located in the endoplasmic reticulum but may also be expressed on the plasma membrane of hepatocytes. Vesicles migrate from the endoplasmic reticulum to the Golgi apparatus and then to the plasma membrane along microtubules. We determined the route followed by cytochrome P4502B to reach the plasma membrane., Methods: Rat hepatocytes were cultured for 2 hours after plating with various inhibitors of cellular trafficking. Detached, uncut, nonpermeabilized hepatocytes were then exposed to a monoclonal antibody specific for cytochrome P4502B and studied by flow cytometry and confocal microscopy., Results: The plasma membrane expression of cytochrome P4502B was markedly decreased after 2 hours of culture with cycloheximide (an inhibitor of protein synthesis), caffeine at 20 degrees C (conditions that decrease vesicular transport from the endoplasmic reticulum to the Golgi apparatus), brefeldin A (which redistributes Golgi components back to the endoplasmic reticulum), monensin (an inhibitor of Golgi functions), and colchicine, vinblastine, or nocodazole (three microtubule inhibitors)., Conclusions: Part of cytochrome P4502B follows a microtubule-dependent vesicular route from the endoplasmic reticulum to the plasma membrane in cultured rat hepatocytes.

Background/aims: Alcohol abuse may lead to microvesicular steatosis, a lesion ascribed to impaired mitochondrial function. Because alcohol abuse leads to reactive oxygen species in the hepatic mitochondria, it may damage mitochondrial DNA. The aim of this study was to look for the presence of the "common" 4977-base pair deletion in the hepatic mitochondrial DNA of alcoholic patients and age-matched, nonalcoholic controls., Methods: Hepatic DNA was subjected to two polymerase chain reactions that amplified non-deleted and deleted mitochondrial DNA, respectively., Results: The deletion was found in 6 of 10 alcoholics with microvesicular steatosis, 2 of 17 alcoholic patients with macrovacuolar steatosis, but in none of 12 patients with acute alcoholic hepatitis, 11 patients with alcoholic cirrhosis, or 62 nonalcoholic patients of comparable ages with various other liver diseases or normal liver histology. In all patients with the deletion, restriction fragments of deleted mitochondrial DNA co-migrated with those of reference Pearson bone marrow-pancreas syndrome patients with the common mitochondrial DNA deletion., Conclusions: The common deletion is frequent in the hepatic DNA of alcoholic patients with microvesicular steatosis. Alcohol-induced mitochondrial DNA damage may contribute to the occurrence of this lesion in some alcoholics.

Background/aims: An epidemic of hepatitis due to germander teas or capsules recently occurred in France. The aim of the present study was to show the hepatotoxicity of germander and determine its mechanism in mice., Methods: A germander tea lyophilisate and a fraction that isolated and concentrated 10-fold the furano neo-clerodane diterpenoids of the lyophilisate were prepared., Results: (1) Intragastric administration of the lyophilisate (1.25 g/kg) or the furano neo-clerodane diterpenoid fraction (0.125 mg/kg) produced similar midzonal liver cell necrosis at 24 hours in mice. (2) Toxicity was prevented by pretreatment with a single dose of troleandomycin (a specific inhibitor of cytochromes P4503A) and enhanced by pretreatment with dexamethasone or clotrimazole (two inducers of cytochromes P4503A). (3) Toxicity was attenuated by pretreatment with butylated hydroxyanisole or clofibrate (two inducers of microsomal epoxide hydrolase) and markedly increased by phorone-induced glutathione depletion., Conclusions: We conclude that germander constituents (probably its furano neo-clerodane diterpenoids) are transformed by cytochromes P450 (particularly P4503A) into hepatotoxic metabolites. The metabolites (probably epoxides) are partly inactivated by glutathione and probably epoxide hydrolase.

Background: Anti-cytochrome P-450 autoantibodies are present in several forms of autoimmune hepatitis. The possibility that cytochromes P-450 are present in the plasma membrane of human hepatocytes was examined., Methods: (1) Plasma membranes with microsomal contamination < 1%, as judged from the activities of glucose-6-phosphatase and NADH-cytochrome c reductase, were prepared. (2) After exposure of uncut, fixed hepatocytes to antibodies, immunofluorescence and immunoperoxidase studies were performed., Results: (1) The specific content of cytochrome P-450 in plasma membrane was 9% of that in microsomes. Plasma membranes showed NADPH-cytochrome c reductase and mono-oxygenase activities; immunoblots showed the presence of cytochromes P-450 1A2, 2C, 2D6, 2E1, and 3A4; cytochromes P-450 1A2, 2D6, and 2C were also recognized by anti-liver microsome and anti-liver/kidney microsome type 1 and type 2 autoantibodies, respectively. (2) Immunofluorescence and immunoperoxidase labeling of the plasma membrane was observed with the three auto-antibodies and with anti-cytochrome P-450 1A2, 2C, 2E1, or 3A4., Conclusions: It is concluded that cytochromes P-450 are present and functional in the plasma membrane of human hepatocytes and that anti-cytochrome P-450 autoantibodies recognize epitopes expressed on the outer surface.

A case of fulminant hepatitis in a patient taking lisinopril for 5 weeks for arterial hypertension is reported. Jaundice, fever, myalgia, and marked increase in serum aminotransferase activities occurred after 2 weeks of treatment. Continuation of lisinopril administration for 3 weeks after the onset of jaundice was associated with the development of grade III encephalopathy and a marked decrease in prothrombin and proaccelerin levels. This case strongly suggests that lisinopril may induce acute hepatitis and that continuation of the treatment after the onset of jaundice can lead to life-threatening hepatic failure.

Tumoral secretions and pathophysiology of diarrhea were studied in 1 patient with pancreatic cholera. High concentrations of vasoactive intestinal peptide were found in both systemic blood and tumoral extracts, together with increased plasma levels of calcitonin and protaglandins E and Falpha. Gastric inhibitory peptide and gastrointestinal and pancreatic hormones were absent from the tumor, except for small amounts of glucagon, and their blood levels were normal. Decreased basal but normal pentagastrin-stimulated gastric acid secretion, normal basal and secretin-stimulated pancreatic secretion, increased volume of gallbladder bile with high bicarbonate, and low bile salt concentrations were observed, but the electrolyte content and flow rate of fluid passing the duodenojejunal junction were within normal limits. Small intestine was found to be the origin of the water and electrolyte fasting losses. Jejunum was the site of bicarbonate secretion. Jejunal glucose and leucine-stimulated water and sodium transports were also strikingly decreased, whereas the absorption rates of the sugar and amino acid were normal. Colon reabsorbed high amounts of water and sodium but increased potassium losses. Biological effects of vasoactive intestinal peptide may explain most of the patient's upper digestive secretion abnormalities and small intestinal function impairments, whereas secondary aldosteronism might explain the modified colonic function.

Liver histology was normal 24 h after the administration of trichloroethylene (1 ml . kg-1) in rats. It was normal, or showed necrosis of a few hepatocytes, after the administration of carbon tetrachloride (64 microliters . kg-1). In rats receiving both solvents, there was extensive centrilobular necrosis. In vitro, trichloroethylene did not initiate lipid peroxidation but potentiated that initiated by carbon tetrachloride; a similar potentiating effect was observed for a wide range of trichloroethylene concentrations (0.19-12 mM). In vivo, a wide range of trichloroethylene doses (0.064-1 ml . kg-1) similarly potentiated the hepatotoxicity of carbon tetrachloride. Administration of trichloroethylene (1 ml . kg-1), 5 h earlier, increased carbon tetrachloride-induced lipid peroxidation in vitro, and increased the hepatotoxicity of a subsequent dose of carbon tetrachloride (64 microliters . kg-1). Previous administration of carbon tetrachloride failed to modify lipid peroxidation and to increase the hepatotoxicity of trichloroethylene. We conclude that trichloroethylene potentiates the hepatotoxicity of carbon tetrachloride, possibly by increasing carbon tetrachloride-induced lipid peroxidation.

We report the case of a patient in whom amitriptyline administration for 5 wk was followed by prolonged cholestasis. Jaundice and pruritus lasted 19 and 20 mo, respectively. Three liver biopsies were performed at different stages of the disease showing the course of liver lesions. Cholestasis initially located in the region of the hepatic venule came to be associated with the progressive development of portal tract lesions consisting of inflammatory infiltration, fibrosis, and disappearance of interlobular bile ducts. Amitriptyline hydroxylation and dextromethorphan O-demethylation are deficient in subjects with the poor metabolizer phenotype of debrisoquine. Drug oxidation phenotyping with dextromethorphan showed that this patient had the extensive metabolizer phenotype. This observation demonstrates that amitriptyline can induce prolonged cholestasis and suggests that the susceptibility to develop liver injury while taking this drug may not be related to a genetic deficiency of its hydroxylation.

Acetaminophen and bromobenzene are transformed in the liver into chemically reactive metabolites that may either bind to glutathione and be detoxified or bind to hepatic proteins and produce liver cell necrosis. Fasting for 42 hr (a) decreased hepatic glutathione concentration, (b) increased the amount of chemically reactive metabolite irreversibly bound to hepatic proteins after administration of 3H-acetaminophen or 14C-bromobenzene, and (c) increased the hepatotoxicity of acetaminophen or bromobenzene. In rats fasted for various lengths of time, there was an inverse relationship between the concentration of glutathione in the liver and the activity of serum glutamic pyruvic transaminases after administration of acetaminophen or bromobenzene. In vitro, there was an inverse relationship between the concentration of glutathione in the incubate and the amount of chemically reactive metabolite bound to microsomal proteins after incubation of 3H-acetaminophen or 14C-bromobenzene with hepatic microsomes. It is concluded that fasting may decrease the inactivation of chemically reactive metabolites by glutathione, increase their binding to hepatic proteins, and enhance the hepatotoxicity of drugs transformed into chemically reactive metabolites that are detoxified by binding to glutathione.

We report the cases of 2 female patients aged 69 and 61 yr, suffering from fulminant hepatitis induced by pirprofen, a new nonsteroidal antiinflammatory drug. The duration of pirprofen administration before the onset of hepatitis was long, 7 and 9 mo, respectively. Hepatitis was not preceded or accompanied by hypersensitivity manifestations. The liver lesion consisted of massive, predominantly centrilobular hepatic cell necrosis and microvesicular steatosis. One patient died of liver failure. Although the risk of fulminant hepatitis is very low, we recommend that, in patients taking pirprofen for more than 2 mo and complaining of asthenia, nausea, or vomiting, serum aminotransferase levels should be measured and administration of the drug should be interrupted as soon as an increased level is noted.

The authors report the cases of 2 patients who died from cirrhosis after receiving perhexiline maleate, a drug widely used in Europe for the treatment of angina pectoris. Perhexiline maleate had been ingested for 24 and 28 mo, respectively. Manifestations of cirrhosis included jaundice, hepatic encephalopathy, ascites, and portal hypertension. Associated manifestations of intolerance to perhexiline maleate included peripheral neuropathy in 1 patient and marked weight loss in both. Histologic lesions resembled those observed in patients with alcoholic liver disease. Ultrastructural lesions included numerous enlarged lysosomes containing myeloid figures. Histochemical stains demonstrated increased phospholipid content of the hepatocytes. These findings are consistent with the view that prolonged administration of perhexiline maleate may induce both histologic lesions resembling those of alcoholic liver disease and ultrastructural and histochemical lesions resembling those of phospholipidosis.

The authors report 6 cases of fulminant hepatitis in patients treated with isoniazid and rifampin. In 4 of these patients, the treatment had been started within 3 days after a general anesthesia. The course of the disease was remarkably similar in all 6 patients: (1) the time interval from the beginning of the isoniazid-rifampin administration to the onset of jaundice was 6 to 10 days; (2) disorders of consciousness appeared less than 3 days after the onset of jaundice; (3) serum transaminases were 26 to 80 times the upper limit of normal; (4) the main liver lesion was centrilobular necrosis; (5) hypersensitivity manifestations were absent; (6) all 6 patients recovered. Fulminant hepatitis might be attributable to a hepatotoxic metabolite of isoniazid, the production of which would be attributable to a hepatotoxic metabolite of isoniazid, the production of which would be increased as a consequence of the enzyme-inducing effect of rifampin and, possibly, other drugs administered for general anesthesia.

The authors report the cases of 3 patients who died from fulminant hepatitis after receiving iproclozide, a hydrazine-containing monoamine oxidase inhibitor. Fulminant hepatitis in these patients resembled that reported in patients receiving other hydrazine-containing monoamine oxidase inhibitors: (1) the 3 patients were women; (2) the monoamine oxidase inhibitor has been ingested for 1 month or more; (3) the main clinical manifestations were jaundice and disorders of consciousness; (4) hypersensitivity manifestations were absent; (5) the predominant liver lesion was necrosis; (6) all 3 patients died. In our 3 patients, jaundice occurred 7 to 10 days after the adjunction to iproclozide of a microsomal enzyme inducer. These observations suggest that concomitant administration of iproclozide and of microsomal enzyme inducers may produce fulminant hepatitis in man. It is speculated that iproclozide could be, like iproniazid, transformed into a hepatotoxic metabolite, the production of which would be increased by microsomal enzyme induction.