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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. GASTROENTEROLOGY 1999;117:181-190

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, @b-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 @b-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 @b-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. GASTROENTEROLOGY 1998;114:764-774

Coincubation of 0.4 mM [3H]glutathione with 4 mM isaxonine , an NADPH-generating system, glutathione S-transferase and mouse liver microsomes, followed by thin-layer chromatography of the incubation mixture, resulted in the appearance of a 3H-labeled peak with characteristics consistent with a glutathione- isaxonine metabolite adduct: this peak was absent if either isaxonine or the NADPH-generating system was omitted and was decreased if the transferase was omitted. In vivo, the concentrations of hepatic glutathione and glutathione disulfide were markedly decreased 2.5 hr after administration of isaxonine (4 mmol X kg-1 i.p.); this depletion of glutathione was prevented essentially by pretreatment with piperonyl butoxide. In vitro, addition of 4 mM glutathione decreased markedly the amount of [14C] isaxonine metabolite that bound to microsomal proteins during incubation of 1 mM [2-14C] isaxonine with hepatic microsomes and an NADPH-generating system. In vivo, pretreatment with diethylmaleate decreased further hepatic glutathione concentration and markedly increased the amount of [14C] isaxonine metabolite covalently bound to hepatic proteins, 2.5 hr after administration of [2-14C] isaxonine (4 mmol X kg-1 i.p.). Administration of isaxonine (4 mmol X kg-1 i.p.) decreased hepatic cytochrome P-450 concentration, but failed to produce liver cell necrosis, even in mice pretreated with phenobarbital, 3-methylcholanthrene or diethylmaleate, despite high levels of in vivo covalent binding in pretreated animals. We conclude that the reactive metabolite of isaxonine may be conjugated with glutathione or may covalently bind to hepatic proteins. The metabolite, however, has limited hepatotoxic potential in mice.

The metabolism of isaxonine was first investigated in mice. Incubation, under air, of [2-14C] isaxonine (1 mM) with mouse liver microsomes and an NADPH-generating system resulted in the irreversible binding of a [14C] isaxonine metabolite to microsomal proteins; binding required active microsomes, NADPH and oxygen, it was inhibited by 4 mM piperonyl butoxide or by a CO-O2 (80:20) atmosphere. In the presence of various concentrations of isaxonine (0.125-2 mM), binding followed Michaelis-Menten kinetics; the Vmax was increased by both phenobarbital and 3-methylcholanthrene pretreatments. In vivo, 2.5 hr after the administration of [2-14C] isaxonine (4 mmol X kg-1 i.p.), a [14C] isaxonine material was irreversibly bound to mouse liver proteins; this binding was decreased by piperonyl butoxide and increased by phenobarbital or 3-methylcholanthrene pretreatments. Irreversible binding also occurred in the kidney. Unlike their effects in the liver, piperonyl butoxide and phenobarbital did not modify significantly in vitro metabolic activation by kidney microsomes and in vivo covalent binding to kidney proteins; pretreatment with 3-methylcholanthrene increased both in vitro and in vivo binding in the kidney. In a second series of experiments, in vitro metabolic activation was demonstrated with human liver microsomes; as in mice, covalent binding required NADPH and was markedly inhibited by piperonyl butoxide. We conclude that isaxonine is activated by mouse and human cytochromes P-450 into a reactive metabolite. In vivo covalent binding to mouse liver and kidney proteins appears to result mainly from the in situ binding of the metabolite formed in each organ.

Repeated administration of clarithromycin (0.5 mmol.kg-1 p.o. daily for 5 days) to rats increased markedly the same cytochrome P-450 isoenzyme (P-450p) as that induced by troleandomycin. Clarithromycin, however, did not form cytochrome P-450 Fe(II)-metabolite complexes in vitro with microsomes from clarithromycin-treated rats or in vivo after repeated doses of clarithromycin. Nevertheless, clarithromycin formed cytochrome P-450 Fe(II)-metabolite complexes with microsomes from dexamethasone-treated rats in vitro, or after administration to dexamethasone-treated rats in vivo. Similar effects were observed with roxithromycin. In contrast, erythromycin and troleandomycin formed metabolic complexes when given alone, whereas josamycin, midecamycin and spiramycin did not form complexes, even in dexamethasone-treated rats. We conclude that clarithromycin and roxithromycin induce cytochrome P-450p, but do not form complexes with this isoenzyme, although they do form complexes with other glucocorticoid-inducible isoenzymes. We propose that macrolides may be classified into three groups, those forming complexes when given alone (e.g., erythromycin and troleandomycin), those forming complexes only in glucocorticoid-pretreated rats (clarithromycin and roxithromycin) and those not forming complexes (josamycin, midecamycin and spiramycin).

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.

The nitroaromatic drug nilutamide has been shown previously to undergo redox cycling in aerobic rat liver microsomes, being reduced by NADPH-cytochrome P-450 reductase to a nitro anion-free radical which reacts with oxygen, to regenerate the parent drug, and form a superoxide anion dismuted to hydrogen peroxide. In the present study, the effects of nilutamide on isolated rat hepatocytes have been determined. After 6 and 8 hr of incubation with 0.5 mM nilutamide, lactate dehydrogenase was released in the incubation medium, and cell viability was decreased markedly. Consistent with a redox cycle producing reactive oxygen species, nilutamide increased nonmitochondrial (cyanide-resistant) oxygen consumption; the toxicity of nilutamide occurred sooner and was more extensive in the presence of sodium azide (an inhibitor of catalase). Consistent with an oxidative stress, the toxicity of nilutamide was associated with depletion of reduced glutathione, increased levels of glutathione disulfide, increased Ca(++)-dependent phosphorylase a activity, oxidation and accumulation of cytoskeleton-associated proteins and formation of blebs; toxicity was prevented by glutathione precursors, thiol reductants and/or antioxidants, such as L-cystine, L-cysteine, N-acetyl-L-cysteine, dithiothreitol, N,N'-diphenyl-p-phenylene-diamine and alpha-tocopherol. Feeding the animals with a diet supplemented with 2% L-cystine increased the initial glutathione stores of hepatocytes and prevented nilutamide toxicity. It is concluded that nilutamide is toxic to isolated rat hepatocytes, as a probable consequence of an oxidative stress due to the redox cycling of this nitroaromatic compound.

The effects of amiodarone on the respiration of isolated mouse liver mitochondria have been determined. Amiodarone (200 microM) had a biphasic effect on state 4 respiration supported by either glutamate plus malate or succinate. Initially, the respiratory rate was increased. This stimulatory effect was not prevented by oligomycin (an inhibitor of ATP synthase). It was associated with marked accumulation of amiodarone in the mitochondria, and with collapse of the mitochondrial membrane potential. This initial uncoupling effect was followed by a progressive decrease in the state 4 respiration rate, leading eventually to marked inhibition. Preincubation for 5 min with amiodarone (200 microM) also decreased markedly ADP-stimulated (state 3) respiration, ATP production and dinitrophenol-stimulated (uncoupled) respiration supported by glutamate plus malate (which donate electrons to complex I), and respiration supported by succinate (which donate electrons to complex II), but did not affect respiration supported by duroquinol (donating electrons to complex III) or by ascorbate plus N,N,N',N'-tetramethyl-p-phenylenediamine (donating electrons to cytochrome c). Preincubation with amiodarone (150-200 microM) decreased markedly respiration mediated by fatty acids of various chain length and respiration mediated by citrate, a tricarboxylic acid cycle substrate. We conclude that amiodarone has a dual effect on mitochondrial respiration. The initial uncoupling effect is probably due to the entry of protonated amiodarone, releasing a proton in the matrix. Accumulation of amiodarone soon leads to inhibition of the respiratory chain at the levels of complex I and complex II and to decreased ATP formation.

Incubation of rat liver microsomes with [3H]methoxsalen and NADPH resulted in the covalent binding of a methoxsalen intermediate to proteins comigrating with cytochromes P-450 UT-A, PB-B/D, ISF-G and PCN-E. Binding was increased by pretreatments with phenobarbital, beta-naphthoflavone (beta NF) and dexamethasone. Such pretreatments also increased the loss of CO-binding capacity either after administration of methoxsalen, or after incubation of hepatic microsomes with methoxsalen and NADPH. Immunoprecipitation of the methoxsalen metabolite-protein adducts in phenobarbital-induced microsomes was moderate with anti-UT-A antibodies, but marked with anti-PB-B/D and anti-PCN-E antibodies. Immunoprecipitation was observed also with anti-ISF-G (anti-beta NF-B) antibodies in beta NF-induced microsomes. Methoxsalen (0.25 mM) inhibited markedly the benzphetamine demethylase activity of phenobarbital-induced microsomes and the erythromycin demethylase activity of dexamethasone-induced microsomes. Whereas methoxsalen itself did not produce any binding spectrum, in contrast either in vivo administration of methoxsalen or incubation in vitro with methoxsalen and NADPH resulted in a low-to-high spin conversion of cytochrome P-450 as suggested by the appearance of a spectrum analogous to a type I binding spectrum. This low-to-high spin conversion was apparently due to a methoxsalen intermediate (probably, covalently bound to the protein and preventing partial sixth ligation of the iron). We conclude that suicide inactivation of cytochrome P-450 by methoxsalen is related to the covalent binding of a methoxsalen intermediate to the protein moiety of several cytochrome P-450 isoenzymes (including UT-A, PB-B/D, PCN-E as well as ISF-G and/or beta NF-B).

The mechanism for trichloroethylene hepatotoxicity was investigated in male Sprague-Dawley rats. Phenobarbital pretreatment increased and CoCl2 pretreatment decreased trichloroethylene hepatotoxicity. After administration of 1(14)C]trichloroethylene, a radioactive material became irreversibly bound to hepatic proteins, while negligible amounts were bound to muscle proteins. When 1(14)C]trichloroethylene was incubated under air with hepatic microsomes and a NADPH-generating system, a radioactive material became irreversibly bound to microsomal proteins; binding was negligible when the NADPH-generating system was omitted; binding was inhibited by carbon monoxide and by piperonyl butoxide; the amount of bound material was greater with microsomes from phenobarbital-pretreated rats and lower with microsomes from CoCl2-pretreated rats than with microsomes from nonpretreated rats. Trichloroethylene administration decreased hepatic glutathione in normal rats but not in piperonyl butoxide-pretreated rats; in vitro, glutathione decreased the amount of trichloroethylene material that bound to microsomal proteins. The reported results are consistent with the view that 1) trichloroethylene is metabolized by cytochrome P-450 into a chemically reactive metabolite which reacts with, and binds to, either proteins or glutathione, 2) binding to proteins produces liver lesions and 3) binding to glutathione decreases the amount of reactive metabolite available for binding to proteins.

Erythromycin, 0.3 mM, elicited a small reverse type I binding spectrum with, and was slowly demethylated by, cytochrome P-450 from control rats. No absorption peak at 456 nm could be detected upon incubation of 0.3 mM erythromycin with NADPH and control microsomes. No complex formed in vivo could be detected in microsomes isolated 2 hr after a single dose of erythromycin, 2 mmol.kg-1 p.o. Repeated administration of erythromycin, 2 mmol.kg-1 p.o. daily for 4 days increased hepatic microsomal protein concentration, NADPH-cytochrome c reductase activity, the amplitude of the reverse type I binding spectrum of erythromycin and erythromycin demethylase activity. Microsomes isolated from rats treated with repeated doses of erythromycin exhibited a marked absorption peak at 456 nm. The absorption at 456 nm was further increased upon incubation with erythromycin and NADPH. It disappeared upon addition of 50 muM potassium ferricyanide. Disruption of the complex with potassium ferricyanide markedly increased the CO-binding capacity of dithionite-reduced microsomes. It further increased the amplitude of the reverse type I binding spectrum of erythromycin and erythromycin demethylase activity and increased ethylmorphine N-demethylase and benzo[a]pyrene hydroxylase activities. It is concluded that erythromycin induces its own transformation into a metabolite which forms a inactive 456-nm absorbing complex with the iron (II) of cytochrome P-450.

Repeated administration of troleandomycin increased bile flow but decreased the biliary secretion of bile acids in rats. The increased bile flow was associated with a parallel increase in the biliary clearance of [14C]erythritol. Analysis of the relationship between bile flow and bile acid secretion indicated that, for any given rate of bile acid secretin, bile flow was higher in troleandomycin-treated rats than in control rats. The increased bile flow was associated with an increased activity of Na+,K+-adenosine triphosphatase in liver plasma membranes. The decreased bile acid secretion into bile was associated with a similar decrease in the bile acid pool size, a decreased bile acid synthesis rate and a decreased activity of microsomal cholesterol 7 alpha-hydroxylase. The concentration of bile acids in serum, the hepatic extraction ratio of [3H]taurocholate and its biliary transport maximum were not modified. It is concluded that repeated administration of troleandomycin increases the canalicular bile acid-independent flow but decreases the activity of cholesterol 7 alpha-hydroxylase, the synthesis, the pool size and the biliary secretion rate of bile acid in rats.

Troleandomycin, a macrolide antibiotic, has been shown to be demethylated and oxidized into a metabolite which forms an inactive complex with the iron(II) of cytochrome P-450. The role of glutathione in the metabolism of troleandomycin was investigated. Administration of troleandomycin (1 mmol X kg-1 p.o.) decreased the concentration of glutathione in the liver. The depletion of glutathione was increased in rats pretreated with phenobarbital and decreased in rats pretreated with CoCl2. In vitro, an inverse relationship was found between the concentration of glutathione in the incubation mixture and the appearance of the cytochrome P-450-troleandomycin metabolite complex. Glutathione, however, did not inhibit the demethylation of troleandomycin and did not destroy the cytochrome P-450-troleandomycin metabolite complex. The in vitro protective effect of glutathione was reproduced by cysteine but not by glycine. In vivo, decreasing the concentration of glutathione in the liver by food deprivation or by the administration of diethylmaleate increased the formation of the cytochrome P-450-troleandomycin metabolite complex. These results indicate that glutathione is depleted by a troleandomycin metabolite in vivo, whereas glutathione protects against the formation of the inactive cytochrome P-450-troleandomycin metabolite complex in vitro and in vivo.

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.

Although acetaminophen is widely used in pregnant women, the effects of pregnancy on its hepatotoxicity remain unknown. We assessed these effects in pregnant mice (17-18 days of gestation). The hepatotoxicity of acetaminophen (300-400 mg X kg-1 i.p.) was increased markedly in pregnant mice, as judged by increased serum glutamic-pyruvic transaminase activity, higher incidence of liver necrosis and greater mortality. In vitro, acetaminophen sulfotransferase activity was increased by 47% in pregnant mice, but acetaminophen glucuronosyltransferase activity was decreased by 54%; the metabolic activation of acetaminophen to covalently bound metabolites was unchanged. Glutathione S-transferase activities were decreased slightly. In vivo, after administration of acetaminophen (300 mg X kg-1 i.p.), the 24-hr urinary excretion of the sulfate conjugate was increased (from 12% of the recovered dose in nonpregnant mice to 21% in pregnant mice), that of the glucuronide was decreased (from 61 to 52%), whereas those of the cysteine and mercapturic acid conjugates and that of acetaminophen were unchanged. Finally, the plasma clearance and the apparent volume of distribution of acetaminophen (both expressed per body weight) remained unchanged. Similarly, in vivo covalent binding to hepatic proteins 4 hr after administration of acetaminophen (300 and 400 mg X kg-1 i.p.) remained unchanged as were in vivo indexes of lipid peroxidation. In contrast, liver glutathione concentration, albeit initially normal, fell to much lower levels after administration of acetaminophen (200-400 mg X kg-1 i.p.) or diethylmaleate (0.5 ml X kg-1 i.p.) in pregnant mice, and recovered more slowly thereafter.(ABSTRACT TRUNCATED AT 250 WORDS)

8-Methoxypsoralen has been shown to both inactivate and induce microsomal enzymes. We report here the effects of other psoralen derivatives in rats. CO-binding cytochrome P-450 decreased when hepatic microsomes were incubated for 10 min with an NADPH-generating system and 8-methoxypsoralen, 5-methoxypsoralen or psoralen (400 microM), but remained unchanged with trioxsalen (400 microM). The destruction of cytochrome P-450 with the former derivatives required NADPH. It was greater with microsomes from phenobarbital-pretreated rats. Monooxygenase activities were decreased by 30 to 60% when 8-methoxypsoralen, 5-methoxypsoralen or psoralen (10 or 25 microM) were added to the incubation mixtures, but remained unchanged upon addition of trioxsalen (10 or 25 microM). In vivo, monooxygenase activities were decreased 4 hr after the administration of a single dose of 8-methoxypsoralen, 5-methoxypsoralen or psoralen (125 mumol X kg-1 p.o.) but remained unchanged after trioxsalen (125 mumol X kg-1 p.o.). During repeated administration of 8-methoxypsoralen or 5-methoxypsoralen (125 mumol X kg-1 p.o. for 3 or 4 days), there was evidence for both induction and inactivation of drug-metabolizing enzymes; monooxygenase activities were high or normal late after a preceding dose, but fell again to normal or low values, respectively, early after a further dose. With psoralen, there was only inactivation, and monooxygenase activities tended to remain low throughout. Monooxygenase activities remained normal with trioxsalen. We conclude that 5-methoxypsoralen, like 8-methoxypsoralen, both inactivates and induces microsomal enzymes. In contrast, psoralen only inactivates them, whereas trioxsalen shows little effects.(ABSTRACT TRUNCATED AT 250 WORDS)

We have reported previously that methoxsalen is a suicide substrate for cytochrome P-450. We now report its effects on the metabolism and toxicity of acetaminophen in mice. Intragastric administration of methoxsalen (125 mumol X kg-1), 30 min before that of acetaminophen (600 mg X kg-1 i.p.), decreased the formation of the mercapturate and cysteine conjugates of acetaminophen, the depletion of glutathione and the in vivo covalent binding of an acetaminophen metabolite to hepatic proteins and prevented the increase in serum glutamic-pyruvic transaminase activity, the appearance of liver lesions and mortality. Methoxsalen (250 mumol X kg-1) also afforded complete protection when given intragastrically 2 hr after acetaminophen (600 mg X kg-1 i.p.). At that time, methoxsalen still decreased in vivo covalent binding measured per whole liver, and permitted a faster recovery of hepatic glutathione. Methoxsalen (180 mumol X kg-1) and N-acetylcysteine (919 mumol X kg-1) exerted additive protective effects when given concomitantly 2 hr after acetaminophen. We conclude that administration of methoxsalen decreases the metabolic activation and the hepatotoxicity of acetaminophen in mice.

Human recombinant interleukin-2 (IL-2) administration is being tested in patients with advanced cancer. Its effects on the expression of cytochromes P-450 were determined in rats. IL-2 administration (1-25 x 10(6) U/kg i.v. twice daily for 1 to 4 days) resulted in a time- and dose-dependent decrease in cytochrome P-450 measured by the absorbance of its Fe(++)-CO complex. After 25 x 10(6) U/kg twice daily for 4 days, cytochrome P-450 decreased 44%; immunoreactive cytochrome P-450 1A1 decreased nonsignificantly (22%); but cytochrome P-450 1A2 decreased 68%; 2B1/2, 50%; 2C11, 75%; 2D1, 36%; and 3A, 70%. Aminopyrine N-demethylase activity decreased 53%, ethoxycoumarin O-deethylase 64%, benzo(a)pyrene hydroxylase 71%, ethoxyresorufin O-deethylase 42%, pentoxyresorufin O-dealkylase 81% and erythromycin N-demethylase 56%. In rats treated with 3-methylcholanthrene for 4 days, IL-2 coadministration (25 x 10(6) U/kg i.v. twice daily for 4 days) did not decrease significantly immunoreactive cytochrome P-450 1A1 and 1A2, whereas cytochromes P-450 2B1/2, 2C11 and 3A decreased 39, 54 and 67%, respectively. In rats treated with phenobarbital for 4 days, IL-2 coadministration decreased immunoreactive cytochromes P-450 2B1/2 29%, whereas cytochromes P-450 1A2, 2C11 and 3A decreased 38, 63 and 67%, respectively. We conclude that administration of high doses of IL-2 decreases the expression of several cytochromes P-450 in rats. Microsomal enzyme inducers appear to limit the effects of IL-2 on the induced forms of cytochromes P-450. Because much lower doses are used in humans, their potential effects on drug metabolism cannot be assessed from present results.

Microvesicular steatosis of the liver has been reported in two subjects receiving amineptine (a tricyclic antidepressant metabolized by beta-oxidation of its acyl chain). A similar disease is observed after ingestion of drugs which inhibit hepatic mitochondrial fatty acid beta-oxidation, or in subjects with various inborn defects in this metabolic pathway. We therefore determined the effects of amineptine on the mitochondrial oxidation of fatty acids in mice. In vitro, the formation of beta-oxidation products during incubation of palmitic acid with mouse liver mitochondria and the various cofactors necessary for beta-oxidation was inhibited by 27, 33, 46 and 57% respectively, in the presence of 0.25, 0.5, 1 and 2 mM of amineptine. Inhibition was reversible. Tricarboxylic acid cycle activity, assessed by the in vitro formation of [14C]CO2 from [1-14C]acetyl coenzyme A by mouse liver mitochondria, was inhibited by 22, 23, 47, 54, 60 and 62%, respectively, in the presence of 0.0625, 0.125, 0.25, 0.5, 1 and 2 mM of amineptine. In vivo, administration of amineptine, 0.5 and 0.75 mmol.kg-1, inhibited by 70 and 84%, respectively, the exhalation of [14C] CO2 during the first 3 hr after the administration of a tracer dose of [U-14C]palmitic acid. Administration of amineptine, 0.0625, 0.25, 0.5 or 1 mmol.kg-1, 6 hr before the measurement, increased hepatic triglycerides by 73, 139, 295 and 320%, respectively. After 1 mmol.kg-1, accumulation of hepatic triglycerides was maximum at 24 hr, reaching 5-fold the control value; liver histology at that time showed microvesicular steatosis.(ABSTRACT TRUNCATED AT 250 WORDS)

Administration of pirprofen may produce microvesicular steatosis of the liver in humans. The effects of pirprofen on the mitochondrial beta-oxidation of fatty acids have been investigated in mice. In vitro, addition of 2 mM pirprofen decreased by 50% the formation of [14C]acid-soluble beta-oxidation products, and decreased by 70% the formation of [14C]CO2 upon incubation of hepatic mitochondria with [14C]palmitic acid, ATP, carnitine and coenzyme A. In vivo, administration of pirprofen (2 mmol . kg-1 i.p.), 1 hr before that of [U-14C]palmitic acid, decreased by 70% the exhalation of [14C]CO2 during the next 6 hr. Administration of pirprofen (2 mmol . kg-1 i.p.), 1 hr before the measurement, decreased plasma beta-hydroxybutyrate by 60%, plasma acetoacetate by 30% and blood glucose by 40%. Administration of pirprofen (2 mmol . kg-1 i.p.) 6 hr before sacrifice, doubled hepatic triglycerides content and produced microvesicular steatosis of the liver. We conclude that pirprofen inhibits the mitochondrial beta-oxidation of fatty acids in mice, thus explaining the microvesicular steatosis observed in mice and in some human subjects.

Administration of methoxsalen (50 mumol X kg-1 i.p.) increased 4-fold the hexobarbital sleeping time in rats; at this low dose, SKF 525-A, piperonyl butoxide and cimetidine had little or no effect. In vitro, the concentration of methoxsalen inhibiting by 50% monooxygenase activities ranged from 10 microM (for benzo(a)-pyrene and hexobarbital hydroxylases] to 25 microM for 7-ethoxy-coumarin deethylase and aminopyrine demethylase); these values were in the range of those observed with SKF 525-A (1-50 microM) or piperonyl butoxide (10-100 microM) but much lower than those for cimetidine (100-500 microM). Methoxsalen (25-1000 microM) decreased cytochrome P-450 in vitro, in the presence of EDTA; this effect required NADPH and oxygen, was decreased by piperonyl butoxide and was increased by phenobarbital pretreatment. Similarly, administration of methoxsalen (125 mumol X kg-1 i.p.) decreased cytochrome P-450 and monooxygenase activities in vivo; the decrease in cytochrome P-450 was enhanced by phenobarbital pretreatment and was prevented by piperonyl butoxide. There was no evidence for lipid peroxidation, denaturation into cytochrome P-420, formation of cytochrome P-450-metabolite complexes, destruction of heme or formation of green pigments. In contrast, a reactive metabolite of methoxsalen covalently bound to microsomal proteins; covalent binding required NADPH and oxygen, was decreased by piperonyl butoxide and was increased by phenobarbital pretreatment. We conclude that methoxsalen is activated into a metabolite which destroys cytochrome P-450.

Administration of either aspirin or salicylic acid (3 mmol.kg-1 b.wt. i.p.) decreased by 50 and 65%, respectively, the in vivo oxidation of [U-14C]palmitic acid to [14C]CO2 in mice; after salicylic acid administration, exhalation of [14C]CO2 from [1-14C]palmitic acid, [1-14C]octanoic acid or [1-14C]butyric acid was decreased by 87, 33 and 38%, respectively. Inhibition lasted 9 hr. It was associated with markedly decreased blood glucose concentrations and increased plasma ketone bodies. Repeated administration of salicylic acid (2 mmol.kg-1 i.p. every 8 hr) tripled hepatic triglycerides and produced mild microvesicular steatosis of the liver at 22 hr in fasted mice. In vitro, salicylic acid (1.5 mM) had no or little effect on the formation of beta-oxidation products from [1-14C]octanoic or [1-14C]palmitoyl-L-carnitine, in the presence of ATP, carnitine (40 microM) and coenzyme A (40 microM), but decreased by 51% that from [1-14C]palmitic acid. In the latter system, increasing the concentrations of coenzyme A and carnitine to 200 microM suppressed the inhibitory effect of salicylic acid. Salicylic acid (1.5 mM) decreased by 80% the in vitro mitochondrial formation of palmitoyl-coenzyme A from [1-14C]palmitic acid and 10 microM coenzyme A; again, increasing the concentration of coenzyme A prevented inhibition. We conclude that salicylic acid decreases the mitochondrial activation and thus beta-oxidation of long chain fatty acids, presumably by sequestering extramitochondrial coenzyme A and possibly carnitine.

Administration of troleandomycin (0.5 mmol.kg-1 p.o. daily for 5 days) decreased by 61% and 36%, respectively, the estradiol and ethinylestradiol 2/4-hydroxylase activities of hepatic microsomes from male Sprague-Dawley rats killed 2 hr after the last dose. This decrease did not appear to be due to the in vivo formation of the inactive cytochrome P-450 p Fe(II)-metabolite complex, since disruption of this complex with potassium ferricyanide did not increase estrogen hydroxylase activities. Troleandomycin administration, however, essentially suppressed cytochrome P-450 UT-A (one of the P-450 forms involved in the hydroxylation of estrogens) and resulted in the appearance of cytochrome P-450 forms whose estradiol hydroxylase activity was inhibitable by troleandomycin in vitro. Similarly, troleandomycin (2 mM) inhibited by 60% estradiol and ethinylestradiol 2/4-hydroxylase activities in microsomes from dexamethasone-treated rats, although it had no inhibitory effect in microsomes from control rats. In contrast, erythromycin and roxithromycin (2 mM) exerted no inhibitory effect, even in microsomes from dexamethasone-treated rats. In vivo, these macrolides (0.5 mmol.kg-1 p.o. daily for 5 days) decreased moderately cytochrome P-450 UT-A levels and estradiol 2/4-hydroxylase activity, and did not modify ethinylestradiol 2/4-hydroxylase activity. We conclude that the administration of troleandomycin, but not that of erythromycin or roxithromycin, decreases ethinylestradiol 2/4-hydroxylase activity in male rat liver microsomes, as a possible consequence of decreased cytochrome P-450 UT-A levels and of the induction of glucocorticoid-responsive P-450 forms whose ethinylestradiol hydroxylase activity is inhibitable by troleandomycin.

The antihypertensive drug dihydralazine may, on rare occasions, cause immunoallergic hepatitis characterized by anti-cytochrome P450 (P450)1A2 autoantibodies. To understand the first steps leading to this immune reaction, we studied the covalent binding fo dihydralazine metabolites to microsomes from rat and human livers. Upon incubation with NADPH and microsomes, dihydralazine formed metabolites that reacted with heme (as evidenced by destruction of heme, formation of 445-nm light-absorbing complexes, and covalent binding of heme to P450 apoprotein) and covalently bound to microsomal proteins. Formation of these metabolites was shown (by NADPH dependence, induction by beta-naphthoflavone, and immunoinhibition by anti-P4501A antibodies) to be mediated by P4501A. Finally, these metabolites appeared to bind to P4501A2, which produced them. These results support the following scheme for the first steps of this autoimmune reaction: P4501A2 metabolizes dihydralazine into reactive metabolites that then bind to it, forming a neoantigen that triggers an immune response characterized by autoantibodies against P4501A2.