Glutathione-dependent factors and inhibition of rat liver microsomal lipid peroxidation

The effects of reduced glutathione (GSH) and glutathione disulfide (GSSG) on lipid peroxidation were investigated in rat liver microsomes containing deficient or adequate amounts of alpha-tocopherol (alpha-TH). Rates of formation of thiobarbituric acid reactive substances (TBARS) as well as rates of consumption of alpha-TH and O2 were decreased by GSH and were more pronounced in the NADPH-dependent assay system than in the ascorbate-dependent system. The GSH-dependent inhibition of lipid peroxidation was potentiated by GSSG in the NADPH-dependent assay system, but it had no effect in the nonenzymatic system. Diphenyliodonium chloride, an inhibitor of NADPH cytochrome P-450 reductase, completely prevented lipid peroxidation in the NADPH-dependent assay system whereas it had no effect on the ascorbate-dependent system. This is further evidenced by the fact that purified rat liver microsomal NADPH cytochrome P-450 reductase (EC 1.6.2.4) was inhibited approximately 24% and 52% by 5 mM GSH and 5 mM GSH + 2.5 mM GSSG, respectively. Glutathione disulfide alone had no effect on reductase activity. Similarly, other disulfides such as cystine, cystamine and lipoic acid were without effect on reductase activity. These results clearly delineate different mechanisms underlying the combined effects of GSH and GSSG on microsomal lipid peroxidation in rat liver. One mechanism involves recycling of microsomal alpha-TH by GSH during oxidative stress via a labile protein, ostensibly associated with "free radical reductase" activity. A second glutathione-dependent mechanism appears to be mediated through the inhibition of NADPH cytochrome P-450 reductase. The enhanced inhibition by GSH + GSSG of microsomal lipid peroxidation in the NADPH-dependent assay system suggests suppression of the initiation phase at the level of NADPH cytochrome P-450 reductase which is independent of microsomal alpha-TH.     

Microsomal function in biliary obstructed rats: effects of S-adenosylmethionine

BACKGROUND/AIMS: S-adenosylmethionine has been reported to have beneficial effects in the treatment of different chronic liver diseases and to protect against different hepatotoxic agents. The aim of this study was to investigate whether S-adenosylmethionine treatment might contribute to improved microsomal function in chronically biliary obstructed rats. METHODS: Secondary biliary cirrhosis was induced by 28 days of bile duct obstruction. Groups of control and cirrhotic animals received S-adenosylmethionine (10 mg/kg per day) through the experimental period. RESULTS: Bile duct obstruction resulted in a marked increase in lipid peroxidation levels and decreases in glutathione concentration, microsomal membrane fluidity, microsomal cytochrome P-450 content, NADPH-cytochrome P-450 reductase activity and the activities of the aniline hydroxylase, aminopyrine demethylase and ethoxycoumarin deethylase. Reductions in glutathione and cytochrome P-450 concentration were not corrected by S-adenosylmethionine, but lipid peroxidation, the decrease in the activities of the various microsomal monooxygenases and the reduction in microsomal membrane fluidity were partially prevented. A significant relationship was found between membrane fluidity and aniline hydroxylase, aminopyrine demethylase or ethoxycoumarin deethylase activities. CONCLUSIONS: S-adenosylmethionine administration partially preserves microsomal function. This effect could be associated to the protection of membrane function by restoring transmethylation reactions.     

Valproate treatment induces lipid globule accumulation with ultrastructural abnormalities of mitochondria in skeletal muscle

The association between an in vivo oxidative stress condition of the liver and hepatic porphyria during HCB intoxication is postulated. After 30 days of treatment, HCB (25 mg/kg b.w.) promotes an induction of microsomal cytochrome P450 system, increase in microsomal superoxide anion generation accompanied by increased levels of liver lipid peroxidation, as measured by the production of thiobarbituric acid reactants and by spontaneous visible chemiluminescence. Concomitantly, liver antioxidant defenses are slightly modified, with decreased activity of glutathione peroxidase, superoxide dismutase and glucose-6-phosphate dehydrogenase contributing to an oxidative stress condition of the liver. These liver biochemical alterations are closely related to increased levels of urinary coproporphyrin, plasma AST and ALT activities and to the onset of liver morphological lesions.     

Changes in lipid metabolism during oxidative stress caused by cobalt chloride injection

The cobalt chloride influence on rat liver and serum blood lipids and lipoproteins content and composition was investigated. The data obtained show that the cobalt chloride injection leads to the oxidative stress development and to activation of lipid transport and metabolism. It has been shown that there is system of lipids homeostasis in the organism and it is activated under conditions of oxidative stress. The system blood lipoproteins and liver lipoproteins was found to participate in metabolism adaptation on oxidative stress and in maintenance of biological membranes structure and functioning.     

Role of quinone reductase in in vivo ethanol metabolism and toxicity

The activation of microsomal glutathione S-transferase in oxidative stress was investigated by perfusing isolated rat liver with 1 mM tert-butyl hydroperoxide (t-BuOOH). When the isolated liver was perfused with t-BuOOH for 7 min and 10 min, microsomal, but not cytosolic, glutathione S-transferase activity was increased 1.3-fold and 1.7-fold, respectively, with a concomitant decrease in glutathione content. A dimer protein of microsomal glutathione S-transferase was also detected in the t-BuOOH-perfused liver. The increased microsomal glutathione S-transferase activity after perfusion with t-BuOOH was reversed by dithiothreitol, and the dimer protein of the transferase was also abolished. When the rats were pretreated with the antioxidant alpha-tocopherol or the iron chelator deferoxamine, the increases in microsomal glutathione S-transferase activity and lipid peroxidation caused by t-BuOOH perfusion of the isolated liver was prevented. Furthermore, the activation of microsomal GSH S-transferase by t-BuOOH in vitro was also inhibited by incubation of microsomes with alpha-tocopherol or deferoxamine. Thus it was confirmed that liver microsomal glutathione S-transferase is activated in the oxidative stress caused by t-BuOOH via thiol oxidation of the enzyme.     

Eosinophil infiltration in nonallergic chronic hyperplastic sinusitis with nasal polyposis (CHS/NP) is associated with endothelial VCAM-1 upregulation and expression of TNF-alpha

New Zealand White rabbits (6 males and 6 females) were fed a diet of high lipid peroxide content (peroxide value: 249.05 meq/kg fat) for 21 days. Twelve rabbits served as controls (peroxide value: 40.3 meq/kg fat). The lipid peroxide loading did not cause clinical signs. The rate of lipid peroxidation, as measured on the basis of thiobarbituric acid reactive substances (TBARS), was significantly (P < 0.05) higher in all of the investigated tissues, in the following order: liver > red blood cells (RBC) > blood plasma. Reduced and oxidised glutathione content was higher in the blood plasma (P < 0.01) and liver (P < 0.001) of rabbits exposed to the peroxide load. Lipid peroxide loading decreased the activity of glutathione peroxidase in the blood plasma, RBC haemolysate and liver and that of glutathione reductase in the liver. The amount of cytochrome P450 (both CO- and metyrapone-reduced) and the activity of cytochrome c (P450) oxidoreductase in the microsomal fraction of the liver homogenate were also lower in the group exposed to lipid peroxide load. Subchronic alimentary lipid peroxide loading in the presence of sufficiently high levels of antioxidants in the complete feed was found to increase the rate of lipid peroxidation and markedly lower the activities of both the glutathione and xenobiotic transforming enzyme systems without causing any clinical signs of toxicity.     

Biochemical and morphological studies on the effects of anthocyans and vitamin E on carbon tetrachloride induced liver injury

The effects of the natural antioxidants-anthocyans and vitamin E (in a solubilized pharmaceutical form) on carbon tetrachloride-induced liver injury in rats are studied. The changes in the activity of serum transaminases (ALAT and ASAT), the content of the reduced glutathione and cytochrome P-450 as well as the intensity of the processes of lipid peroxidation are assessed. The anthocyans exert a protective effect comparable to that of vitamin E on liver cells. The favorable effects of the combination of the antioxidants on the content of the reduced glutathione and on the processes of lipid peroxidation are more intensely expressed. The morphological changes occurring in hepatocytes correlate with the results of the biochemical studies. It is evident that both substances have a marked hepatoprotective activity.     

Particular ability of cytochrome P-450 CYP3A to reduce glyceryl trinitrate in rat liver microsomes: subsequent formation of nitric oxide

Glyceryl trinitrate was denitrated in rat hepatic subcellular fractions, with formation of glyceryl dinitrates and glyceryl mononitrates. Among differently treated-rat liver microsomes, the highest microsomal activity was obtained under anaerobic conditions with microsomal preparations from dexamethasone-treated rats and NADPH. The reaction was inhibited by O2, CO, miconazole, dihydroergotamine and troleandomycin showing that it was catalyzed by cytochrome P-450 CYP3A isoforms. The formation of a transient cytochrome P-450 Fe(II)-NO complex during this reaction was shown by visible spectroscopy. The cytosolic activity was shown to be dependent on glutathione and glutathione transferase and was not inhibited by dioxygen. In the hepatic 9000 x g supernatant containing both NADPH and cytochrome P-450 and glutathione and glutathione transferase, the cytochrome P-450-dependent reaction accounts for 30-40% of the total denitration activity observed under anaerobic conditions, using 100 microM GTN.     

Ascorbic acid and hepatic drug metabolism

Previous in vivo studies indicate that hepatic microsomal drug metabolism decreases in ascorbic acid deficiency and is augmented when high supplements of the vitamin are given to guinea pigs. Kinetic studies with O-demethylase indicate no significant change in the apparent Km of p-nitroanisole in normal, ascorbic acid-deficient animals, or in animals given high supplements of ascorbic acid. The decrease in drug metabolism activity caused by ascorbic acid deficiency is not due to increased lipid peroxidation, nor was phosphatidyl choline significantly altered quantitatively or qualitatively in microsomes from ascorbic acid-deficient animals. Microsomal cytochrome P-450 prepared from ascorbic acid-deficient livers is less stable to sonication, dialysis and treatment with metal chelators. The decrease in cytochrome P-450 and O-demethylase activity associated with dialysis could be prevented by the addition of ascorbic acid. The molar ratio of microsomal ascorbic acid to cytochrome P-450 (plus P-420) is in the order of 2:1. This ratio is maintained during ascorbic acid deficiency in liver and adrenal tissue, during dialysis, on storage and with a partial purification of the cytochrome, which suggests a close association between ascorbic acid and the cytochrome. In addition, ascorbic acid protects cytochrome P-450 and aniline hydroxy lase activity from inhibition by ferrous iron chelators such as alpha, alpha'-dipyridyl. The chelator binds to cytochrome P-450 and prevents formation of the reduced cytochrome P-450-CO spectrum; it in turn gives a reduced spectrum with the cytochrome at 450 nm. These studies suggest that there is an interaction between ascorbic acid and cytochrome P-450 involving the reduced form of the heme iron.     

The effect of certain vitamin deficiencies on hepatic drug metabolism

There is increasing evidence that the liver microsomal drug metabolizing system is affected by various vitamins such as ascorbic acid, riboflavin, and alpha-tocopherol. In regard to ascorbic acid deficiency there is a decrease in the quantity of hepatic microsomal electron transport components such as cytochrome P-450 and NADPH-cytochrome P-450 reductase, as well as decreases in a variety of drug enzyme reactions such as N-demethylation, O-demethylation, and steroid hydroxylation. In addition, young animals given high supplements of vitamin C have increased quantities of electron transport components and overall drug metabolism activities. Kinetic studies indicate no change in the apparent Km of N-demethylase, O-demethylase or hydroxylase for drug substrates in animals depleted or given high amounts of the vitamin. However, there are qualitative changes in both type I and II substrate-cytochrome P-450 binding. Ascorbic acid is not involved in microsomal lipid peroxidation or in any qualitative or quantitative change in phosphatidylcholine. Replenishing vitamin C-deficient animals with ascorbic acid required 3 to 7 days for the electron transport components and drug metabolism activities to return to normal levels. Induction with phenobarbital and 3-methylcholanthrene is not impaired in the deficient animal since drug metabolism activities are induced to the same extent as normal controls; however, the administration of delta-aminolevulinic acid, a precursor of heme synthesis, to deficient animals caused an increase in the quantity of cytochrome P-450. The effects of riboflavin deficiency on electron transport components and drug metabolism activities have been noted only in adult animals after prolonged periods of deficiency. Decreases in drug metabolism activities occur with both type I (aminopyrine and ethylmorphine) and type II (aniline) substrates. As was found with ascorbic acid deficiency, drug enzyme induction occurred to the same extent with phenobarbital in deficient and normal animals. In addition, it required from 10 to 15 days for the drug metabolism activities to return to normal levels when deficient animals were replenished with riboflavin. The effect of vitamin E on drug metabolism is specific in N-demethylase activities decrease while O-demethylase activities are not affected in the deficient state. This vitamin differs from ascorbic acid and riboflavin in that several laboratories have reported no quantitative decrease in cytochrome P-450, although there are some reports that it and delta-aminolevulinic acid dehydratase are lowered quantity of cytochrome in E-deficient animals. The effect of vitamin E, if any, on the P-450 is unresolved; an important question that requires further clarification. As with ascorbic acid there is no difference in the apparent Km of N-demethylase enzymes for varous substrates and the protective effect of vitamin E does not appear to be one of an antioxidant inhibiting microsomal lipid peroxidation.     

Androgen receptor dependent and independent activities of testosterone on hepatic microsomal drug metabolism

Administration of testosterone for 6 days to intact female and castrate male BALB/cJ mice stimulated hepatic microsomal ethylmorphine N-demethylase activity and cytochrome P-450 content by 50-75%. Testosterone also stimulated hepatic microsomal NADPH-oxidase activity, but to a lesser degree. To probe the mechanism of this effect of androgens, two antiandrogens (cyproterone acetate and flutamide) were employed. Since cyproterone acetate was a potent stimulator of hepatic microsomal ethylmorphine N-demethylase activity and cytochrome P-450 content, no antiandrogenic activity of this steroid could be detected. By contrast, flutamide alone had little effect on either ethylmorphine N-demethylase activity or cytochrome P-450 content. However, this drug effectively blocked the stimulatory effects of testosterone on ethylmorphine N-demethylase activity and cytochrome P-450 content but not on NADPH-oxidase activity. This effect was not species specific, since flutamide also prevented androgen stimulation of ethylmorphine metabolism in adult castrate and prepubertal male Fisher rats. The testosterone-induced increase of hepatic weight and microsomal protein content was not affected by the administration of flutamide. The observations are consistent with the hypothesis that androgens have two distinct effects on the liver. First, testosterone may act as a general, nonspecific stimulant of liver weight and microsomal protein content which is independent of the androgen receptor. Secondly, testosterone action in the liver may be expressed via an androgen-specific or androgen receptor-dependent mechanism which controls, in part, the cytochrome P-450-dependent demethylase system.     

Liver microsomal drug-metabolizing enzyme system: functional components and their properties

The liver microsomal drug-metabolizing enzyme system consists of two protein components, cytochrome P-450 and NADPH-cytochrome c reductase, and a lipid, phosphatidylcholine. Cytochrome P-450 serves as the binding site for oxygen and substrate while the reductase acts as an electron carrier shuttling electrons from NADPH to cytochrome P-450. The phospholipid facilitates the transfer of electrons from NADPH-cytochrome c reductase to cytochrome P-450 but itself is not an electron carrier. Different cytochromes P-450 and P-448 have been purified; the spectral, catalytic, and immunological properties as well as the molecular weight (determined by SDS-gel electrophoresis) of all these hemeproteins differ from one another. The presence of multiple cytochrome P-450s may explain the species, strain, age, tissue, and sex differences as well as the effect of inducers and nutritional status in mammlian drug metabolism.     

Alterations of antioxidant enzymes and oxidative damage to macromolecules in different organs of rats during aging

Oxygen free radicals have been hypothesized to play an important role in the aging process. To investigate the correlation between the oxidative stress and aging, we have determined the levels of oxidative protein damage and lipid peroxidation in the brain and liver, and activities of antioxidant enzymes in the brain, liver, heart, kidney, and serum from the Fisher 344 rats at ages of 1, 6, 12, 18, and 24 months. The results showed that the level of oxidative protein damage (measured as carbonyl content) in the brain and liver was significantly higher in older animals than in young animals. No statistical difference was observed in the lipid peroxidation of the liver and brain between young and old animals. The activities of antioxidant enzymes in most tissues displayed an age-dependent decline. Superoxide dismutases in the heart, kidney, and serum, glutathione peroxidase activities in the serum and kidney, and catalase activities in the brain, liver, and kidney, significantly decreased during aging. Cytochrome c oxidase, an enzyme involved in electron transport in mitochondria, initially increased, but subsequently decreased in the aged brain, whereas no significant alteration was observed in the liver mitochondrial antioxidant enzymes. The present studies suggest that the accumulation of oxidized proteins during aging is most likely to be linked with an age-related decline of antioxidant enzyme activities, whereas lipid peroxidation is less sensitive to predict the aging process.     

Inhibition of microsomal lipid peroxidation and monooxygenase activities by eugenol

Previously we reported that eugenol (4-allyl-2-methoxyphenol) inhibits non-enzymatic peroxidation in liver mitochondria (E. Nagababu and N. Lakshmaiah, 1992, Biochemical Pharmacology. 43, 2393-2400). In the present study, we examined the effect of eugenol on microsomal mixed function oxidase mediated peroxidation using Fe+3-ADP-NADPH, carbon tetrachloride (CCL4)-NADPH and cumene hydroperoxide (CumOOH) systems. In the presence of eugenol the formation of thiobarbituric acid reactive substances (TBARS) was decreased in all the systems (IC50 values: 14 microM for Fe+3-ADP-NADPH, 4.0 microM for CCl4-NADPH and 15 microM for CumOOH). Oxygen uptake was also inhibited to a similar extent with Fe+3-ADP-NADPH and CumOOH systems. A comparative evaluation with other antioxidants showed that in Fe+3-ADP-NADPH and CumOOH systems, the antioxidant efficacy was in the order: butylated hydroxytoluene (BHT) > eugenol > alpha-tocopherol, while in CCl4-NADPH system the order was alpha-tocopherol > BHT > eugenol. Time course of inhibition by eugenol indicated interference in initiation as well as propagation of peroxidation. Eugenol did not inhibit cytochrome P-450 reductase activity but it inhibited P-450 - linked monooxygenase activities such as aminopyrine-N-demethylase, N-nitrosodimethylamine demethylase, benzo(a)pyrene hydroxylase and ethoxyresorufin-O-deethylase to different extents. However, CumOOH supported monooxygenases (aminopyrine-N-demethylase and benzo(a)pyrene hydroxylase) required much higher concentrations of eugenol for inhibition. The concentration of eugenol required to inhibit monooxygenase activities was more than that required to inhibit peroxidation in all the systems. Eugenol elicited type 1 changes in the spectrum of microsomal cytochrome P-450. These results suggest that the inhibitory effect of eugenol on lipid peroxidation is predominantly due to its free radical quenching ability. Eugenol significantly protected against the degradation of cytochrome P-450 during lipid peroxidation with all the systems tested. These findings suggest that eugenol has the potential to be used as a therapeutic antioxidant. Further evaluation may throw more light on this aspect.     

Prevention of carbon tetrachloride-induced lipid peroxidation in liver microsomes from dehydroepiandrosterone-pretreated rats

Dehydroepiandrosterone (DHEA), a lipid soluble steroid, administered to rats (100 mg/kg b.wt) by a single intraperitoneal injection, increases to twice its normal level in the liver microsomes. Microsomes so enriched become resistant to lipid peroxidation induced by incubation with carbon tetrachloride in the presence of a NADPH-regenerating system: also the lipid peroxidation-dependent inactivation of glucose-6-phosphatase and gamma-glutamyl transpetidase due to the haloalkane are prevented. Noteworthy, the liver microsomal drug-metabolizing enzymes and in particular the catalytic activity of cytochrome P450IIE1, responsible for the CCl4-activation, are not impaired by the supplementation with the steroid. Consistently, in DHEA-pretreated microsomes the protein covalent binding of the trichloromethyl radical (CCl3 degrees), is similar to that of not supplemented microsomes treated with CCl4. It thus seems likely that DHEA protects liver microsomes from oxidative damage induced by carbon tetrachloride through its own antioxidant properties rather than inhibiting the metabolism of the toxin.     

Effect of phenobarbital treatment on characteristics determining susceptibility to oxidants of homogenates, mitochondria and microsomes from rat liver

This study was designed to investigate the possible oxidative changes associated with alterations in cytochrome P450 levels in rat liver. Accordingly, extent of peroxidative processes, cytochrome and antioxidant content, capacity to face an oxidative stress were determined in liver microsomes, mitochondria, and homogenates from normal and phenobarbital (PB)-treated rats. Liver content of microsomal and mitochondrial proteins was also determined by the values of the activities of marker enzymes (glucose-6-phosphatase and cytochrome oxidase, respectively) in liver homogenate and in two cellular fractions. The increase in the liver content of microsomal and mitochondrial proteins indicated that PB caused proliferation of both smooth endoplasmic reticulum and mitochondrial population. Treatment with PB also gave rise to a general increase in peroxidative reactions (evaluated measuring malondialdehyde and hydroperoxides (HPs)), in the different cell compartments, even though HPs were not found significantly increased in mitochondrial fraction. The increase in peroxidative processes was associated with significant decreases in antioxidant concentration (expressed in terms of equivalent concentration of an antioxidant, such as the desferrioxamine), in all preparations from PB-treated rats. The response to oxidative stress in vitro (evaluated determining the parameters characterizing light emission from preparations stressed with sodium perborate) showed a substantial PB-induced increase in the susceptibility to oxidative challenge only in liver homogenate. The lack of changes in the mitochondrial preparations is likely due to decrease in concentration of both free radical producing species and antioxidants. The lack of changes in microsomal fraction is apparently in contrast with its lower oxidant capacity and higher content of cytochromes which are able to determine sensitivity to pro-oxidants. However, it could be due to the ability of cytochrome P450 to interact with the active oxygen species formed at its active center.     

Effects of motorcycle exhaust on cytochrome P-450-dependent monooxygenases and glutathione S-transferase in rat tissues

The effects of motorcycle exhaust (ME) on cytochrome P-450 (P-450)-dependent monooxygenases were determined using rats exposed to the exhaust by either inhalation, intratracheal, or intraperitoneal administration. A 4-wk ME inhalation significantly increased benzo[a]pyrene hydroxylation, 7-ethoxyresorufin O-deethylation, and NADPH-cytochrome c reductase activities in liver, kidney, and lung microsomes. Intratracheal instillation of organic extracts of ME particulate (MEP) caused a dose- and time-dependent significant increase of monooxygenase activity. Intratracheal treatment with 0.1 g MEP extract/kg markedly elevated benzo[a]pyrene hydroxylation and 7-ethoxyresorufin O-deethylation activities in the rat tissues 24 h following treatment. Intraperitoneal treatment with 0.5 g MEP extract/kg/d for 4 d resulted in significant increases of P-450 and cytochrome b5 contents and NADPH-cytochrome c reductase activity in liver microsomes. The intraperitoneal treatment also markedly increased monooxygenases activities toward methoxyresorufin, aniline, benzphetamine, and erythromycin in liver and benzo[a]pyrene and 7-ethoxyresorufin in liver, kidney, and lung. Immunoblotting analyses of microsomal proteins using a mouse monoclonal antibody (Mab) 1-12-3 against rat P-450 1A1 revealed that ME inhalation, MEP intratracheal, or MEP intraperitoneal treatment increased a P-450 1A protein in the hepatic and extrahepatic tissues. Protein blots analyzed using antibodies to P-450 enzymes showed that MEP intraperitoneal treatment caused increases of P-450 2B, 2E, and 3A subfamily proteins in the liver. The ME inhalation, MEP intratracheal, or MEP intraperitoneal treatment resulted in significant increases in glutathione S-transferase activity in liver cytosols. The present study shows that ME and MEP extract contain substances that can induce multiple forms of P-450 and glutathione S-transferase activity in the rat.     

Bronchoalveolar lavage studies in children without parenchymal lung disease: cellular constituents and protein levels

The hepatotoxicity of acetaminophen overdose depends on the metabolic activation to a toxic reactive metabolite by the hepatic mixed function oxidases. There is evidence that an increase in cytosolic Ca2+ is involved in acetaminophen hepatotoxicity. The effects of the Ca2+-antagonists nifedipine (NF), verapamil (V), diltiazem (DL) and of the calmodulin antagonist trifluoperazine (TFP) on the activity of some drug-metabolizing enzyme systems, lipid peroxidation and acute acetaminophen toxicity were studied in male albino mice. No changes in the drug-metabolizing enzyme activities studied and in the cytochrome P-450 and b5 contents were observed 1 h after oral administration of V (20 mg/kg). DL (70 mg/kg) and TFP (3 mg/kg). NF (50 mg/kg) increased cytochrome P-450 content, NADPH-cytochrome c reductase and ethylmorphine-N-demethylase activities. DL and TFP significantly decreased lipid peroxidation. NF, V, DL and TFP administered 1 h before acetaminophen (700 mg/kg orally) increased the mean survival time of animals. A large increase of serum aspartate aminotransferase(AST), and liver weight and depletion of liver reduced glutathione (GSH) occurred in animals receiving toxic acetaminophen dose. NF, V and DL prevented and TFP decreased the acetaminophen-induced hepatic damage measured both by plasma AST and by liver weight. NF, V, DL and TFP changed neither the hepatic GSH level nor the GSH depletion provoked by the toxic dose of acetaminophen. This suggests that V, DL and TFP do not influence the amount of the acetaminophen toxic metabolite formed in the liver. The possible mechanism of the protective effect of NF, V, DL and TFP on the acetaminophen-induced toxicity is discussed.     

Effects of erythromycin on hepatic drug-metabolizing enzymes in humans

In rats, erythromycin has been shown to induce microsomal enzymes and to promote its own transformation into a metabolite which forms an inactive complex with reduced cytochrome P-450. To determine whether similar effects also occur in humans, we studied hepatic microsomal enzymes from six untreated patients and six patients treated with erythromycin propionate, 2 g per os daily for 7 days. In the treated patients, NADPH-cytochrome c reductase activity was increased; the total cytochrome P-450 concn was also increased but part of the total cytochrome P-450 was complexed by an erythromycin metabolite. The concn of uncomplexed (active) cytochrome P-450 was not significantly modified and the activity of hexobarbital hydroxylase remained unchanged. We also measured the clearance of antipyrine in six other patients; this clearance was not significantly decreased when measured again on the seventh day of the erythromycin propionate treatment. We conclude that the administration of erythromycin propionate induces microsomal enzymes and results in the formation of an inactive cytochrome P-450-metabolite complex in humans. However, the concn of uncomplexed (active) cytochrome P-450 and tests for in vitro and in vivo drug metabolism were not significantly modified.     

Modulation of cytochrome P-450-dependent monooxygenases, glutathione and glutathione S-transferase in rat liver by geniposide from Gardenia jasminoides

Geniposide is an iridoid glycoside extracted from the fruits of Gardenia jasminoides, which are used as a food colorant and as a traditional Chinese medicine for treatment of hepatic and inflammatory diseases. The effects of geniposide and G. jasminoides fruit crude extract on liver cytochrome P-450 (P-450)-dependent monooxygenases, glutathione and glutathione S-transferase were investigated using rats treated orally with the iridoid glycoside (0.1 g/kg body weight/day) or the fruit crude extract (2 g/kg/day) for 4 days. The treatments decreased serum urea nitrogen level but increased liver to body weight ratio, total hepatic glutathione content and hepatic cytosolic glutathione S-transferase activity. Treatments with geniposide and G. jasminoides decreased P-450 content, benzo[a]pyrene hydroxylation, 7-ethoxycoumarin O-deethylation, and erythromycin N-demethylation activities in liver microsomes without affecting aniline hydroxylation activity. The natural products had no effect on glutathione content and monooxygenase activities in kidney microsomes. Immunoblotting analyses of liver microsomal proteins using mouse monoclonal antibody 2-13-1 to rat P4503A1/2 revealed that geniposide and G. jasminoides crude extract decreased the intensity of a P4503A-immunorelated protein. Protein blots probed with mouse monoclonal antibody 1-12-3 to rat P4501A1 and rabbit polyclonal antibody against human P4502E1 showed that both treatments had little or no effect on P4501A and 2E proteins. The present findings demonstrate that geniposide from G. jasminoides has the ability to inhibit a P4503A monooxygenase and increase glutathione content in rat liver.