Induction of cytochrome P450 2E1 (CYP2E1) by ethanol appears to be one of the central pathways by which ethanol generates a state of oxidative stress. Glutathione (GSH) is critical in preserving the proper cellular redox balance and for its role as a cellular protectant. The goal of the present study was to characterize the GSH homeostasis in human hepatocarcinoma cells (HepG2-E47 cells) that overexpress CYP2E1. Toxicity in the E47 cells was markedly enhanced after GSH depletion by buthionine sulfoximine (BSO) treatment. The antioxidant trolox partially prevented the apoptosis and necrosis, while diallylsulfide, a CYP2E1 inhibitor, was fully protective. Damage to mitochondria appears to play a role in the CYP2E1- and BSO-dependent toxicity. CYP2E1-overexpressing cells showed increases in total GSH levels, GSH synthetic rate and in gamma-glutamylcysteine synthetase (GCS) mRNA. This GCS increase was due to transcriptional activation of the GCS gene and could be blocked by certain antioxidants. Activity, protein and mRNA levels for other antioxidants such as catalase, alpha- and microsomal glutathione transferases were also increased in the E47 cells. Up-regulation of these antioxidant genes may reflect an adaptive mechanism to remove CYP2E1-derived oxidants. These oxidants are diffusable and were able to elevate collagen type I protein in a co-culture system consisting of the E47 cells + rat hepatic stellate cells. Such interactions between CYP2E1, mitochondria and altered GSH homeostasis, and elevation of collagen levels, may play a role in alcohol-induced liver injury.

Animals↗

PubMedDate not supplied

Role of cytochrome P-450 IIE1 and catalase in the oxidation of acetonitrile to cyanide.

Acetonitrile is a common industrial solvent and laboratory agent, which can be toxic if ingested. The toxicity of nitriles appears to be due to the production of cyanide, and detailed studies by Freeman and Hayes [(1988) Biochem. Pharmacol. 37, 1153-1159; (1987) Fundam. Appl. Toxicol. 8, 263-271] have shown that microsomes oxidize acetonitrile to cyanide. Treatment of rats with inducers of cytochrome P-450 IIE1 such as pyrazole, 4-methylpyrazole, and ethanol resulted in a 4- to 5-fold increase in cyanide production from acetonitrile by isolated microsomes. Phenobarbital treatment had a small stimulatory effect, whereas 3-methylcholanthrene treatment decreased microsomal oxidation of acetonitrile. Pyrazole treatment increased Vmax per milligram of microsomal protein and per nanomole of P-450 but did not affect the apparent km for acetonitrile, whereas the 4-methylpyrazole treatment increased Vmax and the apparent affinity for acetonitrile. Cyanide production was inhibited by carbon monoxide as well as by substrates and compounds that interact with the P-450 IIE1 isozyme such as ethanol, 2-butanol, DMSO, and 4-methylpyrazole. Oxidation of acetonitrile to cyanide by microsomes from rats treated with pyrazole or 4-methylpyrazole was nearly completely inhibited by anti-P-450 3a IgG. These results implicate a role for P-450 in the oxidation of acetonitrile to cyanide and suggest that P-450 IIE1 may be an especially effective catalyst for this oxidation. Acetonitrile oxidation was not affected by hydroxyl radical scavengers or by desferrioxamine, indicating no role for hydroxyl radicals in the overall mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetonitriles↗

PubMedDate not supplied

Immunoblastic lymphadenopathy presenting as respiratory insufficiency.

The clinical course of a patient is described who experienced respiratory insufficiency as the primary manifestation of immunoblastic lymphadenopathy. Respiratory symptoms preceded the enlargement of lymph nodes, improved spontaneously at first and then after steroid therapy. Subsequently these symptoms reappeared concurrent with lymph node enlargement. Hypergammaglobulinemia was noted with marked elevation of polyclonal immunoglobulin M.

Adult↗

PubMedDate not supplied

Ethanol oxidation by hydroxyl radicals: role of iron chelates, superoxide, and hydrogen peroxide.

Oxygen-derived free radicals such as the hydroxyl radical (.OH) have been shown to mediate the oxidation of ethanol by a variety of oxy radical-generating systems. Among these are microsomal electron transport systems (both intact and purified, reconstituted systems), the coupled oxidation of hypoxanthine or xanthine by xanthine oxidase, and the model iron-ascorbate system. The sequence of reactions leading to the oxy radical-dependent oxidation of ethanol as well as other hydroxyl radical-scavenging agents by these various systems is believed to proceed through the formation of a common intermediate, namely, hydrogen peroxide (H2O2), after dismutation of the superoxide anion radical (O2-.). The presence of iron, especially chelated iron, greatly enhances the production of .OH by serving as an oxidant for O2-. or a reductant for H2O2. Experiments were carried out to evaluate the role of iron, the chelating agent, O2-., and H2O2 in the oxidation of ethanol by a variety of in vitro systems (chemical, enzymatic, and intact membrane bound) that can produce oxy radicals via different mechanisms. The generation of .OH by all the systems studied was sensitive to catalase, which indicates that H2O2 is the precursor of .OH. Superoxide radical appears to be the reducing agent in the hypoxanthine-xanthine oxidase system, indicating an iron-catalyzed Haber-Weiss reaction. In the ascorbate, reductase, and microsomal systems, superoxide radical does not appear to be the reducing agent. However, superoxide radical probably is the precursor of H2O2. While iron plays an important role in the production of .OH by the various systems, the effect of iron depends on the nature of the iron chelate.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMedDate not supplied

Interaction of pyrazole and 4-methylpyrazole with hepatic microsomes: effect on cytochrome P-450 content, microsomal oxidation of alcohols, and binding spectra.

Microsomes isolated from rats treated with either pyrazole or 4-methylpyrazole, potent inhibitors of alcohol dehydrogenase, catalyzed the oxidation of ethanol and 2-butanol at rates 2-3-fold higher than saline controls. Time course experiments and dose-response experiments indicated that an increase in the microsomal oxidation of alcohols could be observed 24 hr after a single treatment with 200 mg/kg body weight of either pyrazole or 4-methylpyrazole, and after 2 or 3 days of treatment with 50 mg/kg of either of these compounds. The pyrazole treatment did not change the activity of NADPH-cytochrome P-450 reductase, the content of cytochrome P-450, or the oxidation of aminopyrine. Hence, microsomal oxidation of alcohols was increased by the pyrazole treatment whether results were expressed "per mg of protein" or "per nmol of P-450." Microsomes from the pyrazole-treated rats displayed an increase in binding spectrum with ethanol as the substrate as compared to controls, as well as type 2 binding spectrum with dimethyl sulfoxide and 2-butanol. These results suggest the possibility that pyrazole may induce an alcohol-preferring P-450 isozyme. By contrast, the 4-methylpyrazole treatment, besides increasing the oxidation of alcohols, also increased the oxidation of aminopyrine and the content of cytochrome P-450. The increase in the oxidation of alcohols and aminopyrine was primarily due to the increase in content of P-450 produced by the 4-methylpyrazole treatment. Binding spectra with dimethyl sulfoxide and 2-butanol were also observed after 4-methylpyrazole treatment; however, the 2-butanol-binding spectrum was a modified type 1 spectrum, not type 2.(ABSTRACT TRUNCATED AT 250 WORDS)

Alcohols↗

PubMedDate not supplied

Mitochondrial catalase and oxidative injury.

Mitochondria dysfunction induced by reactive oxygen species (ROS) is related to many human diseases and aging. In physiological conditions, the mitochondrial respiratory chain is the major source of ROS. ROS could be reduced by intracellular antioxidant enzymes including superoxide dismutase, glutathione peroxidase and catalase as well as some antioxidant molecules like glutathione and vitamin E. However, in pathological conditions, these antioxidants are often unable to deal with the large amount of ROS produced. This inefficiency of antioxidants is even more serious in mitochondria, because mitochondria in most cells lack catalase. Therefore, the excessive production of hydrogen peroxide in mitochondria will damage lipid, proteins and mDNA, which can then cause cells to die of necrosis or apoptosis. In order to study the important role of mitochondrial catalase in protecting cells from oxidative injury, a HepG2 cell line overexpressing catalase in mitochondria was developed by stable transfection of a plasmid containing catalase cDNA linked with a mitochondria leader sequence which would encode a signal peptide to lead catalase into the mitochondria. Mitochondria catalase was shown to protect cells from oxidative injury induced by hydrogen peroxide and antimycin A. However, it increased the sensitivity of cells to tumor necrosis factor-alpha-induced apoptosis by changing the redox-oxidative status in the mitochondria. Therefore, the antioxidative effectiveness of catalase when expressed in the mitochondrial compartment is dependent upon the oxidant and the locus of ROS production.

Apoptosis↗

PubMedDate not supplied

Oxidation of ethylene glycol to formaldehyde by rat liver microsomes. Role of cytochrome P-450 and reactive oxygen species.

Rat liver microsomes oxidized ethylene glycol to formaldehyde in a NADPH-dependent, carbon monoxide-sensitive manner. Formaldehyde production was inhibited by substrates and ligands for cytochrome P-450 such as aniline, p-nitrophenol, pyrazole, and 4-methylpyrazole, and inhibitors such as tryptamine, cimetidine, and miconazole. The apparent Km for ethylene glycol was about 25 mM and the apparent Vmax was about 6 nmol/min/mg protein. Microsomes isolated from rats treated with pyrazole or 4-methylpyrazole to induce cytochrome P-450IIE1 oxidized ethylene glycol at rates which were about twice those found with control microsomes or microsomes isolated from rats treated with phenobarbital or 3-methylcholanthrene, although significant rates were found with all microsomal preparations. Antibody raised against the pyrazole-induced P-450IIE1 inhibited formaldehyde production from ethylene glycol in microsomes from pyrazole-treated rats. H2O2 itself did not oxidize ethylene glycol to formaldehyde; however, the microsomal reaction was inhibited by catalase or glutathione plus glutathione peroxidase and was stimulated by added H2O2 in the presence of NADPH. Nonheme iron also appeared to be required for ethylene glycol oxidation in view of the inhibition of formaldehyde production by desferrioxamine, EDTA, and DTPA. Microsomal oxidation of ethylene glycol was not sensitive to superoxide dismutase, hydroxyl radical scavengers, or Trolox, suggesting that the oxidant derived from H2O2 and iron and responsible for the production of formaldehyde from ethylene glycol was not superoxide, hydroxyl radical, or lipid hydroperoxide. These results suggest that ethylene glycol is oxidized to formaldehyde by an oxidant derived from H2O2 and nonheme iron, and that cytochrome P-450 may function to generate the H2O2 and to catalyze reduction of the nonheme iron.

Animals↗

PubMedDate not supplied

Comparison of the interaction of pyrazole and its metabolite 4-hydroxypyrazole with rat liver microsomes.

Pyrazole is known to interact with and to induce cytochrome P-450 IIE1. Since pyrazole is oxidized by rat liver microsomes to 4-hydroxypyrazole, and several of the actions of pyrazole have been ascribed to its metabolite, experiments were conducted to evaluate the interactions of 4-hydroxypyrazole with microsomes, and to compare these to pyrazole itself. Rats were injected with doses of 4-hydroxypyrazole ranging from 2 to 100 mg/kg body weight/day for 2 days. A slight increase of total cytochrome P-450 was observed at low doses, followed by a decrease at higher concentrations. NADPH-cytochrome P-450 reductase activity was not affected. The oxidation of aniline or dimethylnitrosamine was increased about 50% by the 4-hydroxypyrazole treatment; however, this extent of increase was much less than that produced by pyrazole treatment. In vitro, 4-hydroxypyrazole produced a type II binding spectrum with microsomes, with a peak at about 425 nm and a trough at about 395 nm. The affinity for 4-hydroxypyrazole was increased from a value of about 0.60 mM in control microsomes to a value of about 0.40 mM in microsomes from pyrazole-treated rats. These values are 2-fold greater than those observed with pyrazole as the ligand. 4-Hydroxypyrazole inhibited the microsomal oxidation of ethanol; kinetics of inhibition were mixed. The apparent KI for 4-hydroxypyrazole inhibition of ethanol oxidation by microsomes was about 4 mM, which is about an order of magnitude greater than that for pyrazole. The in vivo and in vitro interactions of 4-hydroxypyrazole with microsomes appear to be similar to those described for pyrazole; however, these interactions are considerably less effective than those of the parent drug, pyrazole. Thus, although some actions of pyrazole may be due to the metabolite 4-hydroxypyrazole, it appears that the induction of P-450 IIE1 and the in vitro interactions of pyrazole with microsomes is not likely to be mediated by prior metabolism of pyrazole to 4-hydroxypyrazole.

Aniline Hydroxylase↗

PubMedDate not supplied

Sensitivity of the rat liver microsomal oxidation of pyrazole to antibody raised against the ethanol-inducible rabbit liver cytochrome P-450 isozyme.

Pyrazole is oxidized to 4-hydroxypyrazole by rat liver microsomes in a cytochrome P-450-dependent reaction and this oxidation can be increased by prior treatment of rats with pyrazole, 4-methylpyrazole, or chronic ethanol feeding. The induction pattern suggests that pyrazole may be an effective substrate for oxidation by P-450 IIE.1. This P-450 isozyme is recognized by antibody (anti-3a IgG) raised against the ethanol-inducible P-450 in rabbits. Experiments were carried out to evaluate the ability of anti-3a IgG to inhibit pyrazole oxidation by microsomes from controls and from rats treated with inducers of P-450 IIE.1. Immunoblots with anti-3a IgG or with the anti-pyrazole P-450 IgG were identical and indicated increased staining of the pyrazole P-450 with microsomes from rats treated with pyrazole, 4-methylpyrazole, or ethanol, relative to saline controls; very little staining occurred with microsomes from pair-fed controls or phenobarbital-treated rats. Rates of pyrazole oxidation were highest with microsomes from rats treated with the inducers of P-450 IIE.1 and lowest with pair-fed controls or rats treated with phenobarbital. Anti-3a IgG produced about a 60% decrease of pyrazole oxidation in microsomes from rats treated with inducers of P-450 IIE.1 and about a 25% decrease with the saline controls; no inhibition was found with microsomes from the phenobarbital-treated rats. The anti-3a IgG-resistant rate of pyrazole oxidation was similar with all the microsomal preparations, and was not due to interaction of pyrazole with hydroxyl radicals.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMedDate not supplied

Oxidation of the alcohol dehydrogenase inhibitor pyrazole to 4-hydroxypyrazole by microsomes. Effect of cytochrome P-450 inducing agents.

Pyrazole and its analogues are widely used to inhibit alcohol dehydrogenase and to block the metabolism of ethanol. Experiments were conducted to demonstrate that pyrazole is oxidized to 4-hydroxypyrazole by isolated rat liver microsomes. A HPLC procedure employing UV and electrochemical detection was developed for the separation and quantitation of 4-hydroxypyrazole. Pyrazole metabolism was NADPH-dependent, sensitive to inhibition by carbon monoxide, and was depressed in the presence of other substrates such as aniline or ethanol. Prior treatment of rats with either pyrazole or 4-methylpyrazole resulted in an increase in pyrazole oxidation to 4-hydroxypyrazole by microsomes. Increases were observed when rates were expressed either per mg of protein or per nmol of P-450. Microsomes from pyrazole- and 4-methylpyrazole-treated rats had Km values for pyrazole of about 0.29 and 0.14 mM, respectively, and Vmax values of about 0.5 and 0.7 nmol of 4-hydroxypyrazole per min per mg of protein, respectively. Chronic consumption of ethanol for 24 days resulted in an increase in pyrazole oxidation (per mg of protein and per nmol of P-450) as compared to pair-fed controls. By contrast, phenobarbital treatment lowered the rate of production of 4-hydroxypyrazole. Treatment with 3-methylcholanthrene resulted in an increase in pyrazole oxidation when rates were expressed per mg of protein, but not per nmol of P-450. These results show that pyrazole is oxidized to 4-hydroxypyrazole by microsomes in a P-450-dependent manner and that this metabolism can be increased by certain inducers, e.g. pyrazole, 4-methylpyrazole, and chronic ethanol treatment.

Alcohol Dehydrogenase↗

PubMedDate not supplied

Regulation of pathways of alcohol metabolism by the liver.

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Alcohol Oxidoreductases↗