Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:1.1.1.1 (alcohol dehydrogenase)
 9,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Lipid peroxidation has been invoked as a mechanism of alcoholic liver injury but its role has been controversial and the mechanism by which it occurs is unclear. Catalytic iron is known to play an important role in cellular injury and is produced during mobilization of ferritin iron. In vivo administration of a large acute dose of ethanol (5 g/kg) which produces hepatic lipid peroxidation in chow-fed rats resulted in mobilization of non-heme iron. The generation of NADH from alcohol metabolism via ADH or superoxide from acetaldehyde-xanthine oxidase mobilized iron from horse spleen ferritin in vitro. Chronic feeding of alcohol as 36% of energy for 6 weeks does not itself produce peroxidation in the rat but potentiates acute effects of ethanol. It produced microsomal induction which enhanced iron-stimulated lipid peroxidation and increased hepatic non-heme iron. Carbon monoxide increased rather than decreased accumulation of microsomal peroxidation products in vitro suggesting that cytochrome P-450 reductase mediates peroxidation but cytochrome P-450 may metabolize products. Incubation at lowered oxygen tensions equivalent to those observed in the perivenular zone (pO2 = 24 mmHg) enhanced in vitro iron mobilization but decreased peroxidation. Lipid peroxidation and its stimulation by iron mobilization and microsomal induction may be an important contributory mechanism of alcohol-induced liver injury.
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PMID:Lipid peroxidation as a mechanism of alcoholic liver injury: role of iron mobilization and microsomal induction. 313 9

Pyrazole and 4-methylpyrazole are potent inhibitors of liver alcohol dehydrogenase and as such have been proposed as potential antidotes to alcohol poisoning. These drugs are also inducers of hepatic cytochrome P-450. We tested pyrazole and four 4-substituted pyrazoles for their potential as inducers of cytochrome P-450 and drug metabolism in mature male rats. Total cytochrome P-450 was significantly increased (p less than 0.05) 1.3 fold by treatment with 4-methylpyrazole. P-nitrophenol hydroxylase (PNPH) activity (nmol/min/mg protein) was increased 1.9 fold following treatment with pyrazole and with 4-methylpyrazole. Treatment with 4-methylpyrazole also resulted in a 2.9 fold increase in ethoxyresorufin demethylase (EROD) activity. In addition, pyrazole treatment led to a significant decrease in the activity of benzphetamine demethylase. 4-Iodopyrazole increased the turnover (nmol/min/nmol P-450) of EROD and PNPH by 1.5 fold each. 4-Nitropyrazole had no significant effect on any of the activities or turnover rates tested. In contrast to results with cultured chick hepatocytes, where induction was directly related to the hydrophobicity of the 4-substituent, the present data indicate that the process of induction of in vivo is more complex.
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PMID:Induction of rat hepatic microsomal drug metabolizing enzymes by pyrazole and 4-substituted pyrazoles. 322 28

(i) Hepatocytes isolated from adult rats were cultured for 2 to 3 weeks on collagen in a modified, serum-free Waymouth medium containing fatty acids and varying concentrations of glucocorticoid, insulin and glucagon. (ii) In the presence of all three hormones, it was possible to maintain the content of DNA, the activity of glucokinase, pyruvate kinase, hexokinase and lactate dehydrogenase at initial levels for 2 to 3 weeks. The activity of glucokinase and pyruvate kinase was affected by the concentration of insulin. (iii) The activity of alcohol dehydrogenase was stable for 3 days and declined to about 25% of the initial level after 2 weeks of culture, irrespective of the presence of hormones. (iv) Maintenance of albumin secretion was dependent on the presence of glucocorticoid, and glucocorticoid and insulin showed an additive or, at some time points, a synergistic effect on its secretion. (v) The content of cytochrome P-450 could be kept at 65% of the initial level, provided that a relatively high concentration of dexamethasone was present (10(-6) M). (vi) In the absence of hormones, urea synthesis was 70% of initial levels throughout the experimental period. With insulin and glucocorticoid present, a high concentration of glucagon (10(-8) M) was required to maintain the synthesis of urea at this level. (vii) It is concluded that hepatocyte cultures as described in the present study may be a useful, well-defined system for long-term metabolic, pharmacologic and toxicologic studies.
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PMID:Long-term culture of hepatocytes: effect of hormones on enzyme activities and metabolic capacity. 327 89

Three cDNAs for chimeras between cytochrome P-450s (pHP3 and pHP2-1) were constructed and inserted between the alcohol dehydrogenase promoter and terminator regions of the yeast expression vector pAAH5 to form expression plasmids, pAH3P2, pAH3E2, and pAH3A2. pAH3P2 contained the entire coding sequence of cytochrome P-450 (pHP2-1) except for the 3rd, the 8th, the 36th, and the 42nd residues of the total of 490 amino acids. Nucleotide sequences of pAH3P2 were replaced with those of cytochrome P-450 (pHP3) in the region coding for the NH2-terminal 210 and 262 amino acid residues to yield pAH3E2 and pAH3A2, respectively. The three expression plasmids were introduced into Saccharomyces cerevisiae AH22 cells and cytochrome P-450 s (3P2, 3E2, and 3A2) were purified from the microsomal fractions of the transformed yeast cells. In the oxidized state either of the cytochromes exhibited a low- and high-spin mixed-type spectrum of cytochrome P-450. The reduced CO complex of the cytochromes showed a Soret absorption maximum at 450 nm. When laurate or caprate was added to ferric cytochrome P-450 s (3P2 and 3E2), the spectrum was converted to that of the typical high-spin type, indicating the binding of the fatty acids to the substrate site of the cytochromes. On the other hand, the addition of the fatty acids to ferric cytochrome P-450 (3A2) induced no spectral change. Only chemicals having a carboxyl group caused such spectral conversion of cytochrome P-450 (3P2) among dodecyl compounds examined.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Characterization of rabbit liver cytochrome P-450 (laurate omega-1 hydroxylase) synthesized in transformed yeast cells. 328 12

Two neuroglial cell lines (U-251 MG and C6) had a substantial capacity to convert ethanol to acetate in vitro largely by an alcohol dehydrogenase (ADH)-independent mechanism and three neuroblastoma cell lines (IMR-32, NB41A3, and Neuro-2a) had a lesser but significant ethanol-metabolizing capacity which was also either partly or largely ADH-independent. The ADH-independent pathway of ethanol metabolism by neural cells appeared to be dependent on one or more isoenzymes of cytochrome P-450. The data emphasize the possibility that the neurotoxicity of ethanol may be related to a relatively high ethanol-metabolizing capability of neural tissue and particularly of neuroglial cells.
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PMID:Neuroglial and neuroblastoma cell lines are capable of metabolizing ethanol via an alcohol-dehydrogenase-independent pathway. 330 84

In vivo administration of the alcohol dehydrogenase inhibitor pyrazole induces a cytochrome P-450 isozyme. The pyrazole-inducible cytochrome P-450 has been purified from rat livers to electrophoretic homogeneity and its biochemical, spectral, and immunological properties characterized. The final preparation had a specific content of 11 nmol of cytochrome P-450/mg of protein. A single band with an apparent molecular weight of 52,000 was observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The absolute spectrum of the isolated pyrazole cytochrome P-450 displayed peaks at 648 and 396 nm, suggestive of a high spin cytochrome. The ethylisocyanide difference spectrum exhibited two maxima, one at 457 nm, the other at 428 nm. Pyrazole and dimethyl sulfoxide produced binding spectra with the purified P-450, with peaks at 425 or 419 nm and troughs at 390 or 386 nm, respectively. K8 values for dimethyl sulfoxide and pyrazole were 21 and 0.04 mM, respectively. The catalytic activity of the pyrazole cytochrome P-450 was elevated with aniline and dimethylnitrosamine (low Km) but not with aminopyrine, benzphetamine, ethoxycoumarin, or ethoxyresorufin as substrates. An antibody against pyrazole cytochrome P-450 recognized a 52,000 molecular weight protein upon reaction with saline microsomes. The intensity of the immunoblot was increased when microsomes isolated from pyrazole, 4-methylpyrazole-, acetone-, or chronic ethanol-treated rats were utilized, but not after phenobarbital or 3-methylcholanthrene treatment. Homology at the amino terminus of 19 amino acids was observed between pyrazole P-450 and the isoniazid-inducible P-450j. Based upon the above catalytic, spectral, and immunological properties, it appears that pyrazole induces a form of cytochrome P-450 which is identical to that induced by ethanol and isoniazid.
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PMID:Characterization and identification of a pyrazole-inducible form of cytochrome P-450. 333 29

The study of the influence of the age of the animals (13 to 53 weeks) on the rate of ethanol metabolism in vivo and the total activity of liver alcohol dehydrogenase and microsomal ethanol oxidizing system showed a progressive decline with age. These effects were observed concomitantly with a diminution in the content of cytochrome P-450 and microsomal functions related to oxidative and free-radical mediated reactions, namely, NADPH oxidase activity, NADPH-dependent oxygen uptake and NADPH-or t-butyl hydroperoxide-induced chemiluminescence. It is concluded that ageing is accompanied by a diminution in the total oxidative activity of the liver tissue, which would explain the depression in basal and ethanol-induced lipid peroxidation found in the oldest group of rats studied.
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PMID:Age-dependent changes in in vivo ethanol metabolism and in the activity of hepatic enzymes involved in ethanol oxidation and microsomal functions. 334 70

4-Hydroxypyrazole has been identified as a major metabolite found in the urine of rats and mice after in vivo administration of pyrazole, a potent inhibitor of alcohol dehydrogenase and of ethanol metabolism. The locus and the enzyme systems responsible for the oxidation of pyrazole have not been identified. In the current report, isolated hepatocytes from fed rats were shown to oxidize pyrazole to 4-hydroxypyrazole. An HPLC procedure employing UV and electrochemical detection was utilized to separate and quantify the 4-hydroxypyrazole. The apparent Km for pyrazole by intact hepatocytes was about 2 mM, whereas the apparent Vmax was about 0.06 nmol 4-hydroxypyrazole per min per mg liver cell protein. The production of 4-hydroxypyrazole was inhibited by carbon monoxide and metyrapone, as well as by competitive drug substrates such as aniline or aminopyrine. These results implicate a role for cytochrome P-450 in the oxidation of pyrazole by the hepatocytes. Ethanol was an effective inhibitor of pyrazole oxidation. Hepatocytes were also isolated from rats treated with acetone and 4-methylpyrazole, to attempt to evaluate whether pyrazole oxidation is induced. The rate of 4-hydroxypyrazole production by hepatocytes after acetone and 4-methylpyrazole treatment was actually lower than that of controls. Kinetic assays suggested the presence of an endogenous inhibitor (perhaps the inducer itself) in the induced hepatocytes. In contrast, hepatocytes isolated from rats fasted for 48 hr showed a 2-fold increase in the oxidation of pyrazole to 4-hydroxypyrazole. The Km for pyrazole was the same in hepatocytes from fasted and fed rats, whereas Vmax was increased after fasting. The locus and enzyme system responsible for the oxidation of pyrazole to 4-hydroxypyrazole, and the site of sensitivity to ethanol, appears to be the cytochrome P-450 system of the hepatocyte.
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PMID:Oxidation of pyrazole to 4-hydroxypyrazole by intact rat hepatocytes. 335 85

Isozyme 3a of rabbit hepatic cytochrome P-450, also termed P-450ALC, was previously isolated and characterized and was shown to be induced 3- to 5-fold by exposure to ethanol. In the present study, antibody against rabbit P-450ALC was used to identify a homologous protein in alcohol dehydrogenase-negative (ADH-) and -positive (ADH+) deermice, Peromyscus maniculatus. The antibody reacts with a single protein having an apparent molecular weight of 52,000 on immunoblots of hepatic microsomes from untreated and ethanol-treated deermice from both strains. The level of the homologous protein was about 2-fold greater in microsomes from naive ADH- than from naive ADH+ animals. Ethanol treatment induced the protein about 3-fold in the ADH+ strain and about 4-fold in the ADH- strain. The antibody to rabbit P-450ALC inhibited the microsomal metabolism of ethanol and aniline. The homologous protein, termed deermouse P-450ALC, catalyzed from 70 to 80% of the oxidation of ethanol and about 90% of the hydroxylation of aniline by microsomes from both strains after ethanol treatment. The antibody-inhibited portion of the microsomal activities, which are attributable to the P-450ALC homolog, increased about 3-fold upon ethanol treatment in the ADH+ strain and about 4-fold in the ADH- strain, in excellent agreement with the results from immunoblots. The total microsomal P-450 content and the rate of ethanol oxidation were induced 1.4-fold and 2.2-fold, respectively, by ethanol in the ADH+ strain and 1.9-fold and 3.3-fold, respectively, in the ADH- strain. Thus, the total microsomal P-450 content and ethanol oxidation underestimate the induction of the P-450ALC homolog in both strains. A comparison of the rates of microsomal ethanol oxidation in vitro with rates of ethanol elimination in vivo indicates that deermouse P-450ALC could account optimally for 3 and 8% of total ethanol elimination in naive ADH+ and ADH- strains, respectively. After chronic ethanol treatment, P-450ALC could account maximally for 8% of the total ethanol elimination in the ADH+ strain and 22% in the ADH- strain. Further, cytochrome P-450ALC appears to be responsible for about one-half of the increase in the rate of ethanol elimination in vivo after chronic treatment with ethanol. These results indicate that the contribution of P-450ALC to ethanol oxidation in the deermouse is relatively small. Desferrioxamine had no effect on rates of ethanol uptake by perfused livers from ADH-negative deermice, indicating that ethanol oxidation by a hydroxyl radical-mediated mechanism was not involved in ethanol metabolism in this mutant.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Identification of P-450ALC in microsomes from alcohol dehydrogenase-deficient deermice: contribution to ethanol elimination in vivo. 339 17

The purpose of this study was to measure rates of catalase-dependent ethanol uptake and rates of H2O2 generation in perfused rat livers in the presence of fatty acids of varying chain length. Rates of ethanol uptake in livers from fasted rats, perfused in a recirculating system, of about 80 mumol g-1 h-1 were decreased to about 10 mumol g-1 h-1 by the addition of an inhibitor of alcohol dehydrogenase (ADH), 4-methylpyrazole. The medium-chain-length fatty acid, laurate (12:0; 1 mM), increased rates of 4-methylpyrazole-insensitive ethanol uptake maximally to 80-85 mumol g-1 h-1. Rates of ethanol uptake diminished as the chain length of fatty acid was decreased [hexanoate (6:0) = 23 mumol g-1 h-1; octanoate (8:0) = 55 mumol g-1 h-1; decanoate (10:0) = 65 mumol g-1 h-1] or increased [myristate (14:0) = 77 mumol g-1 h-1; palmitate (16:0) = 80 mumol g-1 h-1; stearate (18:0) = 29 mumol g-1 h-1; oleate (18:1) = 60 mumol g-1 h-1; erucate (22:3) = 22 mumol g-1 h-1] from 12:0. Oleate did not increase rates of hydroxylation of p-nitrophenol, a substrate for the ethanol-inducible form of cytochrome P-450, indicating that the stimulation of ethanol uptake by fatty acids was not due to increased mixed-function oxidation. The increase of ethanol uptake was also not due to displacement of 4-methylpyrazole from ADH by fatty acids, since oleate stimulated ethanol uptake by about 50% in perfused livers from deermice genetically deficient in ADH. The increase in 4-methylpyrazole-insensitive ethanol uptake by fatty acids was blocked by the catalase inhibitor, aminotriazole, indicating the involvement of catalase. Rates of H2O2 generation by livers perfused in a non-recirculating system with 1.7% albumin were increased from 6 +/- 1 to 23 +/- 5 mumol g-1 h-1 by oleate (1 mM). Because of the discrepancy between rates of ethanol metabolism and H2O2 production, methods were developed to measure H2O2 production in a recirculating perfusion system. H2O2 generation was determined from the time necessary for steady-state level of catalase-H2O2, measured spectrophotometrically (660-640 nm) through a lobe of the liver, to return to basal values after the addition of a known quantity of methanol, which is not metabolized by ADH in the rat.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Catalase-dependent ethanol oxidation in perfused rat liver. Requirement for fatty-acid-stimulated H2O2 production by peroxisomes. 341 82


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