Novel glutathione conjugates formed from epoxyeicosatrienoic acids (EETs).

The catalysis of glutathione (GSH) conjugation to epoxyeicosatrienoic acids (EETs) by various purified isozymes of glutathione S-transferase was studied. A GSH conjugate of 14,15-EET was isolated by HPLC and TLC; this metabolite contained one molecule of EET and one molecule of GSH. Fast atom bombardment mass spectrometry of the isolated metabolite confirmed the structure as a GSH conjugate of 14,15-EET. Studies designed to determine the isozyme specificity of this reaction demonstrated that two isozymes, 3-3, and 5-5, efficiently catalyzed this conjugation reaction. The Km values for 14,15-EET were approximately 10 microM and the Vmax values ranged from 25 to 60 nmol conjugate formed min-1 mg-1 purified transferase 3-3 and 5-5. The 5,6-, 8,9-, and 11,12-EETs were also substrates for the reaction, albeit at lower rates. These results demonstrate that the EETs can serve as substrates for the cytosolic glutathione S-transferases.

Aging selectively alters glutathione S-transferase isozyme concentrations in liver and lung cytosol.

The effects of aging on hepatic and pulmonary cytosolic glutathione S-transferase activities and concentrations were investigated in male Fischer 344 rats 3, 12, and 24 months old. Column isoelectric focusing of liver cytosol indicated that activities, measured with six epoxide substrates, of isozymes E, C, and B increased from 3 to 12 months of age and decreased from 3 to 24 months of age whereas activities of isozyme A were increased in the old group. With lung cytosol, activities of isozymes E, C, and B were decreased whereas isozyme A activities were increased in the old group. Purification to homogeneity of individual isozymes from liver and lung cytosol from each age group indicated that catalytic properties of isozymes were not altered by aging. Immunotitrations of hepatic and pulmonary cytosol from each age group showed that changes in the concentrations of these isozymes occurred with aging: heptic isozymes E, C, and B increased from 3 to 12 months of age and then decreased by 24 months of age, whereas isozymes A and AA were increased by 24 months of age. Pulmonary isozymes E, C, and B followed a pattern of decline from 3 to 24 months of age, whereas isozyme A concentrations were unchanged with increasing age. Analysis of these changes suggested that subunits Ya and Yc as one group and Yb and Y'b as another followed similar increases (from 3 to 12 months) and decreases (from 12 to 24 months) in the liver and that these subunits showed consistent decreases with age in the lung.

Selective inactivation of rat lung and liver microsomal NADPH-cytochrome c reductase by acrolein.

Addition of acrolein to rat lung or liver microsomal suspensions resulted in total inactivation of NADPH-cytochrome c reductase and partial conversion of cytochrome P-450 to P-420 in a concentration- and time-dependent fashion. Acrolein also caused total loss of nonprotein sulfhydryl content in both preparations, whereas protein sulfhydryl content was decreased by 40% and 28% in lung and liver preparations, respectively. Maxima of about 60% of the total lung cytochrome P-450 and 50% of the liver cytochrome P-450 in acrolein-treated microsomes did not support the N-demethylation of benzphetamine or ethylmorphine or hydroxylation of aniline because of the total loss of NADPH-cytochrome c reductase. Addition of purified NADPH-cytochrome c reductase to the acrolein-treated lung or liver microsomal suspension largely restored these monooxygenase activities. Addition of glutathione or dithiothreitol to the lung or liver microsomal suspension prior to the addition of acrolein significantly protected cytochrome P-450 from conversion to cytochrome P-420 as well as NADPH-cytochrome c reductase from inactivation. Thus, selective conjugation of acrolein with lung and liver NADPH-cytochrome c reductase but not cytochrome P-450 was responsible for total loss of these lung and liver monooxygenase activities.

Effects of aging on hepatic and pulmonary glutathione S-transferase activities in male and female Fischer 344 rats.

The effects of aging on cytosolic glutathione S-transferase activities were evaluated with liver and lung cytosol from male and female Fischer 344 rats 3, 12, and 24 months of age. Age-related changes were tissue-, sex-, and substrate-specific. With liver and lung cytosol from both males and females, rates of metabolism of 1,2-epoxy-3-(p-nitrophenoxy)propane and p-nitrobenzyl chloride were lower in the old group than in the young group; however, patterns of decrease differed with tissue and sex. With 1,2-dichloro-4-nitrobenzene, metabolism was affected by aging only in liver and lung cytosol from males. Finally, with 1-chloro-2,4-dinitrobenzene, metabolic rates were altered during aging only with liver cytosol from females. However, the apparent Km was higher with liver cytosol from old males; those values from lung cytosol of males and liver or lung cytosol from females were unchanged. These data indicate that changes in the cytosolic glutathione S-transferase isozymes occurred during aging.

The in vitro dechlorination of some polychlorinated ethanes.

Chlorinated olefins were formed in vitro from hexachloroethane, pentachloroethane, and 1,1,1,2-tetrachloroethane by phenobarbital-induced rat liver microsomes. The production of tetrachloroethylene, trichloroethylene, and 1,1-dichloroethylene, respectively, was quantified by gas chromatographic analysis of headspace samples from the reaction vessels. The reaction showed a pH optimum of 7.0-7.5 under a nitrogen atmosphere; oxygen inhibited the reaction. The formation of olefins required NADPH and it was inhibited by SKF 525-A, metyrapone, and carbon monoxide. The reaction caused an increase in lipid peroxidation as measured by conjugated diene and malondialdehyde formation. The formation of olefins from these polychlorinated ethanes is apparently due to a cytochrome P-450-mediated vic-bisdechlorination reaction which may involve a free radical intermediate.

Hepatic and pulmonary cytosolic metabolism of epoxides effects of aging on conjugation with glutathione.

In liver cytosol from male Fischer 344 rats, glutathione S-transferase specific activities with six epoxide substrates were lower in the 24-month-old (senescent) group than in the 3-month-old (young) group. With lung cytosol from males and liver and lung cytosol from females, specific activities declined with only some of the substrates. Age-related increases in protein content in male and female rat liver occurred by 12 months of age (middle-age) and remained elevated through senescence. In addition, increases in liver weights in males similarly occurred so that total metabolic rates tended to be highest in middle-aged males and similar in young and senescent groups. Few changes similar to these were found in liver cytosol from females or lung cytosol from males or females. Thus, tissue-, sex-, and substrate-specific alterations in epoxide metabolism occurred during aging.

The biotransformation of allyl alcohol and acrolein in rat liver and lung preparations.

The biotransformation of allyl alcohol and acrolein by rat lung and liver preparations was investigated by measuring acrolein, acrylic acid, glycidol, and glycidaldehyde. Acrolein was detected by high-pressure liquid chromatography from incubation mixtures containing allyl alcohol, NAD+, and liver 9000g supernatant fraction or cytosol. Acrolein was not formed when lung fractions were treated similarly. Addition of pyrazole in the incubation mixture inhibited the reaction. The metabolism of acrolein to acrylic acid by liver 9000g supernatant fraction, cytosol, and microsomes has been demonstrated; acrylic acid formation was greater with NAD+ than with NADP+ in all three fractions. Acrylic acid was also formed from allyl alcohol. Disulfiram inhibited the NAD+- and NADP+-dependent reactions. Acrylic acid was not formed when lung preparations were used. Lung and liver microsomal epoxidation products of allyl alcohol and acrolein have been identified. Conversion of glycidol to glycerol and glycidaldehyde to glyceraldehyde by liver epoxide hydrase has been demonstrated. Epoxides, glycidol, and glycidaldehyde were also found to be substrates for lung and liver cytosolic glutathione S-transferase.

Microsomal metabolism of cyclohexene. Hydroxylation in the allylic position.

Hydroxylation of cyclohexene at the allylic position has been shown to occur in hepatic microsomes and 9000 g supernatant fractions of rats and rabbits. The formation of the product, 2-cyclohexen-1-ol, requires the presence of a NADPH-generating system, is inhibited by CO, metyrapone, and SKF 525-A, and is induced by pretreatment with phenobarbital. A small amount of 2-cyclohexen-1-one is also formed in preparations from phenobarbital-pretreated rats. No 2-cyclohexen-1-ol could be detected in the beta-glucuronidase-hydrolyzed urine of rats given cyclohexene orally; however, these rats excreted a small quantity of 2-cyclohexen-1-one.

Metabolism of halogenated ethylenes.

The metabolism of the chlorinated ethylenes may be explained by the formation of chloroethylene epoxides as the first intermediate products. The evidence indicates that these epoxides rearrange with migration of chlorine to form chloroacetaldehydes and chloroacetyl chlorides. Thus, monochloroacetic acid, chloral hydrate, and trichloroacetic acid have been found in reaction mixtures of 1,1-dichloroethylene, trichloroethylene, and tetrachloroethylene, respectively, with rat liver microsomal systems. Rearrangements of the chloroethylene, and glycols formed from the epoxides by hydration may also take place, but would appear, at least in the case of 1,1-dichloroethylene, to be quantitatively less important. The literature on the metabolism of chlorinated ethylenes and its relationship to their toxicity is reviewed.

Effect of experimental diabetes on drug metabolism in the rat.

The effect of experimental diabetes on hepatic drug metabolism was studied in male Holtzman rats. Treatment of animals with streptozotocin and 6-aminonicotinamide, both agents which produce an insulin-deficient animal, caused prolongation of hexobarbital sleeping times and inhibition of the rate of metabolism of both hexobarbital and, to a lesser extent, aniline in vitro. Treatment of animals with N-methylacetamide, a diabetogen which does not cause insulin deficiency in the animal but rather produces an insulin-resistant state, did not affect the metabolism in vitro of either hexobarbital or aniline. Neither insulin nor any of the diabetogenic agents had any direct effect on drug metabolism in vitro. Furthermore, hepatic microsomal protein and cytochrome P-450 contents were not significantly different in any of the diabetic animals from those of the control animals. Hyperglycemia produced by glucose infusion did not affect the metabolism of hexobarbital in vitro. The effects of streptozotocin and 6-aminonicotinamide appeared to be at least partially due to the presence of an inhibitor in the liver cytosol which correlated with elevated hepatic cyclic AMP concentrations.

Further studies of metyrapone effects upon anilide hydroxylation.

The enhancing effect of metyrapone upon the p-hydroxylation of acetanilide has been confirmed with the use of a new gas-chromatographic method for the determination of acetaminophen. This effect has been shown not to be due to inhibition of hydrolysis of acetaminophen or interference with its determination, or to preferential formation of other phenolic metabolites. This effect of metyrapone is remarkably substrate-specific: phenol formation from the homologues of acetanilide, formanilide and propionanilide, and that from the sulfonamide analog of acetanilide, methanesulfonanilide, is inhibited by metyrapone over the concentration range in which acetanilide hydroxylation is enhanced. The same substrate specificity was observed when the modifier was acetophenone. alpha,alpha'-Dipyridyl, however, enhances phenol formation from all three carbonacylanilides, but does not affect that from methanesulfonanilide.

Metabolism and toxicity of styrene.

The absorption, blood levels, distribution, excretion, and biotransformation of styrene in man and experimental animals are briefly reviewed. The acute toxicity of styrene appears to be unrelated to its biotransformation. Reports of organ toxicity upon chronic exposure to styrene are rare; however, since the chief intermediate in styrene metabolism is an epoxide, hepatotoxicity due to covalent binding at the site of formation appears to be a possibility.