Biomedical subjects

G L Kedderis

Publications and source records attributed to G L Kedderis.

At least 55 records · Page 3Linked to original sources

PubMed1985

Peroxidase-catalyzed N-demethylation reactions: deuterium solvent isotope effects.

The effect of D2O on the kinetic parameters for the hydroperoxide-supported N-demethylation of N,N-dimethylaniline catalyzed by chloroperoxidase and horseradish peroxidase was investigated in order to assess the roles of exchangeable hydrogens in the demethylation reaction. The initial rate of the chloroperoxidase-catalyzed N-demethylation of N,N-dimethylaniline supported by ethyl hydroperoxide exhibited a pL optimum (where L denotes H or D) of 4.5 in both H2O and D2O. The solvent isotope effect on the initial rate of the chloroperoxidase-catalyzed demethylation reaction was independent of pL, suggesting that the solvent isotope effect is not due to a change in the pK of a rate-controlling ionization in D2O. The solvent isotope effect on the Vmax for the chloroperoxidase-catalyzed demethylation reaction was 3.66 +/- 0.62. In contrast, the solvent isotope effect on the Vmax for the horseradish peroxidase catalyzed demethylation reaction was approximately 1.5 with either ethyl hydroperoxide or hydrogen peroxide as the oxidant, indicating that the exchange of hydrogens in the enzyme and hydroperoxide for deuterium in D2O has little effect on the rate of the demethylation reaction. The solvent isotope effect on the Vmax/KM for ethyl hydroperoxide in the chloroperoxidase-catalyzed demethylation reaction was 8.82 +/- 1.57, indicating that the rate of chloroperoxidase compound I formation is substantially decreased in D2O. This isotope effect is suggested to arise from deuterium exchange of the hydroperoxide hydrogen and of active-site residues involved in compound I formation. A solvent isotope effect of 2.96 +/- 0.57 was observed on the Vmax/KM for N,N-dimethylaniline in the chloroperoxidase-catalyzed reaction.(ABSTRACT TRUNCATED AT 250 WORDS)

Chloride Peroxidase↗

PubMed1985

Characterization of the oxidation of amine metabolites of nitrotoluenes by rat hepatic microsomes. N- and C-hydroxylation.

The rat hepatic microsomal oxidation of amine metabolites of mono-and dinitrotoluene isomers has been investigated. Microsomes catalyzed the NADPH-dependent oxidation of 2-amino-6-nitrobenzyl alcohol, 2-amino-4-nitrobenzyl alcohol, and the isomeric aminobenzyl alcohols to ethyl acetate-extractable compounds capable of reducing ferric iron. The microsomal metabolism of 2-amino-6-nitrobenzyl alcohol, a metabolite of the hepatocarcinogen 2,6-dinitrotoluene, was characterized in detail. High pressure liquid chromatographic analysis indicated the formation of two metabolites, both of which were reducing agents. One metabolite was identified as 2-hydroxylamino-6-nitrobenzyl alcohol by comparison of its chromatographic properties and mass spectrum with those of the authentic compound. Mass spectral, proton NMR, and UV-visible spectroscopic studies suggested that the other metabolite was 2-amino-5-hydroxy-6-nitrobenzyl alcohol. The microsomal oxidation of 2-aminobenzyl alcohol also resulted in the formation of two reducing agents, one of which was the corresponding hydroxylamine. The formation of 2-hydroxylamino-6-nitrobenzyl alcohol from the microsomal oxidation of 2-amino-6-nitrobenzyl alcohol was linear with respect to time for at least 20 min, while aminophenol formation was only linear for 3 min. The rate of the microsomal oxidation of 2-amino-6-nitrobenzyl alcohol was decreased by known inhibitors of cytochrome P-450, while heat inactivation of microsomal flavin-containing monooxygenase had no effect. The rate of formation of both metabolites was increased 1.5-fold by phenobarbital pretreatment. Pretreatment with beta-naphthoflavone had no effect on the rate of N-hydroxylation, while a small but statistically significant increase in the rate of C-hydroxylation (117% of control) was observed. The rate of oxidation of 2-amino-6-nitrobenzyl alcohol was lower with microsomes from female rats than with those from males, yielding male/female ratios of 1.34 for aminophenol formation and 3.26 for hydroxylamine formation. These data indicate that 2-amino-6-nitrobenzyl alcohol, a metabolite of the hepatocarcinogen 2,6-dinitrotoluene, can be N-hydroxylated by hepatic microsomal cytochrome P-450. The results are consistent with the hypothesis that a hydroxylamine metabolite of 2,6-dinitrotoluene is sulfated in vivo to produce an electrophilic species.

Animals↗

PubMed1984

Peroxidase-catalyzed N-demethylation reactions. Substrate deuterium isotope effects.

Deuterium isotope effects on the kinetic parameters for the hydroperoxide-supported N-demethylation of N,N-dimethylaniline catalyzed by chloroperoxidase and horseradish peroxidase were determined using N,N-di-(trideuteromethyl)aniline. The isotope effect on the Vmax for the chloroperoxidase-catalyzed demethylation reaction supported by ethyl hydroperoxide was 1.42 +/- 0.31. The isotope effects on the Vmax for the horseradish peroxidase-catalyzed reaction supported by ethyl hydroperoxide and hydrogen peroxide were 1.99 +/- 0.39 and 4.09 +/- 0.27, respectively. Isotope effects ranging from 1.76 to 5.10 were observed on the Vmax/Km for the hydroperoxide substrate (i.e. the second order rate constant for the reaction of the hydroperoxide with the peroxidase to form compound I) in both enzyme systems when the N-methyl groups of N,N-dimethylaniline were deuterated. These results are not predicted by the simple ping-pong kinetic model for peroxidase-catalyzed N-demethylation reactions. The data are most simply explained by a mechanism involving the transfer of deuterium (or hydrogen) from N,N-dimethylaniline to the enzyme during catalysis. The deuterium must subsequently be displaced from the enzyme by the hydroperoxide, causing the observed isotope effects.

Aniline Compounds↗

PubMed1984

pH kinetic studies of the N-demethylation of N,N-dimethylaniline catalyzed by chloroperoxidase.

The effect of pH on the kinetic parameters for the chloroperoxidase-catalyzed N-demethylation of N,N-dimethylaniline supported by ethyl hydroperoxide was investigated from pH 3.0 to 7.0. Chloroperoxidase was found to be stable throughout the pH range studied. Initial rate conditions were determined throughout the pH range. The Vmax for the demethylation reaction exhibited a pH optimum at approximately 4.5. The Km for N,N-dimethylaniline increased with decreasing pH, while the Km for ethyl hydroperoxide varied in a manner paralleling Vmax. Comparison of the Vmax/Km values for N,N-dimethylaniline and ethyl hydroperoxide indicated that the interaction of N,N-dimethylaniline with chloroperoxidase compound I was rate-limiting below pH 4.5, while compound I formation was rate-limiting above pH 4.5. The log of the Vmax/Km for ethyl hydroperoxide was independent of pH, indicating that chloroperoxidase compound I formation is not affected by ionizations in this pH range. The plot of the log of the Vmax/Km for N,N-dimethylaniline versus pH indicated an ionization on compound I with a pK of approximately 6.8. The plot of the log of the Vmax versus pH indicated an ionization on the compound I-N,N-dimethylaniline complex, with a pK of approximately 3.1. The results show that chloroperoxidase can demethylate both the protonated and neutral forms of N,N-dimethylaniline (pK approximately 5.0), suggesting that hydrophobic binding of the arylamine substrate is more important in catalysis than ionic bonding of the amine moiety. For optimal catalysis, a residue in the chloroperoxidase compound I-N,N-dimethylaniline complex with a pK of approximately 3.1 must be deprotonated, while a residue in compound I with a pK of approximately 6.8 must be protonated.

Aniline Compounds↗

PubMed1984

Hepatic macromolecular covalent binding of mononitrotoluenes in Fischer-344 rats.

The mononitrotoluenes are important industrial chemicals which display isomeric specificity in their ability to induce hepatic DNA excision repair in Fischer-344 rats. Covalent binding of the structurally related hepatocarcinogen, 2,6-dinitrotoluene, to hepatic DNA is markedly decreased by prior administration of the sulfotransferase inhibitors pentachlorophenol (PCP) and 2,6-dichloro-4-nitrophenol (DCNP). The objectives of this study were to determine whether hepatic macromolecular covalent binding of the mononitrotoluene isomers differed and to determine whether covalent binding of the mononitrotoluenes to hepatic DNA in vivo was decreased by inhibitors of sulfotransferase. Male Fischer-344 rats were given a single oral dose of [ring-U-14C]-2-, 3- or 4-nitrotoluene (2-, 3- or 4-NT) and killed at various times thereafter. Livers were removed and analyzed for total and covalently bound radiolabel. Maximal concentrations of total radiolabel were observed between 3 and 12 h after the dose, and there were no large differences among the 3 isomers in peak concentrations achieved. Covalent binding to hepatic macromolecules was maximal 12 h after administration for all three isomers. Thereafter, concentrations of covalently bound 2-NT-derived material were always 2-6 times higher than those of 3- or 4-NT-derived material. When DNA was isolated from livers of rats given the mononitrotoluenes 12 h previously, only 2-NT was observed to covalently bind at concentrations above the limits of detection of the assay. The covalent binding of 2-NT, but not that of 3- or 4-NT, to both total hepatic macromolecules and DNA was markedly decreased by prior administration of either PCP or DCNP. Covalent binding to hepatic DNA was decreased by greater than 96%. The results of this study correlate well with studies which have demonstrated that 2-NT, but not 3- or 4-NT, induces DNA excision repair. Furthermore, they suggest that 2-NT, like the hepatocarcinogen 2,6-dinitrotoluene, requires the action of sulfotransferase for its conversion to a species capable of covalently binding to hepatic DNA.

Animals↗

PubMed1984

Hepatic macromolecular covalent binding of the hepatocarcinogen 2,6-dinitrotoluene and its 2,4-isomer in vivo: modulation by the sulfotransferase inhibitors pentachlorophenol and 2,6-dichloro-4-nitrophenol.

The sulfotransferase inhibitors 2,6-dichloro-4-nitrophenol and pentachlorophenol were used to investigate the role of sulfate ester formation during the in vivo bioactivation of 2,4- and 2,6-dinitrotoluene (DNT). Male F-344 rats were administered one of the sulfotransferase inhibitors (40 mu mol/kg i.p.) 45 min prior to oral administration of 28 mg/kg [ring-14C]-2,4-DNT or [3-3H]-2,6-DNT and killed 12 h later. Pentachlorophenol had no significant effect on the urinary excretion of the benzyl glucuronide or benzoic acid metabolites of 2,6-DNT. The sulfotransferase inhibitors decreased the total hepatic macromolecular covalent binding of 2,4-DNT by 33%, and of 2,6-DNT by 69%. Purification of hepatic DNA by hydroxylapatite chromatography indicated covalent binding of 2,4- and 2,6-DNT at levels of 45 and 94 pmol equivalents/mg DNA, respectively. The sulfotransferase inhibitors decreased the binding of the hepatocarcinogen 2,6-DNT to hepatic DNA by greater than 95%. 2,6-Dichloro-4-nitrophenol decreased the binding of 2,4-DNT to DNA by greater than 84% while the decrease due to pentachlorophenol was 33%. These results suggest that sulfation is important in the biotransformation of 2,4- and 2,6-DNT to reactive metabolites which covalently bind to DNA. 3H2O was detected in the urine of rats administered [3-3H]-2,6-DNT. Pentachlorophenol decreased 3H2O formation to the same extent as it decreased the total hepatic macromolecular covalent binding of 2,6-DNT, suggesting that 3H exchange at the 3 position of 2,6-DNT occurs following sulfate ester formation. These results are consistent with a nitrenium-carbonium ion resonance of the sulfate ester-derived reactive intermediate of 2,6-DNT.

Animals↗

PubMed1983

The use of intramolecular isotope effects to distinguish between deprotonation and hydrogen atom abstraction mechanisms in cytochrome P-450- and peroxidase-catalyzed N-demethylation reactions.

Intramolecular isotope effects were determined for the N-demethylation of N-methyl-N-trideuteriomethylaniline catalyzed by two isozymes of cytochrome P-450 and several peroxidases in order to differentiate between deprotonation and hydrogen atom abstraction steps. Lactoperoxidase, hemoglobin, myoglobin, and two isozymes of horseradish peroxidase catalyzed the hydroperoxide-dependent N-demethylation at initial rates ranging from 20 to 1700 min-1. These hemeproteins exhibited large and comparable intramolecular isotope effects (kH/kD = 8.6 to 10.1). In contrast, two isozymes of cytochrome P-450 as well as chloroperoxidase (v = 1.5 to 1700 min-1) gave low isotope effects (kH/kD = 1.7 to 3.1) under identical conditions. Catalase exhibited an intermediate intramolecular isotope effect (kH/kD = 5.4). These results have been interpreted to indicate that most of the hemeproteins investigated catalyze N-demethylation reactions via alpha-carbon hydrogen atom abstraction, while the reactions catalyzed by cytochrome P-450 and chloroperoxidase proceed via alpha-carbon deprotonation.

Animals↗

PubMed1983

Steady state kinetics of chloroperoxidase-catalyzed N-demethylation reactions.

The kinetic mechanism of the chloroperoxidase-catalyzed N-demethylation of N,N-dimethylaniline supported by ethyl hydroperoxide was determined from initial rate and inhibition studies. When the concentration of N,N-dimethylaniline was varied in the presence of several different fixed concentrations of ethyl hydroperoxide, a double reciprocal plot of the data gave a series of parallel lines. Parallel lines were also observed when the data were plotted as a function of the concentration of ethyl hydroperoxide. A linear double reciprocal plot was obtained when the concentrations of both substrates were varied in a constant ratio. Competitive substrate inhibition was observed for N,N-dimethylaniline with a KI of 4.78 mM. Competitive substrate inhibition was also observed for ethyl hydroperoxide but the secondary replot of the slopes of the double reciprocal lines versus the concentrations of ethyl hydroperoxide intercepted the origin, indicating that no abortive binary complex between ethyl hydroperoxide and the enzyme was formed. These results suggested that the inhibition of the demethylation reaction by ethyl hydroperoxide was due to the reaction of ethyl hydroperoxide with chloroperoxidase compound I to evolve oxygen. N,N-Dimethylaniline inhibited the chloroperoxidase-catalyzed evolution of oxygen from ethyl hydroperoxide with a KI (0.111 mM) essentially identical with its KM for demethylation (0.122 mM). The inhibition of the demethylation reaction by 2,5-dimethylfuran was competitive with respect to N,N-dimethylaniline and uncompetitive with respect to ethyl hydroperoxide, consistent with dead-end inhibition in a ping-pong system. The results of the initial rate and inhibition studies are consistent with a Ping Pong Bi Bi mechanism as the minimal kinetic model for chloroperoxidase-catalyzed N-demethylation reactions. In this model, ethyl hydroperoxide reacts with chloroperoxidase to form the oxidized enzyme intermediate compound I with the concomitant release of ethanol. N,N-Dimethylaniline then binds to compound I and is oxidized, resulting in the formation of N-methylaniline and formaldehyde and the regeneration of the native peroxidase.

Acetaldehyde↗

PubMed1983

Characterization of the N-demethylation reactions catalyzed by horseradish peroxidase.

The hydroperoxide-supported N-demethylation reactions catalyzed by horseradish peroxidase have been characterized in detail. The ethyl hydroperoxide-supported N-demethylation of N,N-dimethylaniline by horseradish peroxidase resulted in the formation of equimolar amounts of N-methylaniline and formaldehyde with no other products detectable by high performance liquid chromatography analysis of the reaction mixture. One molecule of ethyl hydroperoxide was consumed for each molecule of formaldehyde formed in the reaction. Similar results were obtained for the hydrogen peroxide-supported N-demethylation of N,N-dimethylaniline. The horseradish peroxidase-catalyzed N-demethylation reaction could be supported by a variety of hydroperoxides, peroxides, and peracids. The turnover number for the hydrogen peroxide-supported demethylation reaction (7061) was larger than that for the ethyl hydroperoxide-supported reaction (5382) or for chloroperoxidase- or cytochrome P-450-catalyzed dealkylations. The demethylation reaction exhibited normal Michaelis-Menten saturation kinetics with respect to N,N-dimethylaniline (Km = 0.34 mM) and ethyl hydroperoxide (Km = 0.020 mM), as well as hydrogen peroxide (Km = 0.016 mM). The horseradish peroxidase-catalyzed N-demethylation reaction was not significantly inhibited by reagents which react with the superoxide anion, the hydroxyl radical, or singlet oxygen, suggesting that these activated oxygen species are not free intermediates in the reaction. There was no significant inhibition of the reaction by alpha-phenyl-t-butylnitrone, 5,5-dimethylpyrroline-N-oxide, or other free radical trapping agents. Substitution of D2O for H2O resulted in an inhibition of the reaction with a solvent isotope effect (VH2O/VD2O) of 1.6. Horseradish peroxidase did not catalyze the demethylation of N,N-dimethylaniline-N-oxide, indicating that the reaction does not proceed via N-oxidation of the amine. When the concentrations of both N,N-dimethylaniline and ethyl hydroperoxide were varied in a constant ratio a linear double reciprocal plot was obtained, which is consistent with a ping-pong kinetic mechanism for the horseradish peroxidase-catalyzed demethylation reaction.

Horseradish Peroxidase↗

PubMed1983

Inhibition of the microsomal N-hydroxylation of 2-amino-6-nitrotoluene by a metabolite of methimazole.

Inhibition studies were used to investigate the identity of the microsomal enzyme(s) responsible for the NADPH-dependent N-hydroxylation of 2-amino-6-nitrotoluene. The N-hydroxylation reaction was inhibited by several cytochrome P-450 inhibitors as well as by methimazole, a substrate for flavin-containing monooxygenase. Heat inactivation of flavin-containing monooxygenase had no effect on the rate of the reaction but abolished the inhibition by methimazole. These results indicate that the flavin-containing monooxygenase-mediated metabolism of methimazole produced an inhibitor of the cytochrome P-450-catalyzed N-hydroxylation reaction. When glutathione was included in the incubation the inhibition by methimazole was abolished, presumably due to the reduction of oxygenated metabolites of methimazole. These results show that methimazole inhibition does not necessarily implicate flavin-containing monooxygenase in microsomal N-hydroxylation reactions.

Animals↗

PubMed1983

Source of the oxygen atom in the product of cytochrome P-450-catalyzed N-demethylation reactions.

The source of the oxygen atom in the product of the cytochrome P-450-catalyzed N-demethylation of N-methylcarbazole was determined by mass spectral analysis of the carbinolamine precursor of formaldehyde formed during incubation in oxygen 18-enriched medium. Initial experiments demonstrated that N-(hydroxymethyl)carbazole, the carbinolamine product of the metabolism of N-methylcarbazole, did not exchange oxygen with solvent water. When N-methylcarbazole was incubated in oxygen 18-enriched medium with purified cytochrome P-450 in the presence of either purified NADPH-cytochrome P-450 reductase and NADPH, cumene hydroperoxide, t-butyl hydroperoxide, or peracetic acid, there was no incorporation of oxygen 18 from the medium into N-(hydroxymethyl)carbazole. These results clearly demonstrate that the oxygen atom inserted into N-methylcarbazole by cytochrome P-450 to yield N-(hydroxymethyl)carbazole does not come from the medium and show that the N-demethylation reactions catalyzed by cytochrome P-450 proceed in a manner similar to hydroxylation reactions, with the oxygen atom in the product being derived from the oxidant.

Animals↗

PubMed1980

N-Demethylation reactions catalyzed by chloroperoxidase.

The peroxidase-supported N-demethylations catalyzed by chloroperoxidase, a heme protein isolated from Caldariomyces fumago, have been investigated as models for cytochrome P-450-catalyzed N-dealkylations. The turnover number for the ethyl hydrogen peroxide-supported dealkylation of N,N-dimethylaniline by chloroperoxidase (1476) was much greater than that for cytochrome P-450-catalyzed dealkylations. The dealkylations of N,N-dimethylaniline by chloroperoxidase yielded N-methylaniline and formaldehyde in equimolar amounts with no other products detectable by high pressure liquid chromatography analysis of the reaction mixture. Ethyl hydrogen peroxidase could be replaced by other hydroperoxides, peroxides, or peracids. Chloride ions stimulated the reaction at low pH. The dealkylation reaction exhibited normal Michaelis-Menten saturation kinetics with respect to N,N-dimethylaniline (Km = 0.08 mM) and ethyl hydrogen peroxide (Km = 0.8 mM) at low substrate concentrations. However, substrate inhibition occurred at higher concentrations of N,N-dimethylaniline. The chloroperoxidase-catalyzed demethylations were inhibited by inhibitors of cytochrome P-450 such as azide or n-propyl gallate, but not by metyrapone, SKF-525A, or piperonyl butoxide. Although tiron and DL-epinephrine, trapping agents for the superoxide anion, inhibited the demethylation reactions, superoxide dismutase had no effect. There was no significant inhibition by alpha-phenyl-t-butyl-nitrone or 5,5-dimethyl-pyrroline-N-oxide, which react with free radicals. Diphenylfuran and DL-histidine, which react with singlet oxygen, did not inhibit the reaction. Substitution of D2O for H2O resulted in a marked inhibition with a solvent isotope effect (VH/VD) of 3.6. Chloroperoxidase did not catalyze the demethylation of N,N-dimethylaniline-N-oxide, indicating that the reaction does not proceed via an N-oxide intermediate.

Aniline Compounds↗

PubMedDate not supplied

Covalent interaction of 5-nitroimidazoles with DNA and protein in vitro: mechanism of reductive activation.

Human hepatic microsomal enzymes catalyzed the NADPH-dependent anaerobic reductive activation of [1-14C]metronidazole [1-(2-hydroxyethyl)-2-methyl-5-nitroimidazole] and [4,5-14C]ronidazole [(1-methyl-5-nitroimidazole-2-yl)methyl carbamate] to species that became covalently bound to proteins. Due to the low efficiency of the enzyme-catalyzed covalent binding of metronidazole, the stoichiometry of anaerobic reductive activation was studied with dithionite as the reductant. Two moles of dithionite was consumed per mole of [1-14C]metronidazole for maximal covalent binding to either DNA or immobilized sulfhydryl groups, demonstrating that four electrons are required for the reductive activation of metronidazole. These data implicate the involvement of a hydroxylamine in covalent binding. Maximal covalent binding of [4,5-14C]ronidazole to DNA also required four-electron reduction, consistent with previous studies of the covalent binding of this agent to immobilized sulfhydryl groups [Kedderis et al. (1988) Arch. Biochem. Biophys. 262, 40-48]. Studies of the covalent binding of variously radiolabeled ronidazole molecules to DNA suggested that the imidazole ring was intact while greater than 80% of the 2-carbamoyl group and the C4 proton were not present in the DNA adduct. Studies of both the chemical and human hepatic microsomal reduction of [4-3H]metronidazole demonstrated that covalent binding occurred with the stoichiometric loss of this label, implicating binding at the C4 position. These results suggest that the reductive activation of 5-nitroimidazoles generally proceeds via four-electron reduction to form hydroxylamines followed by nucleophilic attack at C4.

Adult↗

PubMedDate not supplied

Urinary metabolites of [1,2,3-13C]acrylonitrile in rats and mice detected by 13C nuclear magnetic resonance spectroscopy.

Acrylonitrile, a carcinogen in rats, undergoes extensive metabolism via two routes: direct glutathione conjugation or epoxidation. Metabolism to cyanoethylene oxide may mediate the carcinogenic and toxic activity of acrylonitrile. To characterize comprehensively the metabolism in vivo of acrylonitrile, the detection and identification of metabolites in urine of rodents dosed with acrylonitrile have been carried out using NMR spectroscopy. Following administration of [1,2,3-13C]acrylonitrile to male Fisher 344 rats (10 or 30 mg/kg, po) or B6C3F1 mice (10 mg/kg, po), urine samples were collected for 24 h. Carbon-13 NMR spectra were acquired directly on the urine samples after centrifugation and addition of 10-25% D2O. Resonances were assigned to carbons of acrylonitrile metabolites on the basis of chemical shift, proton multiplicity, carbon-carbon coupling, and calculated values of shift, and by comparison with standards. The proton multiplicity of each carbon was determined by heteronuclear 2D J-resolved spectroscopy (HET2DJ), and the carbon-carbon connectivities of resonances were determined using incredible natural abundance double quantum transfer spectroscopy (INADEQUATE). The metabolites identified in rat urine were thiocyanate, N-acetyl-S-(2-cyanoethyl)cysteine, N-acetyl-S-(2-hydroxyethyl)cysteine, N-acetyl-S-(1-cyano-2-hydroxyethyl)cysteine, thiodiglycolic acid, thionyldiacetic acid, and S-(carboxymethyl)cysteine or its N-acetyl derivative. These metabolites were also identified in mouse urine. Metabolites were quantitated by integrating metabolite carbon resonances with respect to that of dioxane added at a known concentration. Thiodiglycolic acid and (carboxymethyl)cysteine (or its N-acetyl derivative) were the major metabolites in the mouse, while N-acetyl-S-(2-cyanoethyl)cysteine and N-acetyl-S-(2-hydroxyethyl)cysteine were the major metabolites in the rat. Metabolites derived from cyanoethylene oxide (CEO) accounted for approximately 60% of the products excreted in rat urine, compared with 80% in the urine from mice. Differences between rat and mouse in the further metabolism of CEO were also observed. The proportion of the dose metabolized via CEO may be an important determinant of the toxicity and carcinogenicity of acrylonitrile.

Acrylonitrile↗

PubMedDate not supplied

Epoxidation of acrylonitrile by rat and human cytochromes P450.

The cytochromes P450 (P450) involved in the epoxidation of the rat carcinogen acrylonitrile (ACN) to the mutagen 2-cyanoethylene oxide (CEO) have been investigated in hepatic microsomes from F-344 rats and humans. Induction of P450 2E1 by acetone treatment increased the Vmax for rat microsomal CEO formation 5-fold, while ACN treatment had little effect. Treatment with beta-naphthoflavone, dexamethasone, and phenobarbital had little effect upon Vmax but increased the KM 3- to 5-fold. The P450 ligand 1-phenylimidazole and substrate ethanol were potent inhibitors of ACN epoxidation after all treatments. 2-(Diethylamino)ethyl 2,2-diphenylvalerate (SKF 525A; 0.1 mM) was not an effective inhibitor with microsomes from untreated or acetone-treated rats, but inhibited approximately 50% following dexamethasone or phenobarbital treatment. Antibodies to P450 2E1 inhibited > 85% of the ACN epoxidation activity in microsomes from untreated or beta-naphthoflavone- or acetone-treated rats, but only produced 40% and 60% inhibition following dexamethasone or phenobarbital treatments, respectively. These results indicate that P450 2E1 is the major catalyst of ACN epoxidation in untreated rats and that other forms of P450 can also epoxidize ACN. Diethyldithiocarbamate (0.1 mM) was a potent irreversible inhibitor of ACN epoxidation after all of the induction treatments, indicating that it is not specific for P450 2E1. Chlorzoxazone (2 mM) produced 75-90% inhibition after all of the induction treatments, indicating that it interacts with several rodent P450 isoforms in addition to 2E1. Human hepatic microsomes (n = 6) epoxidized ACN with Vmax'S ranging from 129 to 315 pmol of CEO formed/(min.mg of protein) and KM's from 12 to 18 microM.(ABSTRACT TRUNCATED AT 250 WORDS)

Acrylonitrile↗

PubMedDate not supplied

Biochemical basis of hepatocellular injury.

The hepatotoxic response elicited by a chemical agent depends on the concentration of the toxicant (parent compound or metabolite) delivered to the hepatocytes across the liver acinus via blood flow. Hepatotoxicants produce characteristic patterns of cytolethality in specific zones of the acinus due to the differential expression of enzymes and the concentration gradients of cofactors and toxicant in blood across the acinus. Most hepatotoxic chemicals produce necrosis, characterized by swelling in contiguous tracts of cells and inflammation. This process has been contrasted with apoptosis, where cells and organelles condense in an orderly manner under genetic control. Biotransformation can activate a chemical to a toxic metabolite or decrease toxicity. Quantitative or qualitative species differences in biotransformation pathways can lead to significant species differences in hepatotoxicity. Fasted rodents are more susceptible to the hepatotoxic effects of many chemicals due to glutathione depletion and cytochrome P-450 induction. Freshly isolated hepatocytes are the most widely used in vitro system to study mechanisms of cell death. Hepatotoxicants can interact directly with cell macromolecules or via a reactive metabolite. The reactive metabolite can alkylate critical cellular macromolecules or induce oxidative stress. These interactions generally lead to a loss of calcium homeostasis prior to plasma membrane lysis. Mitochondria have been shown to be important cellular targets for many hepatotoxicants. Decreasing hepatocellular adenosine triphosphate concentrations compromise the plasma membrane calcium pump, leading to increased cellular calcium concentrations. Calcium-dependent endonucleases produce double-strand breaks in DNA before cell lysis. These biochemical pathways induced by necrosis-causing toxicants are similar to the biochemical pathways involved in apoptosis, suggesting that apoptosis and necrosis differ in intracellular and extracellular control points rather than in the biochemistry involved in cell death.

Animals↗

PubMedDate not supplied

Metabolism of a nitrogen-containing steroid by rat hepatocytes and hepatic subcellular fractions.

Freshly isolated rat hepatocytes metabolized the nitrogen-containing steroid N,N-diethyl-4-methyl-3-oxo-4-aza-5 alpha-androst-1-ene-17 beta- carboxamide (I) to six products analyzed by reverse phase HPLC. The major metabolite, which could account for greater than 50% of the total I-related material, exhibited chromatographic, NMR, and mass spectral properties identical to those of the authentic 4-carbinolamide of I. Thus, the major biotransformation pathway in hepatocytes was hydroxylation of the N-methyl group of I to form a stable carbinolamide intermediate of N-demethylation. Desmethyl-I was observed as a minor metabolite. Another metabolite which accounted for approximately 10% of the total I-related material had chromatographic and spectral properties identical to those of the authentic monoethyl analog of I. The other three metabolites were formed in variable quantities and were unstable when isolated. Mass spectral data suggested that one metabolite was the carbinolamide intermediate of N-deethylation. Treatment of rats with phenobarbital or dexamethasone increased the formation of the monoethyl metabolite of I in hepatocytes but had no effect upon the formation of the 4-carbinolamide metabolite. Rat hepatic microsomes catalyzed the NADPH-dependent metabolism of I to the same metabolites in the same relative amounts as observed with intact hepatocytes. Studies with alternative substrates and inhibitors demonstrated that microsomal cytochrome P-450 was responsible for the metabolism of I. Dog and human hepatic microsomes metabolized I to the same products as rat hepatic microsomes, but monoethyl I was the major metabolite.

Animals↗

PubMedDate not supplied

Loss of rat liver microsomal cytochrome P-450 during methimazole metabolism. Role of flavin-containing monooxygenase.

The role of flavin-containing monooxygenase (FMO) in the decrease in cytochrome P-450 content during the microsomal metabolism of methimazole (N-methyl-2-mercaptoimidazole) was investigated by heat inactivation of FMO. Incubation of liver microsomes from untreated Fischer 344 rats with NADPH and methimazole resulted in a 25% loss of cytochrome P-450 detectable as its ferrous-carbon monoxide complex. The same extent of cytochrome P-450 loss was observed with 1 and 20 mM methimazole, suggesting saturation of the process. There was no significant loss of cytochrome P-450 when microsomal FMO was heat-inactivated prior to incubation with NADPH and methimazole. Heat pretreatment of the microsomes did not affect cytochrome P-450 concentrations and cytochrome P-420 was not observed. These results indicate that FMO-catalyzed metabolism of methimazole is necessary for the loss of cytochrome P-450 in microsomes from untreated rats. Sulfite and N-methylimidazole, the ultimate products of methimazole metabolism, did not cause a significant loss of cytochrome P-450. There was no loss of cytochrome P-450 when glutathione was included in the incubation with methimazole, suggesting that cytochrome P-450 loss was due to an interaction with oxygenated metabolites of methimazole formed by FMO. Losses of cytochrome P-450 were also observed after incubation of microsomes from phenobarbital- (31%) of beta-naphthoflavone-pretreated rats (44%) with NADPH and methimazole. In contrast to microsomes from untreated rats, heat inactivation of FMO did not prevent the loss of cytochrome P-450 in microsomes from the pretreated rats. These results indicate that both phenobarbital and beta-naphthoflavone induce isozymes of cytochrome P-450 capable of directly activating methimazole.

Animals↗