Mixed-function Oxidase Inhibitor Research Articles

Reduction of N-hydroxy-2-acetylaminofluorene by liver microsomes

The reduction of N-hydroxy-2-acetylaminofluorene( N-hydroxy-AAF) to 2-acetylamino-fluorene and 2-aminofluorene by liver microsomes was studied. The reductase preferred NADPH rather than NADH as a cofactor and was strongly inhibited by carbon monoxide and oxygen. Further, the inhibitors of hepatic mixed function oxidase effectively inhibited the reductase activity. Pretreatments of rats with 3-methylcholanthrene and PCB markedly induced the reductase activity. From these results it was suggested that the reduction of N-hydroxy-AAF was catalysed by cytochrome P-450, especially by cytochrome P-448. The reductase activity was also detected in microsomes from lung, kidney and small intestinal mucosa, and the rates of the reduction were about 6, 7 and 27 per cent, respectively, of that in liver microsomes. Species difference in the reductase activity of liver microsomes was also examined. The highest activity of the reductase was found in hamsters, followed by guinea pigs and rabbits.

Hydroxylation of benzo[ a]pyrene and binding of (−)trans 5-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene metabolites to deoxyribonucleic acid catalyzed by purified forms of rabbit liver microsomal cytochrome P-450 : Effect of 7,8-benzoflavone, butylated hydroxytoluene and ascorbic acid

The catalytic activities of hepatic microsornes from untreated, phenobarbital-treated and 3-methylcholanthrene-treated adult rabbits with respect to benzo[a]pyrene hydroxylation and the activation of (−)(rflw-7,8-dihydroxy-7,8-dihydrobenzo[ a]pyrene[(−) trans-7,8-diol] to DNA-binding metabolites were determined in the absence and presence of mixed-function oxidase inhibitors and compared to the corresponding activities of the individual enzyme systems. Treatment of rabbits with phnobarbital led to induction of P-450LM 2 and a concomitant 3-fold enhancement in microsomal benzo[ a]pyrene hydroxylase activity, whereas the conversion of (−) trans-7,8-diol to DNA-binding products was unaffected. Homogeneous phenobarbital-inducible P-450LM 2 exhibited the highest activity and specificity toward benzo[ a]pyrene and the lowest activity toward (−) trans-7,8-diol. Conversely, P-450LM 4 was the major form of cytochrome P-450 induced in rabbit liver by 3-methylcholanthrene or β-naphthoflavone, and this was associated in microsomes with an increase in the metabolism of (−) trans-7, 8-diol but not of benzo[ a]pyrene. Homogeneous P-450LM 4 preferentially Catalyzed the oxygénation of (−) trans-7,8-diol, but was largely ineffective with benzo[ a]pyrene. Partially purified P-450LM 7 lacked substrate specificity, for it metabolized both benzo[a]pyrene and (−) trans-7, S-diol at comparable rates. Additionally, 7,8-benzoflavone strongly inhibited benzo[ a]pyrene hydroxylation by P-450LM 4 and phenobarbital-induced microsomes, as well as (−) trans-7,8-diol metabolism by P-450LM 4 and 3-methyl-cholanthrene-induced microsomes; in contrast, the activity of control microsomes with either substrate, and the activities of P-450LM 4 and LM 2 with benzo[ a]pyrene and (−)trans-7 ,8-diol, respectively, were only partially or slightly decreased by 7,8-benzoflavone. Unlike 7,8-benzoflavone, butylated hydroxytoluene inhibited benzo[ a]pyrene hydroxylation only. Thus, different forms of rabbit liver microsomal cytochrome P-450 were involved in the metabolism of benzo[ a]pyrene and its 7,8-dihydrodiol. The results also demonstrate that the changes in substrate specificity and inhibitor sensitivity seen in phenobarbital- and 3-methylcholanthrene-induced microsomes relative to control rabbit liver microsomes can be accounted for by the catalytic properties of a specific form of cytochrome P-450 that prevails in these preparations, P-450LM 2 and LM 4, respectively.

Biotransformation of 7, 12-dimethylbenz (a) anthracene(DMBA): On the genesis of DMBA- [formula omitted]-dihydrodiols and DMBA- 7- and 12-methyl hydroxylated metabolites

Biotransformation of DMBA in various induced and uninduced S-10 fractions in the presence and absence of hepatic mixed function oxidase inhibitors (α-naphthoflavone, metyrapone, SKF-525A and CS 2) is described. Particular attention is paid to the production of dihydrodiols (DHD) and DMBA-7-and 12-methyl hydroxylated metabolites. It is suggested that DMBA-3, 4-DHD, 8,9-DHD and 5, 6-DHD formation and DMBA-7-and 12-methyl hydroxylations proceed predominantly via different enzyme systems.

Comparative effects of mixed function oxidase inhibitors on adrenal and hepatic xenobiotic metabolism in the guinea pig

Comparative effects of mixed function oxidase inhibitors on adrenal and hepatic xenobiotic metabolism in the guinea pig

Induction of ouabain-resistant mutation and sister chromatid exchanges in Chinese hamster cells with chemical carcinogens mediated by human pulmonary macrophages.

Pulmonary macrophages (PAM) metabolically activated benzo[a]pyrene [B(a)P] and its proximate carcinogenic metabolite, (+/-)trans 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (7,8-diol), to ultimate mutagens that were detected in cocultivated Chinese hamster V79 cells. Increases in the frequency of ouabainresistant (O(r)) mutations and sister chromatid exchanges were found in V79 cells only when they were cocultivated with both PAM and the chemical procarcinogens. 7,8-Diol caused higher frequencies of both O(r) mutations and sister chromatid exchanges than did the parent compound, B(a)P. When metabolically activated by PAM the mean O(r) mutation frequency caused by B(a)P was 9 O(r) mutants/10(6) surviving V79 cells per 10(6) PAM and a 10-fold interindividual variation (range, 2-21) was found. The mean O(r) mutation frequency caused by 7,8-diol was 64 and a ninefold interindividual variation (range, 14-120) was found. In the absence of PAM, the O(r) mutation frequency in V79 cells was one or less O(r) mutant per 10(6) survivors. 7,8-Benzoflavone, an inhibitor of mixed function oxidases, reduced the frequencies of O(r) mutations and of sister chromatid exchanges in V79 cells caused by 7,8-diol and B(a)P. As expected 7,8-benzoflavone did not influence the frequency of O(r) mutations caused by one of the ultimate mutagens derived from B(a)P and 7,8-diol, (+/-)7beta, 8alpha-dihydroxy-9alpha, 10alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene. These data are consistant with the hypothesis that PAM may play a role in the activation of environmental chemical procarcinogens.

A preliminary structure-activity study of the mixed-function oxidase inhibitor 7,8-benzoflavone.

A series of substituted and structural analogues of 7,8-benzoflavone were examined for their ability to inhibit benzo[a]pyrene oxidation by the mixed-function oxidases found in hepatic microsomes prepared from 3-methylcholanthrene- and phenobarbital-induced rats. Of all the benzoflavones tested, only 6-amino-7,8-benzoflavone possessed significant inhibitory activity toward both classes of induced mixed-function oxidases. Parameters which were found to be necessary for maximal inhibitory activity were the maintenance of an unsubstituted or specifically substituted exocyclic phenyl group on position 2, the preservation of the pyran-4-one ring, and a 6 position which is either unsubstituted or substituted with an oxidizable moiety.

Aminopyrine: metabolism and effects in the rat after administration of inhibitors of hepatic monooxygenases.

The administration to the rat of the inhibitors of microsomal mixed function oxidase, SKF 525A and Oxine-5-sulphonic acid (OSA) caused a significant decrease of the hepatic aminopyrine N-demethylase activity, as well as an increase in the plasma levels and antipyretic activity of orally administered aminopyrine. The plasma concentrations of the aminopyrine metabolite 4-aminoantipyrine were reduced in SKF 525-A treated animals while they were slightly increased in those pretreated with OSA. These findings suggest that the in vivo changes of aminopyrine disposition and activity brought about by SKF 525-A were the result of an inhibited hepatic drug metabolism, while the effects produced by OSA were due to a more rapid intestinal absorption of aminopyrine.

Identification of 7,12-dimethylbenz[a]anthracene metabolites that lead to mutagenesis in mammalian cells.

The mutagenicity of 7,12-dimethylbenz[a]-anthracene (DMBA) and 11 of its enzymatically derived metabolites was tested with Chinese hamster V79 cells for identification of mutagenic metabolites. The metabolites consisted of 7-hydroxymethyl-12-methylbenz[a]anthracene, 7-methyl-12-hydroxymethylbenz[a]anthracene, 7,12-dihydroxymethylbenz[a]anthracene, three trans-3,4-diols, two trans-5,6-diols, and three trans-8,9-diols, all of which derived from DMBA or from the hydroxymethyl derivatives. Mutations were characterized by resistance to ouabain and 6-thioguanine. None of the tested metabolites were mutagenic in V79 cells, which do not metabolize polycyclic aromatic hydrocarbons. Therefore, mutagenesis in the V79 cells was tested in the presence of golden hamster cells capable of metabolizing polycyclic aromatic hydrocarbons (cell-mediated assay). In this assay, DMBA, 7-hydroxymethyl-12-methylbenz[a]anthracene, 7-methyl-12-hydroxymethylbenz[a]anthracene, and their trans3,4-diols were mutagenic for both genetic markers, and the mutagenic response increased as a function of the hydrocarbon dose. All other metabolites were either inactive or showed up to a 4-fold higher mutation frequency than the untreated V79 cells for ouabain and 6-thioguanine resistance. The DMBA-trans-3,4-diol was the only metabolite that was more active than DMBA itself; at 0.05 muM it was 6-8 times more active than DMBA itself; at 0.05 muM it was 6-8 times more active than DMBA in inducing both ouabain and 6-thioguanine resistance. This diol was mutagenic at a dose as low as 0.01 muM. Mutagenesis by DMBA and the trans-3,4-diols was inhibited by 7,8-benzoflavone, an inhibitor of mixed-function oxidases. Analysis of DMBA metabolism in intact golden hamster cells indicated that DMBA-trans-3,4-diol is one of the major metabolites produced. Our results therefore suggest that DMBA-trans-3,4-diol may be metabolized to a diol-epoxide, presumably the trans-3,4-diol-1,2-epoxide, which may be a major reactive metabolite responsible for DMBA mutagenicity in mammalian cells.

Inhibition of ethanol and acetate metabolism in mice by chlordimeform and related compounds

1. Since chlordimeform or N′-(4-chloro- o-toly l)- N, N-dimethylformamidine and certain of its mammalian metabolites prolong ethanol induced sleep in mice, their effect on ethanol and acetate metabolism was examined. 2. Intraperitoneal treatment of mice with chlordimeform (50 mg/kg) and demethylchlordimeform or N′-(4-chloro- o-tolyl)- N-methylformamidine (50 mg/kg), the two most potent sleep inducers, appreciably inhibited the metabolism of ethanol to 14CO 2. Another metabolite, 4-chloro- to-formotoluidide (100 mg/kg), a very weak sleep inducer, also markedly inhibited ethanol metabolism. 3. Chlordimeform, demethylchlordimeform, and 4-chloro- o-formotoluidide inhibited the conversion of ethanol to 14CO 2 and the conversion of acetate to 14CO 2 in vitro by mouse liver preparations. 4. The potency of 4-chloro- o-formotoluidide as an inhibitor of acetate metabolism in vitro compared favorably with that of SKF-525A, a known inhibitor of microsomal mixed function oxidases. 5. At a concentration of 1 × 10 −3 M, demethylchlordimeform yielded 27.4% inhibition of yeast alcohol dehydrogenase as compared to 46.3% for thiourea, a known inhibitor of this enzyme. The other compounds gave little, if any, inhibition of yeast alcohol or aldehyde dehydrogenase. 6. Although it is not possible to explain the mechanism for ethanol sleep enhancement in mice by chlordimeform and its metabolite demethylchlordimeform, inhibition of the alcohol dehydrogenase by demethylchlordimeform may be significant in this connection. 7. The decrease in ethanol metabolism apparently was due in large part to the activity of chlordimeform, demethylchlordimeform and especially 4-chloro- o-formotoluidide as inhibitors of microsomal mixed function oxidases.

Inhibition of epoxidation of methyl farnesoate to juvenile hormone III by cockroach corpus allatum homogenates

Radiolabeled methyl farnesoate is epoxidized to juvenile hormone III by an NADPH-dependent reaction occurring in corpus allatum homogenates from the cockroach Blaberus giganteus L. Most of the enzymatically produced juvenile hormone has the 10 R configuration described for previously isolated natural juvenile hormones. The unnatural 2 Z geometrical isomer of methyl farnesoate is epoxidized by the above system faster than the natural 2 E isomer. Several series of chemicals known to be inhibitors of mixed-function oxidases were surveyed as inhibitors of methyl farnesoate epoxidation. The anti-juvenile hormone precocene II caused negligible inhibition at 1 · 10 −4 M, whereas the best inhibitor was o-bromophenoxymethyl-imidazole with an apparent I 50 of 4 · 10 −7 M. None of the inhibitors tested were potent morphogenetic agents on Tenebrio molitor pupae, and they failed to cause precocious development of Oncopeltus fasciatus nymphs. The inhibition of in vitro juvenile hormone biosynthesis suggests the possibility of finding an anti-hormone which acts by blocking juvenile hormone biosynthesis.

Acute inhibition of microsomal drug metabolism by propoxyphene in narcotic tolerant/dependent mice

Propoxyphene (Darvon) was compared to SKF 525-A, a prototypical inhibitor of hepatic microsomal mixed function oxidases, to assess propoxyphene's potential to inhibit drug metabolism in morphine tolerant/dependent mice. In vitro , both propoxyphene (K i = 3.5 × 10 −5M) and SKF 525-A (K i = 4.3 × 10 −6M) inhibited the activity of aminopyrine N-demethylase competitively in hepatic microsomes from tolerant/dependent animals. Propoxyphene and SKF 525-A were weaker, noncompetitive inhibitors of aniline hydroxylase activity. In vivo , equimolar doses (0.24 mmoles/kg, i.p.) of each compound inhibited both of the above monooxygenases in the 10,000 g supernatant fractions of livers from the tolerant/ dependent animals. Propoxyphene was 40–50% as potent an inhibitor of these activities as SKF 525-A. A dose (300 mg/kg) of propoxyphene napsylate, shown to prevent narcotic abstinence signs with no observable toxicity in withdrawing mice, significantly prolonged the blood levels of injected pentobarbital and tripled pentobarbital sleeping time in these animals. When administered at 300 mg/kg chronically, propoxyphene napsylate acted as an inducer of its own metabolism. Propoxyphene napsylate, then, given acutely to narcotic tolerant/dependent mice, is a potent inhibitor of microsomal drug metabolizing capacity. Given chronically, it enhances this capability.

Mechanisms of Resistance to Bromophos-ethyl in Two Strains of the Cattle Tick Boophilus microplus

B. micro plus larvae of the organophosphorus-resistant Biarra and Mt Alford strains were respectively 8 x and 35 x resistant to bromophos-ethyl compared with larvae of the standard organophosphorussusceptible Yeerongpilly strain. Both resistant strains had acetylcholinesterase with decreased sensitivity to inhibition by the oxon of bromophos-ethyl in vitro; this was the only resistance mechanism apparent in the Biarra strain but Mt Alford larvae were protected additionally by increased metabolism of the oxon in vivo to water-soluble products. Total degradation rates for the parent chemical were similar in all strains and relatively slow. Both bromophos-ethyl and its oxon were potent inhibitors of mixed-function oxidase in vivo and it seemed that the slow oxidative metabolism of bromophos-ethyl (the major pathway) could be attributed to substrate and/or product inhibition. No phenolic metabolites were detected and the major water-soluble metabolite was identified electrophoretically as 2,5-dichlorophenyl-o-ethyl phosphate. Some debrominated oxon was detected in all samples of larvae after dosage with bromophos-ethyl or with its oxon, indicating that oxidation of bromophos-ethyl to the oxon, debromination of the oxon followed by deethylation was a major degradative sequence.

Catechol estrogen-forming enzyme of brain: demonstration of a cytochrome p450 monooxygenase.

A catechol estrogen-forming enzyme has been demonstrated in rat brain microsomes using a sensitive radioenzymatic assay. This method is based on conversion of the relatively labile catechol estrogens to their stable O-methylated derivatives. By employing a methyl donor of high specific activity (S-adenosyl-L-[methyl-3H]methionine), a partially purified preparation of catechol- O-methyltransferase (COMT), and selective solvent extraction this method has proven to be extremely sensitive. The specificity of the assay was established by subjecting the O-methylated product to thin layer chromatography in several solvent systems and further confirmed by mass spectral analysis of the chromatographed product. The enzymatic activity of rat brain is optimal using reduced NADP and is inhibited by well known inhibitors of P450-dependent mixed function oxidases, CO and SKF- 525A. The subcellular distribution, cofactor requirements, and the effects of various inhibitors strongly suggest that the enzymatic activity of b...

Inhibitors of hepatic mixed function oxidase. 3. Inhibition of hepatic microsomal aniline hydroxylase and aminopyrine demethylase by 2,6- and 2,4-dihydroxyphenyl alkyl ketones and related compounds.

A series of 2,6- and 2,4-dihydroxyphenyl alkyl ketones has been investigated as inhibitors of hepatic microsomal aniline hydroxylase and aminopyrine demethylase activities. Structural alterations in both series did little to enhance the inhibitory activity of the parent compounds 2,6-dihydroxyacetophenone (3) and 2,4-dihydroxyacetophenone (27). In the 2,6 series activity against both microsomal systems varied only over a relatively narrow range, 6-allyloxy-2-hydroxyacetophenone (19) being the most potent inhibitor. In the 2,4 series, activity against aniline hydroxylase was poor or absent in most cases. tthe most potent inhibitor was 5-ethyl-2,4-dihydroxyacetophenone (31). In contrast, high activity against aminopyrine demethylase was frequently displayed in this series, 3,5-dibromo-2,4-dihydroxypropiophenone (36) showing greatest inhibitory potency. The effects of some compounds on hexobarbital sleeping times and zoxazolamine paralysis times in mice were also examined.

Metabolism [formula omitted] of dioxane: effect of inducers and inhibitors of hepatic mixed-function oxidases

Metabolism [formula omitted] of dioxane: effect of inducers and inhibitors of hepatic mixed-function oxidases

Biosynthesis of Cutin ω-Hydroxylation of Fatty Acids by a Microsomal Preparation from Germinating Vicia faba

omega-Hydroxylation of fatty acids, which is a key reaction in the biosynthesis of cutin and suberin, has been demonstrated for the first time in a cell-free preparation from a higher plant. A crude microsomal fraction (105,000g pellet) from germinating embryonic shoots of Vicia faba catalyzed the conversion of palmitic acid to omega-hydroxypalmitic acid. As the crude cell-free preparation also catalyzes the formation of other hydroxy acids such as alpha- and beta-hydroxy acids, the omega-hydroxylation product was identified by gas chromatography on a polyester column and reverse phase, high performance liquid chromatography, two techniques which were shown to resolve the positional isomers. Gas chromatographic analysis of the dicarboxylic acid obtained by CrO(3) oxidation of the enzymic product also confirmed the identity of the enzymic omega-hydroxylation product. This enzymic hydroxylation required O(2) and NADPH, but substitution of NADH resulted in nearly half the reaction rate obtained with NADPH. Maximal rates of omega-hydroxylation occurred at pH 8 and the rate increased in a sigmoidal manner with increasing concentrations of palmitic acid. This omega-hydroxylation was inhibited by the classical mixed function oxidase inhibitors such as metal chelators (o-phenanthroline, 8-hydroxyquinoline, and alpha,alpha-dipyridyl), NaN(3) and thiol reagents (N-ethylmaleimide and p-chloromercuribenzoate). As expected of a hydroxylase, involving cytochrome P(450), the present omega-hydroxylase was inhibited by CO and this enzyme system showed unusually high sensitivity to this inhibition; 10% CO caused inhibition and 30% CO completely inhibited the reaction. Another unusual feature was that the inhibition caused by any level of CO could not be reversed by light (420-460 nm).

Inhibitors of hepatic mixed function oxidases—II Some benzimidazole, benzoxazole and benzothiazole derivatives

Inhibitors of hepatic mixed function oxidases—II Some benzimidazole, benzoxazole and benzothiazole derivatives

Identification of mutagenic metabolites of benzo(a)pyrene in mammalian cells.

The mutagenicity of benzo[a]pyrene and 15 of its derivatives, which included phenols, the benzo[a]yrene-4,5-epoxide (the K-region epoxide), dihydrodiols, two isomeric 7,8-diol-9,10-epoxides, a 6-methyl derivative, and a 6-hydroxymethyl derivative, were tested with Chinese hamster V79 cells in order to identify the mutagenic metabolites of benzo[a]pyrene. Mutations were characterized by resistance to ouabain or 8-azaguanine. Since V79 cells do not metabolize polycyclic hydrocarbons, mutagenesis was tested both in the presence and absence of benzo[a]pyrene-metabolizing normal golden hamster cells. All the tested phenols, 4,5-diols, trans-9,10-diol, 6-methyl, and 6-hydroxymethyl derivatives of benzo[a]pyrene showed little or no mutagenicity for both genetic markers. The (+/-)7alpha,8beta-dihydroxy-9alpha,10alpha-epoxy-7,8;9,10-tetrahydrobenzo[a]pyrene and K-region 4,5-epoxide exhibited similar and moderate mutagenicity in the absence of benzo[a]pyrene-metabolizing cells, but the (+/-)7alpha,8beta-dihydroxy-9beta,10beta-epoxy-7,8,9,10-tetrahydrobenzo[a]-pyrene showed a 2000- and 270-fold higher mutation frequency for ouabain and 8-azaguanine resistance, respectively, than did the K-region 4,5-epoxide. The trans-7,8-diol which was not mutagenic in the absence of benzo[a]pyrene-metabolizing cells was more mutagenic than benzo[a]pyrene after metabolism and mutagenesis by trans-7,8-diol in these cells was inhibited by 7,8-benzoflavone, an inhibitor of mixed-function oxidases. Metabolically formed trans-7,8-diol was isolated and incubated with rat liver microsomes in the presence of co-factors. High-pressure liquid chromatography analysis indicated that the major metabolite of trans-7,8-diol is 7alpha,8beta-dihydroxy-9beta,10beta-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene. The results indicate that the latter compound is metabolically formed and the major mutagenic intermediate of benzo[a]yrene metabolism.

Microsomal hydroxylation of aniline in the southern armyworm, Spodoptera eridania

Microsomal hydroxylation of aniline in midguts of the southern armyworm, Spodoptera eridania, was found to differ in its requirements for optimal in vitro activity from those of armyworm and rat liver p- chloro N- methylaniline N- demethylase and rat liver aniline hydroxylase. Apparent K m values for armyworm aniline hydroxylase were substantially higher than those for rat liver aniline hydroxylase and the N- demethylase enzymes. The armyworm aniline hydroxylase was inhibited by several mixed-function oxidase inhibitors as well as by aldrin and nicotine and was highly inducible by pentamethylbenzene and phenobarbital.

Plasticizers in the environment: the fate of di-N-octyl phthalate (DOP) in two model ecosystems and uptake and metabolism of DOP by aquatic organisms.

The fate of the plasticizer, di-n-octyl phthalate (DOP) has been examined in 33-day terrestrial-aquatic and three-day aquatic model ecosystems. The five organisms of the two systems contained residues of DOP, demonstrating the propensity of this lipoid soluble organic molecule to be concentrated from the water. The residues in the organisms in the three-day system were higher than in the 33-day system with the exception of the fish, indicating perhaps, that DOP can undergo some degradation before the fish is placed in the system on the 30th day. A half-life of five days for DOP disappearance from the water was calculated from water samples taken periodically. Further, effects of mixed function oxidase and esterase inhibitors were investigated on the metabolism of DOP by various selected organisms and tissues of the two ecosystems.