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W G Levine

Publications and source records attributed to W G Levine.

At least 19 recordsLinked to original sources
PubMed1992

Azoreductase activity by purified rabbit liver aldehyde oxidase.

Our laboratory has investigated the azoreduction of dimethylaminoazobenzene (DAB) and its analogs by hepatic microsomal cytochrome P450. We have extended these studies to the cytosolic fraction of the mammalian liver using the molybdoflavoenzyme, aldehyde oxidase. Purified rabbit liver aldehyde oxidase readily reduced azo dyes which are mainly water soluble and contain charged groups. Lipophilic azo dyes, although readily reduced by microsomal cytochrome P450, were either poor substrates or not reduced at all. Kinetic measurements revealed no relationship between Vmax and Km for all dyes. More extensive studies were conducted on four azo dyes, o-methyl, red, 2'-pyridyl-DAB, sulfonazo III and Orange II, with characteristic functional groups. With each of these substrates, azoreductase activity was greatest when 2-hydropyrimidine (2-OHP) was the electron donor compared to N1-methylnicotinamide (N-MN), propionaldehyde and butyraldehyde. With 2-OHP as the electron donor, o-methyl red and 2'-pyridyl DAB exhibited maximal activity at pH 5.0 while sulfonazo III and Orange II showed maximal activity at pH 9.5 and 7.0, respectively. Km values for o-methyl red and 2'-pyridyl DAB were lower at their pH optima whereas that for sulfonazo III was higher at its pH optimum. There was also no correlation between maximal activity and Km; apparently Km is not a primary determinant for activity. The degree of ionization of function groups depends on pH. Since highest activity is seen at that pH in which maximal ionization of the substrate occurs, it can be concluded that rate of reduction is at least partially dependent on the charged state of the substrate. Azoreduction was inhibited by menadione and SKF 525-A. Sensitivity to inhibition by menadione was greatest at the pH where 2-OHP exhibited considerably higher activity than N-MN, but no differential was seen at the pH where activities with the two-electron donors were similar. On the other hand, sensitivity of azoreductase activity to inhibition by SKF 525-A was the same irrespective of electron donor, indicating that the mechanisms for these two inhibitors were different.

Aldehyde Oxidase↗
PubMed1992

Role of electronic factors in binding and reduction of azo dyes by hepatic microsomes.

The factors which regulate the binding and reduction of azo dyes by rat liver microsomes have been investigated. Azo dyes having both electron-donating and -withdrawing substituents were reduced more readily both enzymically and chemically (dithionite) than those containing electron-donating substituents alone, which is consistent with less negative oxidation-reduction potentials of the former. A linear correlation between Vmax and Km was seen for substrates having only electron-donating substituents, suggesting a possible inverse relation between binding affinity and rate of reduction. This relationship was not apparent for substrates having both electron-donating and -withdrawing substituents. A lower Km was seen with substrates having a greater number of heteroatoms bearing nonbonding electrons in either electron-donating or -withdrawing groups. Furthermore, more basic dyes, which have a higher density of nonbonding electrons, showed an inverse correlation with both Vmax and Km. The requirement of nonbonding electrons was also observed with the binding of the fully reduced amine metabolites to microsomal cytochrome P-450. Type II binding spectra were observed for both aniline and 4-chloroaniline, but not with aniline derivatives bearing electron-withdrawing substituents such as methyl anthranilate and methyl-4-aminobenzoate. Electron-withdrawing substituents increase delocalization of nonbonding electrons on the amino residue; consequently, these are no longer available for binding to the enzyme. The binding constant of the reduced metabolite, aniline (36 microM), relative to the Km values of substrate azo dyes (range 0.31-1.73 microM) implies that the more weakly bound amine metabolites are readily released from the binding/catalytic site.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Protein binding, nuclear translocation and biliary secretion of metabolites of 3'-methyl-N,N-dimethyl-4-aminoazobenzene during hepatocarcinogenesis in rats.

1. The hepatic content, biliary excretion, cytosolic protein binding and nuclear translocation of metabolites of i.v. administered 14C-3'-methyl-N,N-dimethyl-4-aminoazobenzene (3'-methyl-DAB) were investigated in rats at various stages of 2-acetamidofluorene (AAF)-induced hepatocarcinogenesis. 2. At nodular and post-nodular stages biliary excretion of radioactive metabolites was decreased, although hepatic content of radioactivity was similar to controls not dosed with AAF. The secretion in bile of a major azo dye binding protein was also decreased at these stages. 3. Binding of dye metabolites to cytosolic proteins was decreased by 40% at nodular and post-nodular stages compared to controls. 4. Translocation in vitro of dye metabolites from cytosol to nucleus at nodular and post-nodular stages was 40% less than that of controls. Since specific soluble proteins control translocation from cytosol into the nucleus (and bile), this decreased binding of metabolites may explain the diminished translocation of carcinogen metabolites into the nucleus.

2-Acetylaminofluorene↗
PubMed1991

Metabolism of azo dyes: implication for detoxication and activation.

Azo dyes are consumed and otherwise utilized in varying quantities in many parts of the world. Such widely used chemicals are of great concern with regard to their potential toxicity and carcinogenic properties. Their metabolism has been studied extensively and is significant for detoxication and metabolic activation. Both oxidative and reductive pathways are involved in these processes. The majority of azo dyes undergo reduction catalyzed by enzymes of the intestinal microorganisms and/or hepatic enzymes including microsomal and soluble enzymes. The selectivity of substrate and enzyme may to a large extent be determined by the oxygen sensitivity of reduction since a normal liver is mainly aerobic in all areas, whereas the microorganisms of the lower bowel exist in an anaerobic environment. However, it should be pointed out that the pO2 of centrilobular cells within the liver is only a fraction that of air, where pO2 = 150 torr. Therefore, an azo dye reduction experiment performed aerobically may not be an accurate predictor of reductive metabolism in all areas of the liver. Many of the azo dyes in common use today have highly charged substituents such as sulfonate. These resist enzymic attack and for the most part are poorly absorbed from the intestinal tract, providing poor access to the liver, the major site of the mixed-function oxidase system. Lipophilic dyes, such as DAB, which are often carcinogenic, readily access oxidative enzymes and are activated by both mixed-function oxidase and conjugating systems. Reduction of the carcinogenic dyes usually leads to loss of carcinogenic activity. By contrast, most of the highly charged water-soluble dyes become mutagenic only after reduction. Even then, most of the fully reduced amines required oxidative metabolic activation. An outstanding example is the potent human bladder carcinogen benzidine, which derives from the reduction of several azo dyes. Many problems regarding mutagenic and carcinogenic activation remain to be solved. At the present time, it is apparent that both oxidative and reductive pathways yield toxic products. Toxicologic assessment of azo dyes must consider all pathways and particularly the oxygen sensitivity of azoreduction. This is critical in the treatment of waste from chemical plants where there is a great need for soil bacteria which catalyze reduction aerobically. Consideration of secondary pathways are also of great concern. For example, azoreduction of carcinogenic dyes such as DAB removes carcinogenic activity although oxidative metabolism of the primary amines yield mutagenic products. Such apparent dilemmas must be dealt with when considering metabolism/toxicity relationships for azo dyes.

Animals↗
PubMed1990

Characteristics of two classes of azo dye reductase activity associated with rat liver microsomal cytochrome P450.

Azo dyes are reduced to primary amines by the microsomal enzymes NADPH-cytochrome P450 reductase and cytochrome P450. Amaranth, a highly polar dye, is reduced almost exclusively by rat liver microsomal cytochrome P450 and the reaction is inhibited almost totally by oxygen or CO. Activity is induced by pretreatment with phenobarbital or 3-methylcholanthrene. In contrast, microsomal reduction of the hepatocarcinogen dimethylaminoazobenzene (DAB), a lipid soluble, weakly polar compound, is insensitive to both oxygen and CO. However, reconstitution of activity with purified NADPH-cytochrome P450 reductase and a partially purified cytochrome P450 preparation indicates that activity is catalyzed almost exclusively by cytochrome P450. Activity is induced by clofibrate but not phenobarbital, beta-naphthoflavone, 3-methylcholanthrene, isosafrol, or pregnenolone-16 alpha-carbonitrile. These observations suggest the existence of at least two classes of azoreductase activity catalyzed by cytochrome P450. To investigate this possibility, the reduction of a number of azo dyes was investigated using microsomal and partially purified systems and the characteristics of the reactions were observed. Microsomal reduction of azo dyes structurally related to DAB required a polar electron-donating substituent on one ring. Activity was insensitive to oxygen and CO if the substrates had no additional substituents on either ring or contained only electron-donating substituents. Introduction of an electron-withdrawing group into the prime ring conferred oxygen and CO sensitivity on the reaction. Substrates in the former group are referred to as insensitive and substrates in the latter group as sensitive. Inhibitors of cytochrome P450 activity depressed reduction of both insensitive and sensitive substrates. In a fully reconstituted system containing lipid, highly purified NADPH-cytochrome P450 reductase and a partially purified cytochrome P450 preparation, rates of reduction of various insensitive substrates varied several-fold, whereas rates of reduction of sensitive substrates varied by three orders of magnitude. Using purified enzymes, each of the insensitive substrates was shown to be reduced by reductase alone, but only at a fraction of the rate seen in the fully reconstituted system, implying that reducing electrons were transferred to the dyes mainly from cytochrome P450. Conversely, there was substantial, in some cases almost exclusive, reduction of sensitive substrates by purified reductase alone and almost no inhibition by CO. Their reduction, however, was inhibited by CO in microsomal systems.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Studies on the mechanism of reduction of azo dye carcinogens by rat liver microsomal cytochrome P-450.

This laboratory has described the azoreduction of p-dimethylaminoazobenzene (1c) by rat liver microsomal cytochrome P-450. To elucidate the mechanisms involved, the reduction of structurally related azobenzenes by hepatic microsomes was investigated. High substrate reactivity was observed for 1c, its corresponding secondary (1a) and primary (1b) amines and p-hydroxyazobenzene (1d). In contrast, only negligible rates were obtained for unsubstituted azobenzene (1g), hydrazobenzene (2g), p-isopropylazobenzene (1e) and 1f, the benzoylamide derivative of 1b. These results clearly indicate that electron-donating groups, such as hydroxyl or primary, secondary and tertiary amines, are essential for binding of azo dye carcinogens to liver microsomal cytochrome P-450 and, by implication, their enzymic reduction. No inhibition of azoreduction of 1c or 1d was obtained by addition of 1e, 1g, or 2g to the reaction mixture. In the presence of hepatic microsomes, a type I binding spectrum was obtained for 1d and type II binding spectra for 1a, 1b and 1c, the reactive azo dyes. In contrast, very weak binding was observed for the unreactive compounds 1e, 1f, 1g and 2g. Thus, there is good correlation between binding and substrate reactivity. The apparent lack of binding may explain the inability of the non-reactive compounds to inhibit azoreduction. The difference in the reduction rate observed for 1g vs. 1d suggested that hydroxylation would facilitate the reduction of an otherwise non-reactive azo dye. Support for such a mechanism was obtained in two experiments. In the first, marked facilitation of azoreduction of both the inactive compounds, 2g and 2f, was seen when they were incubated with microsomes under aerobic conditions where preliminary hydroxylation can occur. In the second, azobenzene was initially incubated aerobically with microsomes from phenobarbital- or beta-naphthoflavone-induced rats. The hydroxyazobenzene formed was then readily reduced anaerobically by microsomes from untreated rats.

Animals↗
PubMed1987

In vivo and in vitro effects of amrinone and milrinone on hepatic xenobiotic metabolism in rats.

Studies were performed on the response of hepatic xenobiotic metabolizing enzymes to in vitro and in vivo exposure to amrinone and milrinone, two new inotropic compounds used in congestive heart failure. Both drugs exerted selective effects on various cytochrome P-450-dependent metabolic activities as well as conjugating pathways. Aminopyrine N-demethylation was selectively inhibited by in vitro addition of milrinone but not amrinone, and laurate hydroxylation was inhibited by both drugs. Cytosolic glutathione-S-transferase activity was profoundly inhibited by in vitro addition of both drugs. In vivo administration of either drug did not lead to significant inhibition of the pathways studied other than laurate hydroxylation which was depressed 20-30%. Irreversible binding of [14C]-amrinone-derived radioactivity to microsomal protein was partially NADPH-dependent. Inhibition by SKF 525-A, alpha-naphthoflavone and various antioxidants was observed. No binding of [14C]-milrinone-derived radioactivity was seen. It is suggested that amrinone may selectively inhibit certain hepatic drug-metabolizing enzymes through metabolic electrophilic intermediates.

Amrinone↗
PubMed1987

Microsomal azoreduction and glucuronidation in the metabolism of dimethylaminoazobenzene by the rat liver.

1. Enzymic azoreduction of the hepatocarcinogen, N,N-dimethyl-4-aminoazobenzene (DAB) and glucuronidation of its ring-hydroxylation product, 4'-hydroxy-DAB, by hepatic microsomal fractions in vitro were studied during an eight day period of hepatic regeneration following partial hepatectomy in Wistar rats. Azoreduction of DAB and its N-demethylated metabolites did not significantly change during hepatic regeneration in contrast to N-demethylation of these dyes which is profoundly suppressed during regeneration. UDP-Glucuronosyltransferase (UDP-GT) activity towards 4'-hydroxy-DAB was partially depressed during the regeneration period, but the depression was considerably less than that for bilirubin. Transferase activity towards 4-nitrophenol, after initial depression, returned to normal levels after the third day of partial hepatectomy. 2. In Gunn rats, microsomal UDP-GT activity towards bilirubin was undetectable, whereas transferase activity toward 4-nitrophenol was 50% of normal. Addition of diethylnitrosamine (DEN) in vitro restored transferase activity towards 4-nitrophenol to normal levels, but the activity towards bilirubin was unaffected. Gunn rat UDP-GT activity towards 4'-hydroxy-DAB was 25% of normal and was partially activated upon addition of DEN in vitro. 3. Treatment with clofibrate of beta-naphthoflavone induced hepatic microsomal bilirubin- and 4-nitrophenol-specific UDP-GT activities, respectively; both agents induced transferase activity towards 4'-hydroxy-DAB. Triiodothyronine, which induces 4'-nitrophenol-specific UDP-GT and depresses bilirubin-specific UDPG, had little effect on 4'-hydroxy-DAB UDP-GT activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Effect of phenobarbital and beta-naphthoflavone on oxidative metabolism of N,N-dimethyl-4-aminoazobenzene by regenerating rat-liver microsomes and its response to sulphydryl compounds.

The metabolism of the hepatocarcinogen, N,N-dimethyl-4-aminoazobenzene (DAB) is catalysed by selective forms of cytochrome P-450. DAB metabolism has been studied using microsomes from regenerating rat liver prepared 1, 2, 3, 7 and 10 d after partial hepatectomy. Greatly decreased N-demethylation of DAB was seen during liver regeneration, while virtually no effect on ring-hydroxylation was observed. Glutathione stimulated N-demethylation and ring-hydroxylation of DAB, while metabolism of the corresponding secondary amine N-methyl-4-aminoazobenzene (MAB) was not affected. During regeneration, response to the thiol was depressed in the early stages but later returned to normal. beta-Naphthoflavone (BNF) specifically induced N-demethylation of DAB. Induced activity was not depressed during liver regeneration. Phenobarbital (PB) induced total metabolism, which was depressed during regeneration. This indicates greater stability of BNF-induced cytochrome P-450 compared to control and PB-induced cytochrome P-450. The results indicate that during liver regeneration the metabolism of DAB associated with activation (N-demethylation) is depressed, whereas that associated with detoxication (ring-hydroxylation) is only slightly affected. This confirms the involvement of different forms of cytochrome P-450 in DAB metabolism.

Animals↗
PubMed1985

Studies on microsomal azoreduction. N,N-dimethyl-4-aminoazobenzene (DAB) and its derivatives.

The azoreduction of N,N-dimethyl-4-aminoazobenzene (DAB) and N-methyl-4-amino-azobenzene (MAB) by rat liver microsomes was investigated. It was shown that measurement of azoreduction of DAB and structurally related azo dyes by the conventional method of substrate disappearance required an anaerobic environment since N-demethylated and ring-hydroxylated metabolites formed aerobically interfered with the assay system, producing quantitatively inaccurate results. Oxygen partially, but not totally, inhibited azoreduction of DAB. Glutathione (GSH) inhibited the azoreduction of DAB but stimulated the azoreduction of MAB. Dithiothreitol also stimulated azoreduction of MAB but had little effect on azoreduction of DAB. Para-hydroxymercuribenzoate (PHMB) and N-ethylmaleimide (NEM) blocked titratable microsomal thiol groups and inhibited azoreduction of MAB. However, the inhibitory action of NEM was weak with DAB azoreduction although PHMB was a potent inhibitor. These findings suggest that microsomal azoreduction of DAB and MAB may proceed via different mechanisms, possibly through different species of cytochrome P-450 which have selective dependence upon the sulfhydryl environment.

Aerobiosis↗
PubMed1985

Absence of a promoting or sequential syncarcinogenic effect in rat liver by the carcinogenic hypolipidemic drug nafenopin given after N-2-fluorenylacetamide.

The hypolipidemic agent nafenopin, (NF), has been reported to be carcinogenic to rat liver. To determine whether nafenopin exerts a promoting or syncarcinogenic effect in rat liver, its effect on liver carcinogenesis induced by N-2-fluorenylacetamide (FAA) was studied. In two separate experiments, male F344 rats were fed 0.02% FAA for either 10 or 8 weeks to induce preneoplastic liver lesions. Following a recovery period of 1 week, rats were given 0.01 or 0.02% NF in the diet for 23 weeks in one experiment and 0.05 or 0.1% for 24 weeks in the other. The final incidence of neoplasms, and their numbers, size distribution, and degrees of differentiation were not significantly different in groups given NF after FAA compared to those maintained on a basal diet after FAA. In the group treated with the highest dose level of NF following FAA, however, there was a decrease in the number of grossly visible small neoplasms. In contrast, the liver neoplasm promoter phenobarbital increased the multiplicity, although not the incidence, of liver neoplasms when given after FAA. Thus, four different dose levels of NF showed no promoting or syncarcinogenic effect on FAA-induced hepatocarcinogenesis.

2-Acetylaminofluorene↗
PubMed1985

Effect of long-term administration of delta 9-tetrahydrocannabinol on hepatic mixed-function oxidase systems in the rat.

Chronic oral treatment of young female Fischer rats with 25 mg/kg delta 9-tetrahydrocannabinol (THC) per day inhibited aminopyrine demethylation and significantly increased benzo[a]pyrene oxidation by the liver. THC treatment also elevated serum corticosterone levels and produced a significant loss of body weight. The weight loss was not due to vehicle or food intake (pair-feeding). Pair-feeding did, however, produce a stimulation of both mixed function oxidase pathways as well as a marked elevation in serum corticosterone levels. The results indicate that THC has a differential effect on mixed function oxidase pathways in the liver that is not directly related to food intake or corticosterone levels.

Aminopyrine↗
PubMed1984

Effects of butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) on metabolism of N,N-dimethyl-4-aminoazobenzene (DAB) by rat liver microsomes.

The antioxidants, BHA and BHT, inhibited the N-demethylation and ring hydroxylation of N,N-dimethyl-4-aminoazobenzene (DAB) by liver microsomes from untreated and phenobarbital (PB)-treated rats. BHA was somewhat more potent in this regard than was BHT. Microsomal NADPH oxidase from PB-treated rats was stimulated by BHA but control microsomal activity was unaffected. Glutathione did not appreciably reverse the inhibitory effect of BHA on DAB metabolism and had no effect on NADPH oxidase activity. It is concluded that inhibition of DAB metabolism by BHA cannot be accounted for by interaction with NADPH oxidase, particularly in untreated microsomes. It more likely affects one or more species of cytochrome P-450.

Animals↗
PubMed1983

Cytosolic factors that alter the metabolism of N,N-dimethyl-4-aminoazobenzene by rat liver microsomes.

N,N-Dimethyl-4-aminoazobenzene (DAB), an azo dye carcinogen, is N-demethylated and 4'-hydroxylated by rat liver microsomes. Addition of hepatic cytosol to the microsomal system stimulated both pathways. This occurred in the presence of added NADPH or an NADPH-generating system. Cytosol was effective only when present prior to addition of substrate; no stimulation was seen when added after the reaction had begun. This suggested a direct effect on the microsomes rather than a chemical interaction with one or more metabolic intermediates of DAB. The degree of stimulation was somewhat different when using microsomes from phenobarbital- or beta-naphthoflavone-treated animals, implying a selectivity of the cytosolic effect for various isozymes of cytochrome P-450. Some loss of stimulatory activity occurred with dialysis. Activity was restored by adding back glutathione (GSH) which can stimulate DAB metabolism even in the absence of cytosol. DAB metabolism is also stimulated by EDTA. Although both EDTA and cytosol inhibit lipid peroxidation, cytosol stimulated DAB metabolism even in the presence of EDTA. Therefore, suppression of lipid peroxidation does not explain satisfactorily the cytosolic effect. Separation of cytosolic proteins by gel filtration revealed a factor which inhibits N-demethylation but not 4'-hydroxylation of DAB. Heating at 100 degrees partially inactivated the stimulatory activity. However, inhibitory activity was less susceptible to heat inactivation than was stimulatory activity. These results indicate that, in the whole cell, microsomal metabolism of xenobiotics is regulated to an appreciable extent by macromolecular cytosolic substances.

Animals↗