21 results on '"Levine WG"'
Search Results
2. Characteristics of two classes of azo dye reductase activity associated with rat liver microsomal cytochrome P450.
- Author
-
Zbaida S and Levine WG
- Subjects
- Animals, Carbon Monoxide pharmacology, Male, Oxidation-Reduction, Oxygen pharmacology, Rats, Rats, Inbred Strains, Structure-Activity Relationship, Azo Compounds metabolism, Coloring Agents metabolism, Cytochrome P-450 Enzyme System physiology, Microsomes, Liver enzymology, NADPH-Ferrihemoprotein Reductase physiology, p-Dimethylaminoazobenzene metabolism
- Abstract
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)
- Published
- 1990
- Full Text
- View/download PDF
3. Azoreduction of dimethylaminoazobenzene (DAB) in primary cultures of rat hepatocytes. Effect of hypolipidemic agents.
- Author
-
Stoddart AM and Levine WG
- Subjects
- Animals, Cells, Cultured, Clofibrate pharmacology, Cytochrome P-450 CYP4A, Cytochrome P-450 Enzyme System metabolism, Enzyme Induction drug effects, Liver drug effects, Male, Mixed Function Oxygenases antagonists & inhibitors, Mixed Function Oxygenases metabolism, NADH, NADPH Oxidoreductases antagonists & inhibitors, NADH, NADPH Oxidoreductases metabolism, Nafenopin pharmacology, Oxidation-Reduction, Rats, Rats, Inbred Strains, Azo Compounds metabolism, Hypolipidemic Agents pharmacology, Liver metabolism, p-Dimethylaminoazobenzene metabolism
- Abstract
This laboratory has investigated the azoreduction of the hepatocarcinogen, N,N-dimethyl-4-aminoazobenzene (DAB), by hepatic microsomal cytochrome P-450 (P-450) and its specific induction by the hypolipidemic drug, clofibrate. To extend these studies further, a primary hepatocyte culture system was developed as a model. Hepatocytes isolated from male Sprague-Dawley rats were incubated in a basal medium containing fetal calf serum, insulin, and hydrocortisone for up to 96 hr with varying concentrations of clofibrate or nafenopin, a related hypolipidemic agent. Both DAB azoreductase and laurate hydroxylase activities decreased rapidly in control cultures. However, there was gradual marked induction of both activities in medium supplemented with clofibrate: hydrocortisone was required for induction. Nafenopin stabilized and induced DAB azoreductase and laurate hydroxylase activities, respectively. The responses of both activities were dose dependent. DAB azoreductase and laurate hydroxylase activities in control hepatocytes retained their ability to respond to clofibrate for up to 96 hr, although the response gradually diminished after 24 hr. In all cases, maximal induction of both enzyme activities was observed 72 hr after addition of drug. Phenobarbital and beta-naphthoflavone did not induce DAB azoreduction, although the normal induction of other P-450-catalyzed pathways, 7-ethoxycoumarin O-deethylation and ethlymorphine N-demethylation, were seen. Suppression of DAB azoreductase activity by inhibitors of P-450 activity confirmed the involvement of this enzyme in DAB azoreduction. The results demonstrate that a primary culture of rat hepatocytes is a useful model for studying the regulation of DAB azoreductase activity.
- Published
- 1990
4. Uptake and hepatobiliary fate of two hepatocarcinogens, N,N-dimethyl-4-aminoazobenzene and 3'-methyl-N,N-dimethyl-4-aminoazobenzene, in the rat.
- Author
-
Samuels AR, Bhargava MM, and Levine WG
- Subjects
- Animals, Chromatography, High Pressure Liquid, Half-Life, Male, Rats, Rats, Inbred Strains, Tissue Distribution, Bile metabolism, Liver metabolism, Methyldimethylaminoazobenzene metabolism, p-Dimethylaminoazobenzene analogs & derivatives, p-Dimethylaminoazobenzene metabolism
- Abstract
Two radiolabeled hepatocarcinogens, N,N-dimethyl-4-aminoazobenzene (DAB) and 3'-methyl-N,N-dimethyl-4-aminoazobenzene (3'-Me-DAB), were rapidly cleared from the blood of rats after i.v. administration, with half-lives of 40 and 70 sec, respectively. Rates of hepatic uptake and biliary secretion of [14C]-3'-Me-DAB were double that of [14C]DAB within 30 min of administration. Two hr after azo dye injection, the hepatic output into bile of [14C]-3'-Me-DAB-derived radioactivity was three times that of [14C]DAB. Fifty and 75% of the total 3'-Me-DAB-derived radioactivity was recovered in blood, liver, and bile 30 and 120 min after injection while only 30 to 40% of the administered [14C]DAB-derived radioactivity was recovered at these times. We postulate the existence of an extrahepatic azo dye accumulation site which may compete with the ability of the liver to clear azo dye from the circulation and which releases 3'-Me-DAB-derived radioactivity more readily than that of DAB. Azo dye metabolites were isolated from liver, bile, and blood. The chromatographic pattern of liver metabolites generated in vivo by rats which received either hepatocarcinogen was obtained and compared with that of biliary metabolites. With either azo dye, some metabolites were located exclusively in the liver, some were secreted immediately into bile, while others were present in both liver and bile, indicating selectivity in biliary excretion.
- Published
- 1983
5. Clofibrate selectively induces azoreduction of dimethylaminoazobenzene (DAB) by rat liver microsomes.
- Author
-
Levine WG and Raza H
- Subjects
- Aerobiosis, Anaerobiosis, Animals, Biotransformation, Hydroxylation, Kinetics, Lauric Acids metabolism, Male, Microsomes, Liver drug effects, NADPH-Ferrihemoprotein Reductase metabolism, Oxidation-Reduction, Rats, Rats, Inbred Strains, Clofibrate pharmacology, Microsomes, Liver metabolism, p-Dimethylaminoazobenzene metabolism
- Published
- 1986
- Full Text
- View/download PDF
6. 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.
- Author
-
Raza H and Levine WG
- Subjects
- Animals, Enzyme Induction drug effects, Hepatectomy, Kinetics, Male, Microsomes, Liver drug effects, Oxidation-Reduction, Rats, Rats, Inbred Strains, beta-Naphthoflavone, p-Aminoazobenzene metabolism, Benzoflavones pharmacology, Flavonoids pharmacology, Liver Regeneration, Microsomes, Liver metabolism, Phenobarbital pharmacology, Sulfhydryl Compounds pharmacology, p-Dimethylaminoazobenzene metabolism
- Abstract
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.
- Published
- 1986
- Full Text
- View/download PDF
7. Effect of hypolipidemic drugs on the metabolism of lauric acid and dimethylaminoazobenzene by rat liver microsomes.
- Author
-
Raza H and Levine WG
- Subjects
- Animals, Hydroxylation, In Vitro Techniques, Male, Microsomes, Liver drug effects, Oxidation-Reduction, Rats, Rats, Inbred Strains, Hypolipidemic Agents pharmacology, Lauric Acids metabolism, Microsomes, Liver metabolism, p-Dimethylaminoazobenzene metabolism
- Published
- 1987
- Full Text
- View/download PDF
8. Regulation of thiol environment of the N-demethylation and ring hydroxylation of N,N-dimethyl-4-aminoazobenzene (DAB) by rat liver microsomes.
- Author
-
Levine WG
- Subjects
- Animals, Cytochrome P-450 Enzyme System analysis, Dealkylation, Dithionitrobenzoic Acid pharmacology, Dithiothreitol pharmacology, Glutathione pharmacology, Hydroxylation, In Vitro Techniques, Male, NADPH-Ferrihemoprotein Reductase analysis, Phenobarbital pharmacology, Rats, Rats, Inbred Strains, p-Aminoazobenzene analogs & derivatives, p-Aminoazobenzene metabolism, Microsomes, Liver metabolism, Sulfhydryl Compounds pharmacology, p-Dimethylaminoazobenzene metabolism
- Abstract
A study was conducted on the regulation by thiol environment of microsomal metabolism of the azo dye hepatocarcinogen, N,N-dimethyl-4-aminoazobenzene (DAB). Physiological concentrations of glutathione (GSH) stimulated N-demethylation and ring hydroxylation of the dye in normal and phenobarbital (PB)-treated microsomes. However, little effect of GSH was seen with microsomes from beta-naphthoflavone (BNF)-treated rats. The synthetic thiol, dithiothreitol (DTT), stimulated ring-hydroxylation of DAB but inhibited N-demethylation at all concentrations in control nd PB-induced microsomes. A biphasic response to DTT was obtained with BNF microsomes; inhibition of N-demethylation was seen only at low concentrations (0.1 mM) and a return to control values occurred at higher concentrations. DTT inhibition was shown to be specific for the first N-demethylation step, whereas the second was slightly stimulated at concentrations greater than 3.0 mM. Agents which alkylate [N-ethylmaleimide (NEM), p-hydroxymercuribenzoate] or oxidize [5,5'-dithiobis(nitrobenzoic acid) or Ellman's reagent] protein SH groups inhibited DAB metabolism. Inhibition of microsomal NADPH-cytochrome c reductase activity by p-hydroxymercuribenzoate required an order of magnitude more inhibitor than was needed to block DAB metabolism. This suggests that DAB metabolism requires viable SH groups other than those involved in NADPH-cytochrome c reductase activity. NEM, in contrast, inhibited the N-demethylation of DAB and NADPH-cytochrome c reductase at approximately the same concentrations. Ring-hydroxylation was stimulated by high (greater than 1 mM) concentrations of NEM, implying a different enzymic mechanism for this pathway.(ABSTRACT TRUNCATED AT 250 WORDS)
- Published
- 1986
9. Effect of glutathione on the metabolism of N,N-dimethyl-4-aminoazobenzene by rat liver microsomes.
- Author
-
Levine WG and Lee S
- Subjects
- Animals, Cysteine pharmacology, Dithiothreitol pharmacology, Edetic Acid pharmacology, Male, Rats, Rats, Inbred Strains, Glutathione pharmacology, Microsomes, Liver metabolism, p-Dimethylaminoazobenzene metabolism
- Abstract
Rat liver microsomes catalyze both the N-demethylation and the 4'-hydroxylation of the azo dye carcinogen N,N-dimethyl-4-aminoazobenzene (DAB). It was found that addition of glutathione (GSH) to the microsomal system markedly stimulated both metabolic pathways. This occurred in the presence of either added NADPH or an NADPH-generating system. It was necessary that GSH be present when the reaction began; if added later, stimulation did not occur. This suggested a direct effect on microsomes rather than a chemical interaction with metabolic intermediates of DAB. Since stimulation occurred even in the presence of EDTA, the GSH effect cannot be satisfactorily explained in terms of suppression of lipid peroxidation which is totally inhibited by EDTA. Cysteine and cysteamine also stimulated both pathways but were less potent than was GSH; oxidized GSH was without significant effect. Dithiothreitol and beta-mercaptoethanol stimulated 4'-hydroxylation but inhibited N-demethylation, even in the presence of stimulatory concentrations of GSH. Apparently, the synthetic sulfhydryl compounds act through a mechanism different from that of GSH. Inhibition by dithiothreitol is consistent with formation of an N-oxide intermediate during N-demethylation. These observations also support previous findings that N-demethylation and 4' hydroxylation are, in the main, catalyzed by different isozymes of cytochrome P-450.
- Published
- 1983
10. Azoreduction of N,N-dimethyl-4-aminoazobenzene (DAB) by rat hepatic microsomes. Selective induction by clofibrate.
- Author
-
Raza H and Levine WG
- Subjects
- Animals, Benzoflavones pharmacology, Cytochrome P-450 Enzyme System biosynthesis, Enzyme Induction, Hydroxylation, In Vitro Techniques, Male, Nafenopin pharmacology, Oxidation-Reduction, Proadifen pharmacology, Rats, Rats, Inbred Strains, beta-Naphthoflavone, Clofibrate pharmacology, Microsomes, Liver metabolism, p-Dimethylaminoazobenzene metabolism
- Abstract
Metabolism of the hepatocarcinogen, N,N-dimethyl-4-aminoazobenzene (DAB) by rat liver microsomes proceeds via N-demethylation, ring hydroxylation, and azoreduction. DAB azoreduction was induced in microsomes from rats treated with the hypolipidemic drug, clofibrate, whereas oxidative metabolism of the carcinogen was inhibited. In contrast, treatment with nafenopin, another hypolipidemic drug, inhibited microsomal azoreduction of DAB, whereas oxidative pathways were only slightly affected. No direct effect of either drug on azoreductase activity was observed. Both drugs markedly induced microsomal laurate hydroxylation. DAB azoreduction was increased slightly in microsomes from rats treated with beta-naphthoflavone while treatment with phenobarbital led to partial inhibition. Pretreatment with isosafrol or pregnenolone-16 alpha-carbonitrile did not significantly alter DAB reduction. Metyrapone, added in vitro, inhibited microsomal DAB azoreductase activity only in phenobarbital-treated microsomes, whereas alpha-napthoflavone and SKF 525-A inhibited activity in control and all induced microsomes. DAB azoreduction proceeds readily in air and is not sensitive to carbon monoxide. Neither clofibrate nor nafenopin affected NADPH-cytochrome c reductase activity. It is concluded that clofibrate-induced azoreductase activity is probably attributable to a specific isoform of cytochrome P-450 which can be distinguished from those which catalyze oxidative pathways of DAB or laurate hydroxylation.
- Published
- 1986
11. Noncovalent binding of 3'-methyl-N,N-dimethyl-4-aminoazobenzene and its metabolites to liver cytosolic proteins and its role in their nuclear translocation.
- Author
-
Srinivasan K, Levine WG, and Bhargava MM
- Subjects
- Animals, Biological Transport, DNA metabolism, In Vitro Techniques, Male, Protein Binding, Rats, Rats, Inbred Strains, Cell Nucleus metabolism, Cytosol metabolism, Liver metabolism, Methyldimethylaminoazobenzene metabolism, p-Dimethylaminoazobenzene analogs & derivatives
- Abstract
When liver cytosol prepared from rats administered [14C]3'-methyl-N,N-dimethyl-4-aminoazobenzene was subjected to Sephadex gel chromatography, four peaks (I-IV) of radioactivity containing proteins and one peak (V) of radioactivity devoid of protein were obtained. Forty to fifty-five per cent of the radioactivity in the protein peaks was butanol-extractable. When the protein peaks were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, over 90% of the radioactivity was separated from the proteins, indicating lack of covalent binding. Several differences in the metabolite patterns were seen when the butanol-extractable metabolites from the five chromatographic peaks were analyzed by TLC. When pooled fractions of the peaks were incubated with isolated rat liver nuclei, only radioactivity associated with peak II was translocated into the nucleus. Translocation was time- and temperature-dependent and was maximal at 40 min at 37 degrees C. Only 10 to 12% of the radioactivity associated with peak II could be translocated even in the presence of an excess of nuclei, indicating that specific protein metabolite adduct(s) present in this fraction is/are translocated. Five per cent of translocated radioactivity was irreversibly bound to DNA, 3% to RNA, 67% to non-histone proteins, and 7.5% to histones; the remaining was not associated with any of these macromolecules.
- Published
- 1987
12. Role of isozymes of cytochrome P-450 in the metabolism of N,N-dimethyl-4-aminoazobenzene in the rat.
- Author
-
Levine WG and Lu AY
- Subjects
- Animals, In Vitro Techniques, Male, Methylcholanthrene pharmacology, Metyrapone pharmacology, Microsomes, Liver enzymology, Microsomes, Liver metabolism, Phenobarbital pharmacology, Piperonyl Butoxide pharmacology, Pregnenolone Carbonitrile pharmacology, Proadifen pharmacology, Rats, Rats, Inbred Strains, Cytochrome P-450 Enzyme System metabolism, Isoenzymes metabolism, p-Dimethylaminoazobenzene metabolism
- Abstract
The metabolism of N,N-dimethyl-4-aminoazobenzene (DAB) was investigated in vitro by use of hepatic 10,000g supernatant fraction, microsomes, and purified cytochromes P-450 prepared from rats. Position-selective metabolism was studied in response to induction by 3-methylcholanthrene (MC), phenobarbital (PB), beta-naphthoflavone (BNF), and pregnenolone-16 alpha-carbonitrile (PCN) as well as inhibition by SKF 525-A, metyrapone, alpha-naphthoflavone, and piperonyl butoxide. The principal phase I pathways are demethylation of the tertiary (DAB) and secondary (MAB) amines and ring hydroxylation. When metabolism was measured with 10,000g supernatant fractions, each pathway responded differently and often independently to the inducers and inhibitors, suggesting that they are catalyzed preferentially by different isozymes of cytochrome P-450. Microsomes from PB-treated animals demethylated and hydroxylated DAB at the same rate as did control microsomes, based on cytochrome P-450 content, whereas microsomes from BNF- or MC-treated animals demethylated more rapidly and hydroxylated more slowly. Microsomes from PB-treated animals demethylated the secondary amine, MAB, more rapidly than the tertiary amine, DAB. Purified cytochrome P-448 from MC-treated animals catalyzed DAB demethylation very readily but hydroxylation very poorly. The turnover number was 10 times that seen in microsomes from MC-treated animals. Only one of the four cytochrome P-450 fractions isolated from PB-treated animals had significant activity with DAB and the turnover number of one of these (fraction B) was approximately that seen in microsomes. This study supports the concept of selectivity of various isozymes of cytochrome P-450 for the different steps in phase I metabolism of DAB. Furthermore, it is apparent that the association of certain inhibitors with specific isozymes of cytochrome P-450 is a generalization that requires qualification in terms of the substrates(s) involved.
- Published
- 1982
13. Cytosolic factors that alter the metabolism of N,N-dimethyl-4-aminoazobenzene by rat liver microsomes.
- Author
-
Levine WG and Lee SB
- Subjects
- Animals, Dialysis, Edetic Acid pharmacology, Glutathione metabolism, In Vitro Techniques, Male, Metabolic Clearance Rate drug effects, NADP metabolism, Rats, Rats, Inbred Strains, Serum Albumin, Bovine pharmacology, Cytosol metabolism, Microsomes, Liver metabolism, p-Dimethylaminoazobenzene metabolism
- Abstract
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.
- Published
- 1983
- Full Text
- View/download PDF
14. Metabolism and biliary excretion of NN-dimethylaminoazobenzene: a possible role for liver glutathione.
- Author
-
Levine WG
- Subjects
- Animals, Mixed Function Oxygenases metabolism, Oxidation-Reduction, Rats, Bile metabolism, Glutathione metabolism, Liver metabolism, p-Dimethylaminoazobenzene metabolism
- Published
- 1978
- Full Text
- View/download PDF
15. Effects of butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) on metabolism of N,N-dimethyl-4-aminoazobenzene (DAB) by rat liver microsomes.
- Author
-
Levine WG
- Subjects
- Animals, Cytochrome P-450 Enzyme System analysis, In Vitro Techniques, NADH, NADPH Oxidoreductases analysis, NADPH Oxidases, Rats, Anisoles pharmacology, Butylated Hydroxyanisole pharmacology, Butylated Hydroxytoluene pharmacology, Microsomes, Liver metabolism, p-Dimethylaminoazobenzene metabolism
- Abstract
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.
- Published
- 1984
16. Biliary excretion of N,N-dimethyl-4-aminoazobenzene (DAB) in the rat. Effects of pretreatment with inducers and inhibitors of the mixed-function oxidase system and with agents that deplete liver glutathione.
- Author
-
Levine WG and Finkelstein TT
- Subjects
- Animals, Bile drug effects, Enzyme Induction drug effects, In Vitro Techniques, Liver drug effects, Male, Mixed Function Oxygenases antagonists & inhibitors, Rats, Time Factors, Bile metabolism, Glutathione metabolism, Liver metabolism, Mixed Function Oxygenases metabolism, Oxidoreductases metabolism, p-Dimethylaminoazobenzene metabolism
- Published
- 1978
17. Studies on microsomal azoreduction. N,N-dimethyl-4-aminoazobenzene (DAB) and its derivatives.
- Author
-
Levine WG
- Subjects
- Aerobiosis, Animals, Carbon Radioisotopes, Male, Oxidation-Reduction, Rats, Rats, Inbred Strains, Structure-Activity Relationship, Microsomes, Liver metabolism, p-Dimethylaminoazobenzene analogs & derivatives, p-Dimethylaminoazobenzene metabolism
- Abstract
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.
- Published
- 1985
- Full Text
- View/download PDF
18. Mechanism of azoreduction of dimethylaminoazobenzene by rat liver NADPH-cytochrome P-450 reductase and partially purified cytochrome P-450. Oxygen and carbon monoxide sensitivity and stimulation by FAD and FMN.
- Author
-
Levine WG and Raza H
- Subjects
- Animals, Clofibrate pharmacology, Diet, Flavin Mononucleotide pharmacology, Flavin-Adenine Dinucleotide pharmacology, Liver enzymology, Male, Oxidation-Reduction, Rats, Rats, Inbred Strains, Azo Compounds metabolism, Carbon Monoxide pharmacology, Cytochrome P-450 Enzyme System metabolism, Liver metabolism, NADPH-Ferrihemoprotein Reductase metabolism, Oxygen pharmacology, p-Dimethylaminoazobenzene metabolism
- Abstract
We have reported that the hepatocarcinogen dimethylaminoazobenzene (DAB) is reduced by rat liver microsomes in an oxygen- and carbon monoxide-insensitive manner and that activity is induced by clofibrate but no other recognized inducers of cytochrome P-450 activity. In the present study we have shown that the reaction proceeds in a partially purified reconstituted cytochrome P-450 system as well as with purified NADPH-cytochrome P-450 reductase alone. In the latter system, activity is totally inhibited in air whereas the former system is active in air as well as in a carbon monoxide atmosphere. Although clofibrate induces both DAB azoreductase and laurate hydroxylase activities, the suicide substrate 10-undecynoic acid blocks the latter but not the former, implying catalysis by distinct enzymes. FAD and FMN stimulate DAB azoreduction 40-50-fold by both NADPH-cytochrome P-450 reductase alone and by the reconstituted cytochrome P-450 system. However, it was shown that these flavins facilitate electron flow to DAB only from reductase and not from cytochrome P-450. The fact that the reconstituted system, which contains NADPH-cytochrome P-450 reductase, is oxygen insensitive suggests that there is an obligatory electron flow through cytochrome P-450 to DAB, bypassing the oxygen-sensitive step.
- Published
- 1988
19. A role for liver glutathione in the hepatobiliary fate of N,N-dimethyl-4-aminoazobenzene.
- Author
-
Levine WG and Finkelstein TT
- Subjects
- Animals, Biotransformation, Dealkylation, Hydrocarbons, Iodinated pharmacology, In Vitro Techniques, Male, Maleates pharmacology, Rats, Bile metabolism, Glutathione metabolism, Liver metabolism, p-Dimethylaminoazobenzene metabolism
- Published
- 1979
20. Microsomal azoreduction and glucuronidation in the metabolism of dimethylaminoazobenzene by the rat liver.
- Author
-
Raza H, Levine WG, Chowdhury NR, and Chowdhury JR
- Subjects
- Animals, Biotransformation, Enzyme Induction, Glucuronates metabolism, Glucuronosyltransferase biosynthesis, Glucuronosyltransferase metabolism, Hepatectomy, In Vitro Techniques, Male, NADH, NADPH Oxidoreductases biosynthesis, NADH, NADPH Oxidoreductases metabolism, Nitroreductases, Rats, Rats, Gunn, Rats, Inbred Strains, p-Dimethylaminoazobenzene pharmacology, Azo Compounds metabolism, Microsomes, Liver metabolism, p-Dimethylaminoazobenzene metabolism
- Abstract
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)
- Published
- 1987
- Full Text
- View/download PDF
21. Induction and inhibition of the metabolism and biliary excretion of the azo dye carcinogen, N,N-dimethyl-4-aminoazobenzene (DAB), in the rat.
- Author
-
Levine WG
- Subjects
- Animals, Dealkylation, Enzyme Activation drug effects, Hydroxylation, In Vitro Techniques, Male, Methylcholanthrene pharmacology, Mixed Function Oxygenases metabolism, Phenobarbital pharmacology, Proadifen pharmacology, Rats, Time Factors, Bile metabolism, p-Dimethylaminoazobenzene metabolism
- Abstract
The azo dye carcinogen, N,N-dimethyl-4-aminoazobenzene (DAB), is metabolized initially by N-demethylation and 4'-hydroxylation. The metabolites appear in the bile principally as sulfates and glucuronides, and to a lesser extent as glutathione conjugates. Pretreatment with 3-methylcholanthrene (MC) or phenobarbital (PB) accelerates biliary excretion of metabolites. The responses are different for each inducing agent. PB induces N-demethylation and 4'-hydroxylation whereas MC induces N-demethylation but inhibits 4'-hydroxylation. These effects are evident in the in vitro metabolism of DAB and in vivo through analysis of biliary metabolites. MC induction accelerates N-demethylation of both the tertiary amine, 4'-OH-DAB, and the secondary amine, N-methyl-4-amino-4'-hydroxyazobenzene (4'-OH-MAB), whereas PB induction accelerates N-demethylation of 4'-OH-MAB but not of 4'-OH-DAB. A further distinction between the responses to MC and to PB is seen in the relative lack of effect of SKF 525-A on MC-induced metabolism and biliary excretion of DAB, whereas PB-induced and noninduced metabolism is markedly inhibited. Previous observations indicated a depressed N-demethylation and biliary excretion of DAB after glutathione depletion. These studies lead to the conclusion that N-demethylation is the major rate-determining factor for biliary excretion. 4'-Hydroxylation, although a prominent pathway, appears to be less critical to rates of biliary excretion.
- Published
- 1980
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.