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We have previously established that microsomal reduction of azo dyes requires polar electron-donating ring substituents [e.g. -OH, -NH2, -NHCH3, or -N(CH3)2]. Reduction of dyes substituted exclusively with such moieties on either phenyl ring is insensitive to both O2 and CO (I-substrates). In contrast, azoreduction of compounds containing additional electron-withdrawing substituents (e.g. -SO3H, -COOH, -COOCH3, and -AsO3H2) on the opposite (prime) phenyl ring is sensitive to both O2 and CO (S-substrates). We have recently shown that Hammett aromatic substituent constants and redox potentials of the dyes, determined by cyclic voltammetry (CV), distinguish between I- and S-substrates. Dyes that exhibit positive Hammett sigma substituent constants on 1 of the 2 rings are S-substrates, and undergo immediate quenching of their one electron-reduced intermediates upon exposure to air, as observed by CV. In contrast, dyes that have negative Hammett sigma values on one or both rings are I-substrates, and their one electron-reduced intermediates are relatively stable in air. In this study, we have investigated the susceptibility to microsomal azoreduction of monosubstituted dyes, with ring substituents possessing a wide range of Hammett sigma values. We have observed a direct correlation between the susceptibility of these compounds to microsomal azoreduction and the Hammett sigma constant of the aromatic ring substituent, and the presence of a positive potential in the dyes observed by CV. A Hammett sigma value of -0.37 or lower is required for substrate activity. Dyes with two substituents in the prime ring confirmed the previous correlation of negative and positive Hammett sigma values with I- and S-substrate activities, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

1. Microsomal reduction of azo dyes related to dimethylaminoazobenzene (DAB) is catalysed by at least two types of cytochrome P-450. The first is selectively induced by clofibrate. The second is induced by phenobarbital, beta-naphthoflavone, isosafrole, and pregnenolone-16 alpha-carbonitrile, as well as clofibrate. 2. Azoreduction by the first type of P-450 is insensitive to both O2 and CO and involves dyes with only electron-donating substituents (I substrates). 3. Azoreduction by the second type of P-450 is inhibited by both O2 and CO and involves dyes with electron-withdrawing as well as donating substituents (S substrates). 4. All azo dye substrates exhibit two negative and one positive redox potential, as measured anaerobically by cyclic voltammetry. The negative potentials reflect one- and two-electron reductions while the positive potential permits electron transfer from microsomal P-450, the redox potential for which is reported to be negative (approximately 0.35 V). The positive potential is associated with a polar electron-donating group para to the azo linkage, which is an absolute requirement for microsomal reduction. Dyes without this functional group do not exhibit positive potentials and are not reduced. 5. The first negative potential of S substrates is quenched upon admitting air to the system, whereas this potential is unaffected in I substrates. The relative stability of the one-electron reduced state may be an explanation for the differential O2 sensitivity of I and S substrate reduction.

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)

We have established that reduction of azo dyes structurally related to 4-(dimethylamino)-azobenzene (DAB) by rat liver microsomal cytochrome P-450 requires a polar electron-donating substituent on one ring. Reduction of azo dyes containing only electron-donating substituents is insensitive to both oxygen and CO (I substrates). However, reduction of azo dyes containing electron-withdrawing substituents as well is sensitive to both oxygen and CO (S substrates). Positive, irreversible potentials were observed by cyclic voltammetry (CV) in anhydrous solutions for both I and S substrates but not for the nonreducible azo dyes. This positive potential permits electron transfer to dyes from NADPH-cytochrome P-450 reductase and from cytochrome P-450, both of which have negative potentials. Reduction products retaining electron-donating groups (amino, phenolic) also exhibited positive potentials, implying that these groups contribute the positive potential in the dye molecule. All substrates also exhibited two negative potentials with a clear distinction between I and S substrates. The latter exhibited, on average, potentials that were less negative than the former by about 0.6 V. This is consistent with the more rapid reduction of S substrates by liver microsomes [Zbaida and Levine (1990) Biochem. Pharmacol. 40, 2415-2423]. Admitting air to the system quenched the first potential for S but not for I substrates, which is consistent with the oxygen sensitivities of their reduction. Addition of water significantly shifted the second negative potential to a more positive value, ultimately leading to single negative potential. The water permits rapid protonation of the two-electron-reduced intermediate, facilitating further reduction.(ABSTRACT TRUNCATED AT 250 WORDS)

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)

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.

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)

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.

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.

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.

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.

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.

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.

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)