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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)

In previous studies on azoreduction by microsomal cytochrome P-450, we identified two classes of substrates structurally related to 4-dimethylaminoazobenzene. Both require polar electron-donating groups for binding to enzyme and are differentiated by their structure, their redox potentials, their rates of chemical and enzymic reduction, and the influence on their metabolism of inducing agents, CO and O2. Azo compounds whose reductions are insensitive to CO and O2 (I-substrates) contain electron-donating substituents on either ring. Azo compounds whose reductions are O2- and CO-sensitive (S-substrates) also contain electron-withdrawing groups on the opposite (prime) ring. For all dyes, NMR studies revealed minor differences in the chemical shifts of the protons attached to the phenyl ring substituted with electron-donating substituents (ring A). This is consistent with the narrow range of pKa's (basicity) and KM values for all substrates. However, there are significant differences in the chemical shifts of the aromatic protons of the prime ring (ring B). The difference in chemical shifts is most pronounced for aromatic protons adjacent to the prime ring substituents, showing a clear distinction between I and S substrates. Furthermore, the Hammett sigma substituent constants on the prime ring clearly distinguish between the two classes of dyes. I- and S-substrates have negative and positive sigma Hammett values, respectively. This implies that the mechanism of microsomal azoreduction is critically dependent on the charge and redox potentials of the dyes and is exclusively determined by the nature of the substituents on the prime ring.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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.

Pretreatment of rats for 2 days with the hypolipidemic drug, nafenopin, 0.5 g/kg, results in an increase in liver weight and bile flow. Despite these changes, hepatic transport of ouabain is reduced. This was demonstrated in the isolated perfused liver as well as in vivo. Although net uptake into liver is diminished in treated rats, the initial rapid phase of the plasma disappearance curve is unaffected. This suggests that the primary uptake process is unaffected by nafenopin or is altered in a manner that is not reflected in this phase of plasma disappearance. Alternatively, nafenopin may increase ouabain efflux from liver to plasma. Biliary excretion of ouabain is markedly suppressed after nafenopin pretreatment and higher liver levels of ouabain were encountered after the first 20 min in treated animals. The inference is drawn that liver to bile transport of ouabain is also suppressed by nafenopin pretreatment.

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.

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.

In rats treated with the hypolipidemic drug, nafenopin (NP), for 2 days the biliary excretion of phenolphthalein glucuronide (PPG) was markedly decreased, while in contrast that of chlorothiazide (CTZ) was enhanced. This suggests the existence of independent hepatic transport mechanisms for these two anions. For both PPG and CTZ blood disappearance curves showed an initial, rapid phase followed by a second, slow phase. The rapid phase for both compounds is affected only slightly by pretreatment with NP. Therefore, it is inferred that suppression of biliary excretion was attributable mainly to impairment of the liver-to-bile transport process. The increased bile flow induced by NP treatment was previously shown to be related to the biliary excretion of NP and its metabolites. Inhibition of NP choleresis by PPG may involve competition for biliary transport of these compounds. The marked hepatomegaly and choleresis seen after NP pretreatment was more evident in male rats than in females.

The hepatic uptake and biliary excretion of 3H-taurocholate and 3H-ouabain was studied in the rat during saline (control) and dehydrocholate infusions. Dehydrocholate (140 mumol/hr) did not influence the plasma disappearance nor the biliary excretion of taurocholate after a single iv injection (37 mumol/kg). Bile production in the dehydrocholate experiment was increased 2- to 3-fold compared with controls. The biliary transport maximum for exogenously administered taurocholate was determined by constant infusion to be 135.0 +/- 3.0 mumol/hr (22 mumol/min/g of liver). Concomitant infusions of 140 mumol of dehydrocholate per hr did not alter the maximal taurocholate output. The effects of the two bile salts on bile flow were additive. Dehydrocholate (140 mumol/hr) reduced the biliary excretion of 3H-ouabain (0.8 mumol/kg) and elevated the secondary slow component of the plasma disappearance of the cardiac glycoside. The hepatic levels of ouabain were increased compared with controls. It is concluded that dehydrocholate interferes with ouabain transport at the canalicular level but not with primary hepatic uptake. Taurocholate (140 mumol/hr) failed to influence the total biliary output of ouabain. These differences and the lack of interaction between dehydrocholate and taurocholate suggest a hepatic transporting pathway for taurocholate which differs from that for taurocholate which differs from that for dehydrocholate and/or its metabolites.

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.

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.