Uptake and hepatobiliary fate of two hepatocarcinogens, N,N-dimethyl-4-aminoazobenzene and 3'-methyl-N,N-dimethyl-4-aminoazobenzene, in the rat.

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.

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.

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.

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.

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)

Effect of nafenopin (SU-13,437) on liver function: influence on the hepatic transport of organic anions.

Rats treated with hypolipidemic agent, nafenopin (SU-13, 437) exhibit a higher plasma retention and a markedly reduced biliary excretion of organic anions, such as sulfobromophthalein (BSP) and its dibromo analog (DPSP), indocyaninegreen (ICG), succinylsulfathiazole (SST) and polar metabolites of bilirubin and the carcinogens 7, 12-dimethylbenzanthracene (DMBA) and 3,4 benzpyrene (BP), despite an increase in liver mass and a profound choleresis. However, taurocholate is not affected in this manner, which supports the idea of a transport mechanism for taurocholate that differs from that of other organic anions. A pharmacokinetic study was made for DBSP in vivo. After nafenopin treatment, primary hepatic uptake (k12) and transport from liver into bile (k23) are reduced in vivo. Infusion studies indicate that biliary transport maximum (Tm) for DBSP is also decreased although the calculated hepatic storage (S) is only moderately affected. In the isolated perfused liver, hepatic clearance and biliary excretion of BSP are reduced by two-thirds. The time course of anion transport inhibition and the hepato-biliary disposition of 14C-nafenopin suggest a direct effect of the drug. The extra liver mass induced by nafenopin appears to be hypo- or nonfunctional with respect to hepatic transport of organic anions.

Effect of nafenopin (SU-13,437) on liver function: mechanism of choleretic effect.

Administration of nafenopin (SU-13-437) to male rats for two days leads to a doubling of bile production and a 50% increase in liver weight. These two effects have been shown not to be directly interrelated. A marked decrease in biliary salt concentration suggests that the bile salt independent flow is stimulated. The extra bile produced is probably of canalicular origin since bile to plasma concentration ratios of erythritol are unchanged. At least three polar metabolites of nafenopin have been observed in rat bile. Observations in rats with partial biliary fistulas indicate that the drug and its metabolites undergo extensive entero-hepatic circulation. Our studies support the view that much of the enhanced bile flow is associated with the presence of nafenopin and/or its metabolites within the hepatobiliary system. However, the response is too extensive to be explained merely by osmotic choleresis. Induced structural changes in the liver may also account forsome of this effect.

Glutathione, lipid peroxidation and regulation of cytochrome P-450 activity.

Depletion of hepatic glutathione leads to an increase in lipid peroxidation and depression of cytochrome P-450-catalyzed metabolism of the azo dye carcinogen, N,N-dimethyl-4-aminoazobenzene. This contributes to the marked decrease in biliary excretion of N-demethylated metabolites of the dye. Parallel time courses are seen for decreased hepatic glutathione, enhanced lipid peroxidation and depressed excretion of dye metabolites. In vitro metabolism of DAB by hepatic 10,000 g supernatant fractions is depressed by iron only after glutathione depletion. In view of the iron requirement for microsomal lipid peroxidation, it is proposed that glutathione depletion leads to an increase in the intracellular iron available for activation of lipid peroxidation. In this way, glutathione may contribute to the regulation of cytochrome P-450 activity.

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.

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.