The enzymatic activation of a promutagenic pyrolysate, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), was studied using the Ames mutagenesis test system. The enzyme catalyzing the mutagenic activation of MeIQx is mainly localized in the microsomal fraction. A large number of revertants was observed in the presence of hepatic microsomes obtained from 3-methylcholanthrene (3-MC)- or polychlorinated biphenyl (PCB)-treated rats but only a minimal number with the hepatic microsomes from untreated or phenobarbital (PB)-treated rats. In addition, the microsomal activation was reduced efficiently by known inhibitors of cytochrome P-450-mediated reactions such as 7,8-benzoflavone, ellipticine and flavone. Among five forms of purified rat cytochrome P-450, the highest sp. act. (no. of revertants induced/nmol cytochrome P-450) for the activation of MeIQx was observed with a high-spin form of cytochrome P-450, P-448-H, followed by the low-spin form, P-448-L, and to a lesser extent by PB-inducible forms, P-450b and P-450e. P-450-male, which is a main constitutive form of cytochrome P-450 in male rat livers, showed considerable catalysis for the mutagenic activation of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and MeIQx. These results indicate that the metabolic activation of MeIQx is catalyzed mainly by two forms of cytochrome P-450, P-448-H and P-488-L, in the livers of PCB- or 3-MC-treated rats, but also that P-450-male may play an important role in the activation in livers of intact male rats.

Metabolic activating capacity of human livers for carcinogenic heterocyclic arylamines has been studied using a Salmonella mutagenesis test. A large individual variation was observed among 15 liver samples in the capacities of activation of Glu‐P‐1(2‐amino‐6‐methyldipyrido[1,2‐α:3′,2′‐d]imidazole), IQ (2‐amino‐3‐methylimidazo[4,5‐f]quinoline) and MeIQx (2‐amino‐3,8‐dimethyl‐3H‐imidazo[4,5‐f]quinoxaline). The average numbers of revertants induced by the three heterocyclic arylamines were nearly the same or rather higher in the presence of hepatic microsomes from human than those from rat. In high‐performance liquid chromatography, formation of N‐hydroxy‐Glu‐P‐1 was detected and accounted for more than 80% of the total mutagenicity observed in the human microsomal system with Glu‐P‐1, indicating that, similarly to experimental animals, N‐hydroxylation is a major activating step for heterocyclic arylamines in human. Addition of flavone or 7,8‐benzoflavone to human liver microsomes showed effective inhibition of the mutagenic activation of Glu‐P‐1, although the treatment rather enhanced microsomal benzo[a]pyrene hydroxylation in human livers. Mutagenic activation of Glu‐P‐1 by human liver microsomes was also decreased by the inclusion of anti‐rat P‐448‐H IgG, and was well correlated with the content of immunoreactive P‐448‐H in livers, suggesting the involvement of a human cytochrome P‐450, which shares immunochemical and catalytic properties with rat P‐448‐H, in the metabolic activation of heterocyclic arylamines in human livers.

In mammalian hepatic cytosol both acetyltransferase and sulfotransferase are involved in the activation of N-hydroxy derivatives of arylamines and arylamides. The role of acetyltransferase is also shown in Salmonella, whereas no rigid evidence is provided on the role of sulfotransferase in Salmonella. In Ames mutagenesis test without S9-mix, the number of revertants of Salmonella typhimurium TA98 induced was 10-fold higher with 2-hydroxyamino-3-methylimidazo[4,5-f] quinoline (N-hydroxy-IQ) than with 2-hydroxyamino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (N-hydroxy-Glu-P-1). The extents of the binding to calf thymus DNA of N-hydroxy-Glu-P-1 were, however, 3.9 to 8.6-fold higher than that of N-hydroxy-IQ in both acetyl CoA- and PAPS-fortified rat hepatic cytosol systems. To understand the mechanism causing the apparent discrepancy between the results of the mutation and DNA binding, the activating capacities of cytosols of S. typhimurium TA98 and TA98/1,8-DNP6 strains on the binding of N-hydroxy-Glu-P-1 and N-hydroxy-IQ have been examined in comparison with those of rat livers. Although both N-hydroxyarylamines were activated by hepatic cytosols in the presence of PAPS, no significant DNA binding of these N-hydroxyarylamines was detected in the presence of PAPS and either one of the two strains of bacterial cytosols. In addition, both cytosols of TA98 and TA98/1,8-DNP6 strains showed no measurable activity on the sulfation of p-nitrophenol, suggesting no capacity for sulfotransferase-mediated activation of N-hydroxyarylamines in Salmonella. On the contrary, the extents of the acetyl CoA-dependent binding of N-hydroxy-IQ in cytosols of TA98, but not of TA98/1,8-DNP6, were respectively 6- and 9-fold higher than those in hepatic cytosols of male and female rats, although the extents of the binding of N-hydroxy-Glu-P-1 were rather higher in hepatic than in bacterial cytosols. In addition, the covalent binding of N-hydroxy-2-acetylaminofluorene to DNA was detected in hepatic, but not in bacterial cytosols, although the binding of N-hydroxy-2-aminofluorene was detectable in both hepatic and bacterial cytosols in the presence of acetyl CoA. These results indicate that the metabolic activating capacities of Salmonella and rat liver cytosols differ qualitatively, and the difference in the substrate specificity of acetyltransferase between Salmonella and rat livers may be involved, in part, in the difference of their DNA damage in bacteria and mammals.

Characteristics of cytosolic sulfotransferase-mediated binding of carcinogenic N-hydroxyarylamines(amide) have been investigated and compared among experimental animal species and humans in vitro. Human cytosols exhibited significant sulfating activities towards 2-hydroxyamino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (N-hydroxy-Glu-P-1), N-hydroxy-2-aminofluorene (N-hydroxy-AAF) and N-hydroxy-2-acetylaminofluorene (N-hydroxy-AAF), but had no detectable activity toward 2-hydroxyamino-3-methyl-imidazo[4,5-f]quinoline (N-hydroxy-IQ). Although the extent of the covalent binding of these N-hydroxyarylamines(amide) differed significantly among individuals, clear correlations were observed among the sulfation of N-hydroxyarylamines (amide) and also with p-nitrophenol sulfation. Hepatic cytosols from mouse, rat, guinea-pig, hamster, rabbit, dog and monkey also mediated the binding of N-hydroxy-Glu-P-1, N-hydroxy-AF and N-hydroxy-AAF, while only rat cytosols showed detectable DNA binding of N-hydroxy-IQ. Among the species examined, rat showed the highest capability for activating these N-hydroxyarylamines(amides). Significant sex-related differences were detected in rat, dog and monkey for all substrates examined, except N-hydroxy-IQ. Clear correlations were observed in the animal species between N-hydroxyarylamines(amide), but not with p-nitrophenol. Using an ion-exchange chromatographic system, sulfating activity of p-nitrophenol in human livers was separated into two fractions and the PAPS-dependent DNA binding of N-hydroxy-AF was supported mainly by the later fraction. On Western blots, an immunoreactive protein was detected in these fractions using an antibody raised against rat hepatic N-hydroxy-AAF sulfotransferase. The band was also detected in human hepatic cytosols with considerable individual variation in their amounts. These results indicate the involvement of a closely related form(s) of sulfotransferase in the PAPS-mediated activation of N-hydroxyarylamines(amide) in human as well as in the experimental animal species.

Two forms of cytosolic acetyltransferases, AT-I and AT-II, have been purified from hamster livers, and a comparison made of their chemical and catalytic properties and genetically expressed difference. Homogeneous AT-I and AT-II were 31 and 30 kd respectively on SDS-PAGE and catalyzed efficiently various N- and O-acetylations in their reconstitution systems. AT-I used both acetyl CoA and arylhydroxamic acids as acetyl donors, while AT-II did not utilize arylhydroxamic acids as acetyl donors. In the reconstitution system, purified AT-I, but not AT-II, catalyzed acetyl CoA-dependent O-acetylation of 2-N-hydroxyamino-6-methyldipyrido[1,2-alpha:3', 2'-d]imidazole (N-OH-Glu-P-1) and arylhydroxamic acid-dependent N-acetylation of 4-aminoazobenzene (AAB). On the other hand purified AT-II showed high activities of acetyl CoA-dependent N-acetylation of 2-aminofluorene (AF) and p-aminobenzoic acid (PABA). Polyclonal antibodies raised against AT-I inhibited cytosolic acetylations of N-OH-Glu-P-1 and AAB, and to a lesser extent of AF, while PABA N-acetylation was only marginally inhibited. Using Western blots, both AT-I and AT-II were recognized by the antibodies. AT-I was detectable in all the livers examined, and the content did not differ among the individuals (monomorphic distribution). In contrast, AT-II was distributed polymorphically, and the trimodal distribution of AT-II (high, intermediate and low) was correlated with the phenotype identified by cytosolic N-acetylations of AF and PABA (rapid, intermediate and slow). In addition, cross-mating experiments with intra- and inter-phenotype animals confirmed that hepatic AT-II isozyme is inherited by a Mendelian co-dominant trait. These results indicate that the polymorphic appearance of an acetyltransferase, AT-II, is responsible for the N-acetylation polymorphism in individual hamsters.