Masakuni Degawa

Mutagenicity of carcinogenic azo dyes and their derivatives

The mutagenicity of N,N-dimethyl-4-aminoazobenzene and N-methyl-4-aminoazobenzene and their derivatives was shown on Salmonella typhimurium TA100 and TA98. S-9 Mix, obtained from rat liver after injection of polychlorinated biphenyl, was abligatory for their mutagenic action. N-Acetoxy-N-methyl-4-aminoazobenzene and N-benzoyloxy-N-methyl-4-aminoazobenzene and their 4-methoxycarbonyl derivatives were also mutagenic on TA100 and TA98 and did not require metabolic activation by S-9 Mix. It is suggested that the carcinogenic effects of azo dyes may involve modification of DNA.

Mutagenic activation of urinary bladder carcinogens by CYP4B1 and the presence of CYP4B1 in bladder mucosa

We investigated the mutagenic activation of 2-naphthylamine (2-NA), 3,2-dimethyl-4-aminobiphenyl (DMAB), and 3,3-dichlorobenzidine (DCB), bladder carcinogens, by renal and bladder microsomes and by purified P450s using the umu gene expression system, which detects DNA damage. Mouse renal microsomes had high mutagenic activation toward DCB and low activity toward 2-NA. Purified mouse Cyp4b1 also had high mutagenic activity toward DCB. Anti-Cyp4b1 antibody efficiently inhibited DCB bioactivation by mouse renal microsomes with a high Cyp4b1 content. Lauric acid, a substrate of Cyp4b1, efficiently inhibited DCB bioactivation by renal and bladder microsomes of the mouse and by purified Cyp4b1. To assess the contribution of CYP4B1 to bladder carcinoma, further investigation was done with the umu test and an immunochemical study. Ten forms of purified rat P450s including rat CYP4B1 were used in the umu test for 2-NA, DMAB, and DCB. CYP4B1 had the highest activity toward DMAB and DCB. Other P450s had activities of less than 20% that of CYP4B1. CYP4B1 also activated 2-NA, but its activity was about 10% of that toward DMAB or DCB. Rat bladder epithelium was stained specifically with anti-Cyp4b1 antibody, indicating the presence of CYP4B1 in the rat bladder mucosa. Also, CYP4B1 mRNA was detected by northern blotting and reverse transcription-polymerase chain reaction (RT-PCR). These findings suggested that CYP4B1 could contribute to the initiation of carcinogenesis in rat and mouse bladder by activation of aromatic amines.

Mutagenicity of metabolites of carcinogenic aminoazo dyes.

The mutagenicity of 8 azo dyes and 6 p-phenylenediamine derivatives, which comprised the metabolites of carcinogenic 4-aminoazobenzene derivatives, was studied on Salmonella typhimurium TA98 and TA100. 4-Hydroxy-N-methyl-4-aminoazobenzene and its O-sulfate and O-glucuronide, and 3-hydroxy-4-aminoazobenzene were mutagenic on TA98 in the presence of S-9 mix. p-Phenylenediamine and its o-methoxyl derivative were definitely mutagenic on TA98 with the addition of S-9 mix. All metabolites tested were non-mutagenic on TA100, although the mother azo dyes were mutagenic both on TA98 and TA100 in the presence of S-9 mix. These results rule out a possibility that the mutagenicity, at least on TA100 microbes, of carcinogenic 4-aminoazobenzene derivatives may be mediated by any of the ring-hydroxyl or azo reduction metabolites and their conjugates produced from the azo dyes by incubation with S-9 mix.

Heterocyclic amine mixture carcinogenesis and its enhancement by caffeine in F344 rats

In order to elucidate whether mixed exposure to environmental carcinogens and caffeine increases the risk of cancer induction, we investigated the relationship between preneoplastic lesion development in the liver and colon and drug metabolizing enzyme induction and DNA adduct formation, in rats treated with a mixture of heterocyclic amines (HCAs) and caffeine. In Experiment 1, male F344 rats were administered 3 different HCAs, the food carcinogens, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), alone or in combinations of 2 or 3 at 50 ppm in the diet for 16 weeks. The numbers of hepatic glutathione-S-transferase P form positive (GST-P+) foci and colonic aberrant crypt foci (ACF) were greater in the IQ + MeIQx group than expected from simple summation and increased levels of HCA-DNA adducts were noted. However, no summation was obtained when combined with PhIP, which rather caused inhibition. In Experiment 2, the effects of concurrent caffeine administration on the PhIP carcinogenicity were assessed. Caffeine at 1000 and 500 ppm in the drinking water for 2 weeks significantly increased levels of CYP1A2. Ten weeks concurrent administration of caffeine (1000 ppm) and PhIP (400 ppm) resulted in significant increase of colon ACFs and CYP1A2 expression. Thus, concurrent administration of IQ and MeIQx caused elevation of their carcinogenicity but other mixtures with PhIP did not enhance carcinogenicity. However, a non-carcinogen, caffeine, enhanced PhIP colon carcinogenesis, possibly due to induction of CYP1A2.

A simple method for assessment of rat cytochrome P-448 isozymes responsible for the mutagenic activation of carcinogenic chemicals

Cytochrome P-448H/L-enriched and cytochrome P-448L-enriched microsomes were prepared from the livers of Sprague-Dawley rats treated with 3-methylcholanthrene (MC) and with a combination of MC and carbon tetrachloride, respectively, and their activities for mediating mutagenic activation of 9 carcinogenic aromatic amines and benzo[a]pyrene, which are found to be different from cyt. P-450 isozymes as to mutagenic activation, were compared on the basis of microsomal cytochrome P-450 content using Salmonella typhimurium TA98 as a tester bacterium. With regard to the substrate-specificity of cytochrome P-448 isozymes, the present results reflected the reported results with use of a cytochrome P-450-reconstituted system. These findings indicate that the mutation test with cytochrome P-448H/L-enriched and cytochrome P-448L-enriched microsomes could be used as a simple method for the determination of the cytochrome P-448 isozymes responsible for the mutagenic activation of carcinogens and mutagens without the use of a cytochrome P-450-reconstituted system.

Species Difference among Experimental Rodents in Induction of P450IA Family Enzymes by 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine

Rats, mice, hamsters and guinea pigs were given an i.p. injection of 2‐amino‐1‐methyl‐6‐phenyl‐imidazo[4,5‐b]pyridine (PhIP), a protein‐derived pyrolysate component present in cooked foods, and inductions of cytochrome P450 (P450) in the liver and kidney of these animals were examined. The activity and amount of P450s corresponding to the rat P4S0IA1 and P450IA2 were assessed by means of a bacterial mutation test using 3 carcinogenic heterocyclic aromatic amines including PhIP as substrates and by Western blotting with a monoclonal antibody reactive with both P4S0IA1 and P450IA2. In rats, PhIP induced P450IA1, P450IA2 and a new but unspecified P450 isozyme in the liver, and induced P450IA1 in the kidney. However, PhIP induced none of these P450 isozymes in mice, hamsters and guinea pigs.

Different effects of DNA adducts induced by carcinogenic and noncarcinogenic azo dyes on in vitro DNA synthesis

M13mp10 phage DNA modified with the carcinogen 3-methoxy-4-aminoazobenzene (3-MeO-AAB) or the noncarcinogen 2-methoxy-4-aminoazobenzene (2-MeO-AAB) was used as a template for E.coli DNA polymerase I. Analysis of the reaction products on DNA sequencing gels showed that with both types of compound the induced lesions blocked DNA synthesis, mainly at one base prior to guanine adducts, but that the inhibition by 3-MeO-AAB-adducts was substantially greater than that by 2-MeO-AAB-adducts. Thus different effects on DNA replication between 3-MeO-AAB- and 2-MeO-AAB-adducts might be a reflection of differences in their carcinogenic potency.

Induction of a high spin form of microsomal cytochrome P-448 in rat liver by 4-aminoazobenzene derivatives☆

Male Sprague-Dawley rats were treated with 4-aminoazobenzene derivatives or other drug metabolizing enzyme inducers such as phenobarbital, 3-methylcholanthrene and isosafrole. The expression of hepatic microsomal cytochrome P-450 of the rats, principally that of a high spin form of cytochrome P-448 (cytochrome P-448H), was assessed by a bacterial mutation test and by immunological methods. The results of the mutation test with use of Salmonella typhimurium TA9 and 3 aromatic amine substrates showed that 2-methoxyl, 3-methoxyl and 2,3-dimethoxyl derivatives of AAB and methyl derivatives of AAB such as o-aminoazotoluene, N-methyl-4-aminoazobenzene and N,N-dimethyl-4-aminoazobenzene have a large capacity for the selective induction of cytochrome P-448H. Activity of the cytochrome increased by 6 hr after an azo dye treatment, reached a maximum after 24 hr, and then declined. In contrast, 4-methoxy-AAB has a small, and AAB has no, capacity for the cytochrome induction. The aminoazo dye-induced enzymes differ in their substrate specificities from those induced with 3-methylcholanthrene or phenobarbital, and the induced enzyme was identified to be cytochrome P-448H, as determined by an enzyme-linked immunosorbent assay and immunoblotting with use of anti-cytochrome P-448 monoclonal antibodies. These observations indicate that several methoxyl and methyl derivatives of 4-aminoazobenzene are potent and selective inducers of cytochrome P-448H in the rat.

Decreased levels of 2-amino-3-methylimidazo[4,5-f]quinoline-DNA adducts in rats treated with β-carotene, α-tocopherol and freeze-dried aloe

To assess mechanisms of chemoprevention of hepatocarcinogenesis by trans‐β‐carotene (β‐C), DL‐α‐tocopherol (α‐T), and freeze‐dried whole leaves of Kidachi aloe (Aloe), formation of 2‐amino‐3‐methylimidazo[4,5‐f]quinoline (IQ)‐DNA adducts was measured by 32P‐post‐labeling analysis, and CYP1A1 and CYP1A2 protein levels were analyzed by ELISA. Group 1 rats were fed diet containing 0.02%β‐C, 1.5%α‐T or 30% Aloe over an 8‐day period, while group 2 was given basal diet alone. On day 7, all animals were subjected to two‐thirds partial hepatectomy (PH). Twelve hours after PH, they received a single dose of the carcinogenic food pyrolysate IQ (100 mg/kg) intragastrically, to initiate hepatocarcinogenesis. Rats were killed 6, 12, 24 and 48 h after IQ administration. The levels of adducts, expressed as relative adduct labeling values in rats treated with β‐C, α‐T and Aloe, were decreased as compared with the control group at hour 24 (36 h after PH), with a significant difference in the case of the β‐C group (46.4% of the control value). Similarly, all showed a tendency for decrease at hour 48. Furthermore, the levels of CYP1A2, known to be responsible for activation of IQ, showed a significant reduction at hour 24. It is concluded that β‐C, and possibly also α‐T and Aloe, have the potential to reduce IQ‐DNA adduct formation, presumably as a result of decreased formation of active metabolites. The results may explain, at least in part, the previously observed inhibitory effects of these compounds on induction of preneoplastic hepatocellular lesions.

2-Methoxy-4-nitroaniline and its isomers induce cytochrome P4501A (CYP1A) enzymes with different selectivities in the rat liver

We reported previously that 2-methoxy-4-nitroaniline (2-MeO-4-NA) is a selective inducer of cytochrome P4501A2 (CYP1A2) in the rat liver, and its molecular size is the smallest among known CYP1A2-selective inducers. In the present study, a structure-activity relationship on the CYP1A2-selective induction has been investigated using isomeric nitroanisidines and their related chemicals. Western blot analyses revealed that the chemicals removed a substituent (amino, methoxyl or nitro group) from a 2-MeO-4-NA molecule had no capacity for inducing CYP1A enzymes in rat livers. On the other hand, isomeric nitroanisidines such as 2-MeO-4-NA, 2-MeO-5-NA and 4-MeO-2-NA induced both CYP1A2 and CYP1A1 enzymes with different selectivities. As judged from the induced levels of CYP1A proteins, 2-MeO-4-NA (CYP1A2/CYP1A1 ratio; 9.5) and 4-MeO-2-NA (0.3) were the most selective inducers of CYP1A2 and CYP1A1, respectively, among the isomeric nitroanisidines (0.44 mmol/kg) used. The induced level of CYP1A2 protein was in the order 2-MeO-4-NA > 2-MeO-5-NA > 4-MeO-2-NA, although no significant difference was observed on their CYP1A2 mRNA level. On the contrary, increases in the levels of CYP1A1 mRNA and protein were in the order 4-MeO-2-NA > 2-MeO-5-NA > 2-MeO-4-NA. The present findings indicate that all three substituents (amino, methoxyl and nitro groups) are necessary components of nitroanisidines for induction of CYP1A enzymes, and also show that regio-isomeric positions of these substituents determine the selectivity in the induction of CYP1A enzymes.