Structural determination of a mutagenic aminophenylnorharman produced by the co-mutagen norharman with aniline

Abstract

Norharman (9H-pyrido[3,4-b]indole), widely distributed in our environment, including cigarette smoke and cooked foodstuffs, is not mutagenic to Salmonella strains, but becomes mutagenic to S.typhimurium TA98 and YG1024 with S9 mix in the presence of non-mutagenic aromatic amines such as aniline and o-toluidine. To elucidate the mechanisms of co-mutagenicity, we tried to isolate the mutagen(s) produced by a reaction between norharman and aniline with S9 mix. By HPLC purification, two mutagenic compounds (I and II), one (I) showing mutagenicity with and the other (II) without S9 mix, were isolated. The structure of compound I was deduced to be a coupled compound of norharman and aniline, 9-(4'-aminophenyl)-9H-pyrido[3,4-b]indole (aminophenylnorharman), by a variety of spectrometry techniques and this was confirmed by its chemical synthesis. The mutagenic activity of this novel heterocyclic amine was tested using the pre-incubation method and was found to induce 187,000 revertants in TA98 and 1,783,000 revertants in YG1024 per microg with S9 mix. Compound II was shown to be hydroxyaminophenylnorharman. Formation of the same DNA adducts was observed in YG1024 when aminophenylnorharman or a mixture of norharman plus aniline was incubated with S9 mix. The hydroxyamino derivative also yielded the same DNA adducts in YG1024. Thus, the appearance of mutagenicity by norharman with aniline in the presence of S9 mix suggests that the coupled mutagenic compound, aminophenylnorharman, is formed from norharman and aniline, then converted to the hydroxyamino derivative and forms DNA adducts to induce mutations in TA98 and YG1024.

Detection of Aminophenylnorharman, a Possible Endogenous Mutagenic and Carcinogenic Compound, in Human Urine Samples

“…APNH is thought to be metabolically converted to a hydroxyamino derivative by CYP1A2 and further activated to form esters by acetyltransferase, then covalently binding to DNA bases, with N 4 ¶-(2 ¶-deoxyguanosin-8-yl)-9-(4 ¶-aminophenyl)-9H-pyrido [3,4-b] indole (dG-C8-APNH) as the main adduct (8). Mutagenic activity of APNH in S. typhimurium strains proved comparable with those of 2-amino-3,8-dimethylimidazo [4,5-f]quinoxaline (MeIQx) and 3-amino-1-methyl-5H-pyrido [4,3-b] indole (Trp-P-2), food-borne carcinogenic heterocyclic amines (5,9). In a long-term carcinogenicity experiment using F344 rats, APNH induced tumors in the liver, colon, thyroid, hematopoietic systems, and clitoral gland (10).…”

“…We have reported that the appearance of mutagenicity derived from norharman and aniline in the presence of S9 mix is due to the formation of a mutagenic compound 9-(4 ¶-aminophenyl)-9H-pyrido [3,4-b]indole [aminophenylnorharman (APNH)], shown in Fig. 1 (5,6). Moreover, it has been shown that enzymes mainly responsible for APNH formation from norharman and aniline are cytochrome P450 3A4 (CYP3A4) and CYP1A2 (7).…”

LACK OF COMBINATION HEPATOCARCINOGENICITY OF HARMAN, NORHARMAN AND AMITROLE WHEN GIVEN WITH NaNO2 IN THE RAT

“…Both compounds do demonstrate mutagenic activity when in combination with other nonmutagens, such as aniline and o-toluidine (Nagao et al, 1977). It is likely that humans are exposed to these compounds in daily life and both chemicals, while lacking mutagenicity themselves, possess comutagenic potential in several in vitro assays (Umezawa et al, 1978;Nagao et al, 1977;Totsuka et al, 1998). Another compound of interest is 2-amino-1,3,4-triazole (amitrole), a pesticide whose use is now limited to non-crop applications because of its induction of thyroid tumors in mice and rats by a non-genotoxic mechanism which involves interference with the functioning of thyroid peroxidase (IARC, 2001).…”

Tumor‐initiating potency of a novel heterocyclic amine, aminophenylnorharman in mouse colonic carcinogenesis model

“…5 A novel HCA, 9-(4 0 -aminophenyl)-9H-pyrido [3,4-b]indole (aminophenylnorharman, APNH) can be formed by this reaction, then converted to the N-hydroxyamino derivative which produces DNA adducts after esterification to induce mutations in S. typhimurium TA98 and YG1024. [6][7][8] APNH is able to induce sister chromatid exchanges and chromosome aberrations. 9 We have recently demonstrated that APNH forms DNA adducts at the C-8 position of guanine residues in the various tissues including liver and colon of F344 rats after administration of APNH.…”