Quantification of the co-mutagenic β-carbolines, norharman and harman, in cigarette smoke condensates and cooked foods
Introduction
The β-carboline compound, norharman (9H-pyrido[3,4-b]indole), is itself not mutagenic to Salmonella typhimurium strains, either with or without an S9 mix, but shows mutagenicity in TA98 and YG1024 with an S9 mix in the presence of non-mutagenic aromatic amines including aniline and o-toluidine [1], [2], [3]. It has therefore been termed a ‘co-mutagen’. Moreover, DNA adduct formation by norharman with aromatic amines has been demonstrated to be related to the co-mutagenic action of norharman in S. typhimurium TA98 [4]. Another β-carboline compound, harman (1-methyl-9H-pyrido[3,4-b]indole), has also been reported to show co-mutagenic activity, although its activity is less than that of norharman [3].
By HPLC purification, we have isolated two mutagenic compounds, produced by the reaction of norharman with aniline, one showing mutagenicity with, and the other without, an S9 mix. The former was determined to be a coupled compound of norharman and aniline, 9-(4′-aminophenyl)-9H-pyrido[3,4-b]indole (aminophenylnorharman), inducing 1 783 000 revertants in YG1024 per μg with an S9 mix. The latter was hydroxyaminophenylnorharman. The same DNA adducts were formed when aminophenylnorharman alone or a mixture of norharman with aniline was incubated with an S9 mix and YG1024. Thus, the appearance of mutagenicity in this case was suggested to be due to the formation of the coupled compound, as shown in Fig. 1. [5]. Aminophenylnorharman is converted to the hydroxyamino derivative which further activated to yield esters, and finally the ultimate forms react with DNA bases to induce mutation in Salmonella.
Aromatic amines including aniline and o-toluidine are present in cigarette smoke condensate and some kinds of vegetables [6], [7]. In addition, norharman and harman have been found in cigarette smoke condensate and cooked foods [8], [9]. Thus, it is very likely that humans are simultaneously exposed to β-carbolines and aromatic amines. To clarify the significance of compounds like norharman and harman in the presence of aromatic amines to humans, much detailed data on levels of these compounds in the environment are required. In the present study, we therefore estimated the amounts of norharman and harman in mainstream and sidestream smoke of various brands of cigarettes, and also in various cooked foods.
Section snippets
Materials
Six Japanese brands of cigarettes (non-filter, brand I, and five filter-tipped, brands II–IV) were smoked in an automatic smoking machine under standard conditions as described previously [10]. Samples of mainstream and sidestream cigarette smoke from 10 cigarettes in each case were collected on quartz-fiber filters.
Meats used in the present study were purchased at a local market and cooked in a routine fashion. In the case of broiled beef, chicken and mutton, slices were broiled for about 3 min
Results and discussion
Norharman and harman were detected in mainstream and sidestream smoke condensates of all the samples. The recoveries of norharman and harman during the purification process were 67.5 and 63.8%, respectively. In addition, the compounds detected by HPLC were confirmed to be norharman and harman by their UV and fluorescence emission spectra.
By correcting the amounts of these β-carbolines, norharman and harman, detected by HPLC for their recoveries, the levels in mainstream and sidestream smoke
Acknowledgements
This study was supported by a Grant-in-Aid for Cancer Research from the Ministry of Health and Welfare of Japan, and grants from the Organization for Pharmaceutical Safety and Research (OPSR), and the Smoking Research Foundation. Y. Totsuka was the recipient of Research Resident Fellowships from the Foundation of Cancer Research during the performance of this work.
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