Identification of methylglyoxal as a major mutagen in wood and bamboo pyroligneous acids.

To identify the major mutagen in pyroligneous acid (PA), 10 wood and 10 bamboo pyroligneous acids were examined using the Ames test in Salmonella typhimurium strains TA100 and TA98. Subsequently, the mutagenic dicarbonyl compounds (DCs), glyoxal, methylglyoxal (MG), and diacetyl in PA were quantified using high-performance liquid chromatography, and the mutagenic contribution ratios for each DC were calculated relative to the mutagenicity of PA. Eighteen samples were positive for mutagens and showed the strongest mutagenicity in TA100 in the absence of S9 mix. MG had the highest mutagenic contribution ratio, and its presence was strongly correlated with the specific mutagenicity of PA. These data indicate that MG is the major mutagen in PA.

[Examination of original plant of Jamaica quassia extract, a natural bittering agent, based on composition of the constituents].

Jamaica quassia extract is a natural bittering agent used as a food additive in Japan. The main constituents of the extract have already been reported to be quassin and neoquassin. In this study, the differences in composition of the constituents among four Jamaican quassia extract products were analyzed by LC/MS. The results showed that the four products have similar compositions of their minor constituents, as well as their main constituents. We isolated four of the minor constituents that were commonly included in the four products, and identified them as 11-dihydro-12-norneoquassin, canthin-6-one, 4-methoxy-1-vinyl-beta-carboline and 4,9-dimethoxy-1-vinyl-beta-carboline. The List of Existing Food Additives in Japan mentions that Jamaica quassia (Picrasma excelsa) is the original plant from which Jamaica quassia extract is produced. However, we presume that Jamaica quassia extract may actually be made from appropriate plants other than Picrasma excelsa, since P. excelsa is listed as an endangered species by the International Union for Conservation of Nature and Natural Resources. We prepared hot water extracts from two other species of plants, Quassia amara (American quassia, Surinam quassia) and P. quassioides ('Nigaki' in Japanese), and investigated their constituents by LC/MS. The results showed that the compositions of the constituents in the Jamaica quassia extract products resembled those in the extract derived from Q. amara. These findings suggest that Jamaica quassia extract products are probably made from Q. amara.

Mutagenicity of surface soil from residential areas in Kyoto city, Japan, and identification of major mutagens.

To clarify the mutagenic potential of surface soil in residential areas in Kyoto city, surface soil samples were collected twice or three times from 12 sites, and their organic extracts were examined by the Ames/Salmonella assay. Almost all (>92%) samples showed mutagenicity in TA98 without and with S9 mix, and 8/25 (32%) samples showed high (1000-10,000 revertants/g of soil) or extreme (>10,000 revertants/g of soil) activity. Moreover, to identify the major mutagens in surface soil in Kyoto, a soil sample was collected at a site where soil contamination with mutagens was severe and continual. The soil extract, which showed potent mutagenicity in TA98 without S9 mix, was fractionated by diverse column chromatography methods. Five major mutagenic constituents were isolated and identified to be 1,6-dinitropyrene (DNP), 1,8-DNP, 1,3,6-trinitropyrene (TNP), 3,9-dinitrofluoranthene (DNF), and 3,6-dinitrobenzo[e]pyrene (DNBeP) by co-chromatography using high performance liquid chromatography and spectral analysis. Contribution ratios of 1,6-DNP, 1,8-DNP, 1,3,6-TNP, 3,9-DNF, and 3,6-DNBeP to total mutagenicity of the soil extract in TA98 without S9 mix were 3, 10, 10, 10, and 6%, respectively. These nitroarenes were detected in surface soil samples collected from four different residential sites in other prefectures, and their contribution ratios to soil mutagenicity were from 0.7 to 22%. These results suggest that surface soil in residential areas in Kyoto was widely contaminated with mutagens and there were some sites where surface soils were heavily polluted. 1,6-DNP, 1,8-DNP, 1,3,6-TNP, 3,9-DNF, and 3,6-DNBeP may be major mutagenic constituents that contaminate surface soil in Kyoto and other residential areas.

Isolation and identification of non-chlorinated phenylbenzotriazole (non-ClPBTA)-type mutagens in the Ho River in Shizuoka Prefecture, Japan.

We previously identified 2-[2-(acetylamino)-4-amino-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA) congeners as major mutagens in water concentrates from several rivers that flow in three different areas, i.e. Kyoto, Aichi, and Fukui Prefectures, in Japan. In synthesis studies, these PBTAs were shown to be formed from corresponding dinitrophenylazo dyes via non-chlorinated derivatives (non-ClPBTAs). However, only non-ClPBTA-1, i.e. 2-[2-(acetylamino)-4-[bis(2-methoxyethyl)amino]-5-methoxyphenyl]-6-amino-4-bromo-2H-benzotriazole, had been detected as a minor contaminant in the Nishitakase River in Kyoto. In this study, analysis of mutagens in water concentrate from the Ho River, which flows through an area with a textile dyeing industry in Shizuoka Prefecture, Japan, allowed the isolation of four compounds (I, II, III, and IV). These four mutagens were identified as 2-[2-(acetylamino)-4-[N-(2-cyanoethyl)ethylamino]-5-methoxyphenyl]-6-amino-4-bromo-2H-benzotriazole (non-ClPBTA-2), 2-[2-(acetylamino)-4-[(2-hydroxyethyl)amino]-5-methoxyphenyl]-6-amino-4-bromo-2H-benzotriazole (non-ClPBTA-3), 2-(2-acetylamino-4-amino-5-methoxyphenyl)-6-amino-4-bromo-2H-benzotriazole (non-ClPBTA-4), and 2-[2-(acetylamino)-4-(diethylamino)-5-methoxyphenyl]-6-amino-4-bromo-2H-benzotriazole (non-ClPBTA-7) by spectral data and co-chromatography using synthesized standards. Non-ClPBTA-3 and -7 were highly mutagenic in Salmonella typhimurium YG1024, inducing 159,000 and 178,000 revertants/microg, respectively, in the presence of S9 mix. Like PBTAs, non-ClPBTAs might have been produced from azo dyes during industrial processes in dyeing factories and released into rivers.

Detection of a novel mutagen, 3,6-dinitrobenzo[e]pyrene, as a major contaminant in surface soil in Osaka and Aichi Prefectures, Japan.

We previously identified 1,3-, 1,6-, and 1,8-dinitropyrene (DNP) isomers as major mutagens in surface soil in three metropolitan areas of Japan. In the present study, an organic extract from surface soil collected at a park in Takatsuki in Osaka Prefecture, which showed extremely high mutagenicity in Salmonella typhimurium TA98 in the absence of mammalian metabolic system (S9 mix), was investigated to identify major mutagens. A new powerful bacterial mutagen, as well as 1,6- and 1,8-DNP isomers, was isolated from the organic extract (1.8 g) of the soil sample (2.2 kg) by column chromatography. On the basis of mass spectra, the new mutagen, which accounted for 15% of the total mutagenicity of the soil extract, was thought to be a dinitrated polycyclic aromatic hydrocarbon with a molecular weight of m/z 342. The mutagen was synthesized from benzo[e]pyrene by nitration and was determined to be 3,6-dinitrobenzo[e]pyrene (DNBeP) based on its 1H NMR spectrum. The mutagenic potency of 3,6-DNBeP in the Ames/Salmonella assay was extremely high, in that it induced 285,000 revertants/nmol in TA98 and 955,000 revertants/nmol in YG1024 without S9 mix and was comparable to those of DNP isomers, which are some the most potent bacterial mutagens reported so far. In addition to the soil sample from Takatsuki, 3,6-DNBeP was also detected in surface soil samples collected at parks in four different cities, i.e., Izumiotsu and Takaishi in Osaka Prefecture and Nagoya and Hekinan in Aichi Prefecture, and accounted for 22-29% of the total mutagenicity of these soil extracts in TA98 without S9 mix. These results suggest that 3,6-DNBeP is a major mutagen in surface soil and may largely contaminate the surface soil in these two regions in Japan.