Chem. Res. Toxicol. 2000, 13, 165-169
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Articles Isolation and Chemical-Structural Identification of a Novel Aromatic Amine Mutagen in an Ozonized Solution of m-Phenylenediamine Hiroshi Kami,*,†,‡ Tetsushi Watanabe,‡ Saeko Takemura,† Yoichiro Kameda,† and Teruhisa Hirayama‡ Shiga Research Laboratories, Schering-Plough Kabushiki Kaisha, 1-4 Sasagaoka, Minakuchi-cho, Koga-gun, Shiga 528-0061, Japan, and Kyoto Pharmaceutical University, 5 Nakauchi-cho, Misasagi, Yamashina-ku, Kyoto 607-8414, Japan Received August 24, 1999
The mutagenicity of a m-phenylenediamine (m-PD) solution was markedly enhanced by oxidation with ozone. The ethyl acetate extracts from a m-PD solution ozonized at pH 10.7 were fractionated by normal-phase and reversed-phase column chromatography to isolate mutagens by monitoring mutagenic activities on Salmonella typhimurium TA98 in the presence of a mammalian metabolic activation system (S9 mix). From fraction 5-3-2, which exhibited the strongest mutagenicity (308 000 revertants/mg), a major mutagenic compound was isolated. On the basis of the high-resolution EI-mass, 1H NMR and 13C NMR spectral, and X-ray crystallography data, the structure of this compound was determined to be 2-amino-5-[(3aminophenyl)amino]-4-[(3-aminophenyl)imino]-2,5-cyclohexadien-1-one (PDT-1), which is a novel compound. PDT-1 is a newly identified frame-shift type mutagen, inducing 65 400 revertants and 295 000 revertants of S. typhimurium TA98 and YG1024 per micromole, respectively, in the presence of S9 mix. When a m-PD solution was oxidized with 1 or 2 mol of ozone at pH 4.0, 7.0, and 10.7, the contribution of PDT-1 to the mutagenicity of ethyl acetate extracts from the ozonized m-PD solution was 5-23%.
Introduction Chlorination has been performed commonly in the chemical treatment of wastewater, but the chlorination of wastewater containing organic compounds produces various mutagenic and/or carcinogenic byproducts, for example, trihalomethanes, chloropropanones, and MX [3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone] (13). Ozonation has recently been utilized as an alternative to chlorination since ozone is a very strong oxidizing agent and is an effective bactericide and virucide. Burleson et al. (4, 5) and Caulfield et al. (6) reported that ozonation inactivated or reduced the mutagenicity of aromatic amines in aqueous solution. However, Chan et al. (7) reported that azobenzenes, azoxybenzenes, benzidines, and phenazines were formed from anilines by ozone treatment, and Sayato (8) reported that highly genotoxic products such as formaldehyde, acetaldehyde, glyoxal, and methylglyoxal were detected in the sewage or humic solution after ozonation. Phenylenediamine (PD)1 derivatives are used widely as ingredients of dyes, paints, and resins in industrial organic synthesis. Ames et al. (9) reported that nine kinds of PD derivatives, such * To whom correspondence should be addressed. † Schering-Plough Kabushiki Kaisha. ‡ Kyoto Pharmaceutical University. 1 Abbreviations: DAA, 2,4-diaminoanisole; DMSO, dimethyl sulfoxide; PD, phenylenediamine; PDT-1, 2-amino-5-[(3-aminophenyl)amino]-4-[(3-aminophenyl)imino]-2,5-cyclohexadien-1-one.
as o-PD, m-PD, 2,4-diaminotoluene, and 2,4-diaminoanisole (DAA), exhibited various degrees of mutagenicity, and industrial wastewater containing these chemicals should therefore be properly treated. PD derivatives cannot be degraded by biological purification (10), and so chemical methods are required for wastewater treatment. Andreozzi et al. (11) investigated the ozonation of o-phenylendiamine from the standpoint of chemical mechanisms, but the reaction products from other PD derivatives with ozone and the mutagenicity of ozonized PD derivatives have not been clarified. In our previous paper (12), we reported that the mutagenicity of m-PD and DAA solutions was markedly enhanced by oxidation with 1-2 mol of ozone. Aminophenazines, which were isolated as the major mutagenic compounds from PD derivatives oxidized with hydrogen peroxide (13, 14), were also observed in ozonized m-PD and DAA solutions. However, the contribution of the observed aminophenazines to overall mutagenicity was low, suggesting that other mutagenic compounds were formed by ozonation. The purpose of this study was to isolate and identify the major mutagenic compounds in ozonized m-PD solutions. Here we report the identification of the structure of a novel mutagenic compound and its mutagenicity in Salmonella typhimurium TA98, TA100, and YG1024, which is an O-acetyltransferase-overproducing strain derived from TA98 (15). Furthermore, we have quantified
10.1021/tx9901534 CCC: $19.00 © 2000 American Chemical Society Published on Web 02/17/2000
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Chem. Res. Toxicol., Vol. 13, No. 3, 2000
this compound in a solution of m-PD ozonized at pH 4.0, 7.0, and 10.7 to investigate the effect of pH on the formation of this compound.
Experimental Procedures Chemicals. m-Phenylenediamine dihydrochloride (m-PD), silica gel (#7747, Merck), and ODS gel (COSMOSIL 40C18PREP) were purchased from Nacalai Tesque (Kyoto, Japan). Spectrophotometric-grade dimethyl sulfoxide (DMSO) was purchased from Wako Pure Chemicals (Osaka, Japan). All other chemicals that were used were analytical grade. Ozonation of m-PD and Isolation of the Mutagenic Compound from an Ozonized m-PD Solution. One gram of m-PD was dissolved in 1 L of 0.01 M phosphate buffer (pH 10.7), and ozone was applied directly to the solution for 20 min. Ozone was generated by an ozone generator (type O-3-2, Nihon Ozone K. K.) at an air flow rate of 1 L/min. After ozonation, the remaining ozone was removed from the solution by nitrogen gas induction for 1 min. The ozonized solution was extracted twice with 1 L of ethyl acetate, and the extract was evaporated to dryness. In total, 3 g of m-PD was ozonized as described above. The extract was fractionated to isolate mutagens by monitoring the mutagenic activity on S. typhimurium TA98 with S9 mix. Ethyl acetate extract (2.8 g) was applied to a silica gel (#7747) column (400 mm × 26 mm i.d.) and eluted with 250 mL of n-hexane/ethyl acetate (19/1, v/v), n-hexane/ethyl acetate (9/1, v/v), n-hexane/ethyl acetate (4/1, v/v), and n-hexane/ethyl acetate (2/1, v/v), 1400 mL of n-hexane/ethyl acetate (1/1, v/v) and n-hexane/ethyl acetate (1/2, v/v), and 500 mL of n-hexane/ ethyl acetate (1/4, v/v) and methanol. Each eluate was evaporated to dryness and designated as Fr.1-Fr.7, respectively. HPLC analysis of each fraction was performed on a STR ODS-H column (5 µm particle size, 150 mm × 4.0 mm i.d., Shimadzu Techno Research, Inc.) with a mobile phase of 40% acetonitrile at a flow rate of 0.7 mL/min. This HPLC analysis was carried out at ambient temperature, and the eluate was monitored for absorbance at 254 nm. Fr.5 (220 mg) was applied to a silica gel (#7747) column (400 mm × 11 mm i.d.) and eluted with 900 mL of n-hexane/ethyl acetate (2/1, v/v), n-hexane/ethyl acetate (1/1, v/v), n-hexane/ ethyl acetate (1/2, v/v), n-hexane/ethyl acetate (1/4, v/v), ethyl acetate, and methanol. Each eluate was evaporated to dryness and designated as Fr.5-1-Fr.5-5, respectively. Fr.5-3 (70 mg) was applied to an ODS gel (COSMOSIL 40C18-PREP) column (400 mm × 11 mm i.d.) and eluted with 20% acetonitrile. Each eluate was evaporated to dryness and designated as Fr.5-3-1Fr.5-3-3, respectively. Mutagenic compound I was isolated from Fr.5-3-2 by recrystallization with dioxane/H2O. Spectral Measurement of Compound I. UV absorption spectra were measured with a Shimadzu SPD-M10AV photodiode array detector. High-resolution electron impact mass spectra were recorded with a JEOL JMS-SX 102A QQ apparatus with a direct inlet system. 1H NMR and 13C NMR spectra were recorded for solutions in DMSO-d6 with a VARIAN XL-300 spectrometer operated at 300 MHz, and chemical shifts are reported in parts per million using tetramethylsilane as an internal standard. X-ray crystallographic analysis was carried out on a Rigaku AFC7R diffractometer with filtered CuKd radiation and a rotating anode generator. Quantification of PDT-1 in Ethyl Acetate Extracts from an Ozonized m-PD Solution at pH 4.0, 7.0, and 10.7. Each 40 mL of a 2.5 mM m-PD solution in 0.01 M phosphate buffer (pH 4.0, 7.0, or 10.7) was oxidized with ozone for 15 or 30 s. After ozonation, the remaining ozone was removed from the solution by nitrogen gas induction for 1 min. The m-PD solution ozonized at pH 7.0 or 10.7 was extracted twice with 40 mL of ethyl acetate. The m-PD solution ozonized at pH 4.0 was neutralized with sodium hydroxide and extracted with ethyl acetate as described above. The ethyl acetate layers were evaporated to dryness, and the extracted materials were redissolved in methanol for the mutagenicity test and HPLC analysis.
Kami et al. Table 1. Yields and Mutagenicities of Ethyl Acetate Extracts and Fractions of Ozonized m-PD Solutions
sample ethyl acetate extractb first silica gel columnc Fr.1 Fr.2 Fr.3 Fr.4 Fr.5 Fr.6 Fr.7 second silica gel columne Fr.5-1 Fr.5-2 Fr.5-3 Fr.5-4 Fr.5-5 ODS gel columnf Fr.5-3-1 Fr.5-3-2 Fr.5-3-3
yield (mg)
TA98 with S9 mix (revertants/ mg)
recovery of total mutagenicity (%)a
2897
70600
287 21 22 621 256 24 821
NDd ND 27800 10900 377000 330000 21500
0 0 0.3 4 51 4 9
34 2 82 9 34
43100 60000 361000 91400 45900
0.9 0.1 18 0.5 1.0
8 44 13
8100 308000 20400
