Mutagenesis, Vol. 17, No. 4, 293-299,
July 2002
© 2002 UK Environmental Mutagen Society/Oxford University Press
Mutagenicity of two 2-phenylbenzotriazole derivatives, 2-[2-(acetylamino)-4-(diethylamino)-5-methoxyphenyl]-5-amino- 7-bromo-4-chloro-2H-benzotriazole and 2-[2-(acetylamino)-4-(diallylamino)-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole and their detection in river water in Japan
Department of Public Health, Kyoto Pharmaceutical University, 5 Nakauchicho, Misasagi, Yamashina-ku, Kyoto 607-8414, Japan, 1 Graduate School of Nutritional and Environmental Sciences and 2 School of Pharmaceutical Science, University of Shizuoka, 52-1 Yada, Shizuoka 422-8526, Japan, 3 Cancer Prevention Division, National Cancer Center Research Institute, 1-1 Tsukiji 5-chome, Chuo-ku, Tokyo 104-0045, Japan, 4 Faculty of Pharmaceutical Sciences, Hokuriku University, Kanagawa-machi, Kanazawa 920-1181, Japan and 5 Department of Food and Nutrition Science, Kyoto Women's University, Kitahiyoshi-cho, Imakumano, Higashiyama-ku, Kyoto 605-8501, Japan
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We recently detected five 2-phenylbenzotriazole (PBTA)-type mutagens (PBTA-1, PBTA-2, PBTA-3, PBTA-4 and PBTA-6) in concentrates from several rivers that flow in geographically different areas in Japan containing textile-related industries. On the basis of synthesis studies, these five PBTA derivatives were deduced to have originated from the corresponding dinitrophenylazo dyes, which are industrial chemicals used in textile dyeing, via reduction and chlorination. 2-[(2-Bromo-4,6-dinitrophenyl)azo]-5-(diethylamino)-4-methoxyacetanilide (Color Index name Disperse Blue 291, CAS registry no. 56548-64-2) and 2-[(2-bromo-4,6-dinitrophenyl)azo]-5-(diallylamino)-4-methoxyacetanilide (Color Index name Disperse Blue 373, CAS registry no. 51868-46-3) are used in textile dyeing and have 2-[(2-bromo-4,6-dinitrophenyl)azo]-4-methoxyacetanilide moieties in their structures, which are thought to be essential for their conversion to mutagenic PBTA derivatives. In the present study we have synthesized 2-[2-(acetyl-amino)-4-(diethylamino)-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-7) and 2-[2-(acetylamino)-4-(diallylamino)-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-8) from Disperse Blue 291 and Disperse Blue 373, respectively, by reduction with iron powder and subsequent chlorination with sodium hypochlorite. Both PBTA-7 and PBTA-8 exerted strong mutagenicity in Salmonella typhimurium TA98 and YG1024 in the presence of S9 mix (43 000 and 1 430 000 revertants/nmol for PBTA-7 and 40 700 and 2 213 000 revertants/nmol for PBTA-8 in TA98 and YG1024). To clarify whether PBTA-7 and PBTA-8 exist in the environment, water samples were collected at seven sites in six rivers flowing through two different regions where textile dyeing industries are located. All water samples were mutagenic in Salmonella typhimurium YG1024 with S9 mix and their potencies ranged from 108 000 to 1 990 000 revertants/g blue rayon. PBTA-7 and PBTA-8 were detected in water samples from both regions at levels of <0.1–101.4 ng/g blue rayon and <0.1–48.9 ng/g blue rayon, respectively. In some samples PBTA-7 and PBTA-8 could contribute up to 15% of the water mutagenicity.
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Numerous chemicals are released into surface waters such as rivers from industrial and domestic sources directly or after treatment at municipal wastewater treatment facilities. Many studies have shown that river waters have been contaminated by genotoxic substances and the mutagenic potency of industrial wastewaters from pulp and paper mills, steel foundries and organic chemical manufacturing facilities have been reported to be especially high (Stahl, 1991
; Houk, 1992). Municipal wastewater is generally a complex mixture of domestic water, industrial water, groundwater infiltration and surface run-off. Some researchers have suggested that the genotoxicity of municipal wastewater is proportional to the industrial contribution (Meier and Bishop, 1985; Meier et al., 1987). On the other hand, potent heterocyclic amine mutagens such as 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), which are excreted in human urine and feces, have been identified as major mutagens in river water (Ohe, 1997; Kataoka et al., 2000). Trp-P-2 was detected in processed municipal wastewater (Segawa et al., 1993; Ono et al., 1996). These findings suggest that major genotoxic compounds and their sources may vary among rivers. Therefore, identifying major genotoxic compounds in river waters is important to clarify their sources and efficiently minimize contamination.
We recently identified five 2-phenylbenzotriazole (PBTA)-type compounds as major mutagens in concentrates from several rivers in Japan (Nukaya et al., 1997, 2001; Oguri et al., 1998; Shiozawa et al., 2000; Watanabe et al., 2001). These concentrates were taken at sites below sewage plants where abundant effluent from textile dyeing factories is treated along with domestic wastewater or at sites downstream from textile dyeing factories. Based on synthesis studies, four of these five PBTA-type compounds, i.e. 2-[2-(acetylamino)-4-[bis (2-methoxyethyl)amino]-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-1) (Nukaya et al., 1997), 2-[2-(acetylamino)-4-[N-(2-cyanoethyl)ethylamino]-5-meth-oxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-2) (Oguri et al., 1998), 2-[2-(acetylamino)-4-[(2-hydroxyethyl)amino]-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-3) (Shiozawa et al., 2000) and 2-[2-(acetylamino)-4-amino-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-4) (Nukaya et al., 2001), were suggested to be formed from the corresponding dinitrophenylazo dyes, which are used in the textile industry, via reduction with sodium hydrosulfite and subsequent chlorination with sodium hypochlorite (Oguri et al., 1998; Shiozawa et al., 1998, 2000; Nukaya et al., 2001). The fifth compound, 2-[2-(acetylamino)-4-[bis(2-hydroxyethyl)amino]-5-methoxy-phenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-6), is a hydrolyzed product of 2-[4-[bis(2-acetoxyethyl)amino]-2-(acetylamino)-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-5) with sodium hydroxide (Watanabe et al., 2001). PBTA-5 was synthesized from 2-[(2-bromo-4,6-dinitrophenyl)azo]-5-[bis(2-acetoxyethyl)amino]-4-methoxyacetanilide (Color Index name Disperse Blue 79:1, CAS registry no. 75497-74-4) by reduction and subsequent chlorination, like the other PBTA-type compounds described above. Disperse Blue 79:1 and the precursors of PBTA-1, PBTA-2, PBTA-3 and PBTA-4, i.e. 2-[(2-bromo-4,6-dinitro-phenyl)azo]-5-[bis(2-methoxyethyl)amino]-4-methoxyaceto-anilide (Shiozawa et al., 1998), 2-[(2-bromo-4,6-dinitrophenyl) azo]-5-[N-(2-cyanoethyl)ethylamino]-4-methoxyacetoanilide (Oguri et al., 1998), 2-[(2-bromo-4,6-dinitrophenyl)azo]-4-methoxy-5-[(2-hydroxyethyl)amino]acetanilide (Shiozawaet al., 2000) and 2-[(2-bromo-4,6-dinitrophenyl)azo]-5-amino-4-methoxyacetanilide (Nukaya et al., 2001), have 2-[(2-bromo-4,6-dinitrophenyl)azo]-4-methoxyacetanilide moieties in their structures and these moieties are thought to be essential for the formation of mutagenic PBTA derivatives.
2-[(2-Bromo-4,6-dinitrophenyl)azo]-5-(diethylamino)-4-methoxyacetanilide (Color Index name Disperse Blue 291, CAS registry no. 56548-64-2) and 2-[(2-bromo-4,6-dinitro-phenyl)azo]-5-(diallylamino)-4-methoxyacetanilide (Color Index name Disperse Blue 373, CAS registry no. 51868-46-3) are used in the textile dyeing industry (Murakami and Tanaka, 1996a,b). Since Disperse Blue 291 and 373 also have a 2-[(2-bromo-4,6-dinitrophenyl)azo]-4-methoxyacetanilide moiety in their structures, these azo dyes might also be converted to mutagenic PBTA-type compounds by reduction and subsequent chlorination, like the other precursors of PBTA-type mutagens, and the resulting PBTA mutagens could contaminate river waters in areas where there are textile-related industries.
In the present study we have synthesized 2-[2-(acetylamino)-4-(diethylamino)-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-7) and 2-[2-(acetylamino)-4-(diallylamino)-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-8) from Disperse Blue 291 and Disperse Blue 373, respectively, and determined the mutagenicity of these PBTA derivatives in the Salmonella assay. Moreover, to clarify whether PBTA-7 and PBTA-8 exist in the environment, we have analyzed river water samples collected from two geographically different areas of Japan with textile-related industries. The contribution of PBTA-7 and PBTA-8 to the mutagenicity of the river water samples is also discussed.
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Chemicals
Blue rayon was obtained from Funakoshi Co. Ltd. (Tokyo, Japan). HPLC grade acetonitrile and methanol were purchased from Wako Pure Chemical Industries (Osaka, Japan). All other chemicals were of analytical grade.
Spectral measurement
UV absorption spectra were measured using a Shimadzu SPD-M10AV photodiode array detector and a Beckman DU 640 spectrophotometer. 1H and 13C NMR spectra of solutions in chloroform-d or DMSO-d6 were taken with a JEOL JNM-GSX270 (1H, 270 MHz; 13C, 67.5 MHz) or a JEOL JNM-GSX500 (1H, 500 MHz; 13C, 125 MHz) Fourier transform spectrometer. The following abbreviations are used: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad. Chemical shifts are shown in p.p.m. using tetramethylsilane as an internal standard. Electron impact mass spectra (EI-MS) were measured with a JEOL JMS-GC mate mass spectrometer. Fast atom bombardment mass spectra (FAB-MS, matrix m-nitrobenzyl alcohol) were measured with a JEOL JMS-SX103 mass spectrometer. All melting points were obtained on a Yazawa micro-melting point apparatus (5Y-1) and are given as uncorrected values.
Synthesis of 3-(diethylamino)-4-methoxynitrobenzene
Ethyl bromide (25 ml, 0.3 mol) was added dropwise to a stirred mixture of 3-amino-4-methoxynitrobenzene (16.8 g, 0.1 mol) and sodium hydride (60% dispersion in mineral oil, 12 g) in N,N-dimethylformamide (200 ml) at room temperature. The mixture was then stirred for 1 h at room temperature, heated gradually to 80°C and stirred for 2 h at the same temperature. The mixture was poured over ice in a beaker and extracted with ether. The ether solution was washed with brine and dried over magnesium sulfate. After removal of the ether under reduced pressure, an oily residue was subjected to column chromatography on silica gel (eluent 1% methanol in chloroform) to give an oily product (21.1 g, 94%). MS m/z: 224 (M+). 1H NMR (CDCl3) 
: 1.07 (6H, t, J = 7.1 Hz, 2xCH3), 3.22 (4H, q, J = 7.1 Hz, 2x-CH2-), 3.96 (3H, s, OCH3), 6.88 (1H, d, J = 8.8 Hz, ArH), 7.77 (1H, d, J = 2.7 Hz, ArH), 7.90 (1H, dd, J = 2.7, 8.8 Hz, ArH).
Synthesis of 3-(diethylamino)-4-methoxyacetanilide
A mixture of 3-(diethylamino)-4-methoxynitrobenzene (11.2 g, 0.05 mo1) and 5% palladium charcoal (1 g) in ethano1 (200 ml) was stirred for 2 h at room temperature under a hydrogen atmosphere. After removal of the catalyst by filtration, the filtrate was concentrated under reduced pressure to give an oily residue, which was treated with acetic anhydride (7.6 g, 0.075 mol) and potassium carbonate (10 g) in dichloromethane (200 ml). The mixture was poured into ice–water and the organic layer was separated. The aqueous layer was extracted with dichloromethane. The combined organic solution was washed with brine and dried over magnesium sulfate. After removal of the solvent, the residual solid was recrystallized with hexane/ethyl acetate to give a pure product (9.9 g, 83%). m.p. 99–100°C. MS m/z: 236 (M+). 1H NMR (CDC13) : 1.03 (6H, t, J = 7.1 Hz, 2xCH3), 2.15 (3H, s, COCH3), 3.15 (4H, q, J = 7.1 Hz, 2x-CH2-), 3.83 (3H, s, OCH3), 6.78 (1H, d, J = 9.4Hz, ArH), 7.05 (1H, br, CONH), 7.05–7.10 (2H, m, ArH).
Synthesis of 2-[(2-bromo-4,6-dinitrophenyl)azo]-5-(diethylamino)-4-methoxyacetanilide (Disperse Blue 291)
The azo coupling reaction of 2-bromo-4,6-dinitrobenzenediazonium sulfate [prepared from 2-bromo-4,6-dinitroaniline (7.86 g, 0.03 mo1), sodium nitrite (2.0 g, 0.03 mo1) and concentrated sulfuric acid (30 ml)] with 3-(diethylamino)-4-methoxyacetanilide (7.0 g, 0.03 mo1) was carried out as described previously (Shiozawa et al., 1998) to yield a green powder (11.2 g, 75%). m.p. 180–180–183°C. FAB-MS m/z: 508 (M+, 79Br), 510 (M+ + 2, 8lBr). lH NMR (DMSO-d6) : 1.23 (6H, t, J = 6.8 Hz, 2xCH3), 2.51 (3H, s, COCH3), 3.57 (4H, q, J = 6.8 Hz, 2x-CH2-), 3.82 (3H, s, OCH3), 7.18 (1H, s, ArH), 7.92 (1H, s, ArH), 8.68 (1H, d, J = 1.7 Hz, ArH), 8.70 (1H, d, J = 1.7 Hz, ArH), 9.25 (1H, br, CONH).
Synthesis of 2-[2-(acetylamino)-4-(diethylamino)-5-methoxyphenyl]-6-amino-4-bromo-2H-benzotriazole (non-ClPBTA-7)
Iron powder (5.6 g) and magnesium chloride·6H2O (13.2 g) were added to a solution of Disperse Blue 291 (5.1 g, 10 mmol) in 200 ml of mixed solvent (tetrahydrofuran/water 1/1). The mixture was stirred at 70°C for 1 h. Insoluble materials were filtered off and washed with tetrahydrofuran. The filtrate was concentrated to 1/3 of its original volume under reduced pressure and extracted with dichloromethane. The organic solution was washed with brine and dried over magnesium sulfate. Removal of the solvent under reduced pressure yielded a dark oil, which was subjected to medium pressure liquid chromatography on an ODS silica gel column (eluent 70% methanol) to yield a yellow powder (260 mg, 6%). m.p. 123–125°C. FAB-MS m/z: 446 (M+, 79Br), 448 (M+ + 2, 8lBr). 1H NMR (CDCl3) : 1.13 (6H, t, J = 7.1 Hz, 2xCH3), 2.26 (3H, S, COCH3), 3.29 (4H, q, J = 7.1 Hz, 2x-CH2-), 3.96 (3H, s, OCH3), 3.98 (2H, br, NH2), 6.90 (1H, d, J = 1.8 Hz, ArH), 7.15 (1H, d, J = 1.8 Hz, ArH), 7.74 (1H, s, ArH), 8.26 (1H, s, ArH), 11.06 (1H, br, CONH). 13C NMR (CDCl3) : 12.17 (2C), 25.45, 45.89 (2C), 55.96, 95.40, 105.68, 110.60, 113.71, 121.60, 123.61, 124.56, 138.95, 140.64, 144.81, 146.50, 148.66, 168.56.
Synthesis of 2-[2-(acetylamino)-4-(diethylamino)-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-7)
An aqueous solution of sodium hypochlorite (1 ml, available chlorine >1%) was added dropwise to a stirred solution of non-ClPBTA-7 (100 mg, 0.22 mmo1) in dichloromethane (30 ml) at room temperature. After stirring for 5 min, the organic layer was separated, washed with brine and dried over magnesium sulfate. The solvent was removed under reduced pressure to yield a pale yellow solid, which was subjected to medium pressure liquid chromatography on an ODS silica gel column (eluent 70% methanol) to yield a yellow powder (76 mg, 72%). m.p. 200–202°C. UV (MeOH) max (
): 226 (48 500), 265 (29 200), 392 (28 900). FAB-MS m/z: 480 (M+, 79Br, 35Cl), 482 (M+ + 2, 81Br, 35Cl and 79Br, 37Cl), 484 (M+ + 4, 81Br, 37Cl). 1H NMR (CDCl3) : 1.13 (6H, t, J = 7.1 Hz, 2xCH3), 2.27 (3H, s, COCH3), 3.30 (4H, q, J = 7.1 Hz, 2xCH2), 3.97 (3H, s, OCH3), 4.36 (2H, br, NH2), 7.22 (1H, s, ArH), 7.78 (1H, s, ArH), 8.26 (1H, s, ArH), 11.05 (1H, br, CONH). 13C NMR (CDCl3) : 12.24 (2C), 25.45, 45.86 (2C), 56.05, 99.83, 105.80, 109.06, 113.47, 121.23, 122.79, 124.79, 138.63, 141.16, 142.36, 142.60, 148.59, 168.57.
Synthesis of 3-(diallylamino)-4-methoxyacetanilide
A mixture of allyl iodide (5.0 g, 0.3 mol), 3-amino-4-methoxyacetanilide (18.1 g, 0.1 mol) and potassium carbonate (4 g) in acetone (300 ml) was refluxed for 30 min. After the mixture had been concentrated under reduced pressure, water (300 ml) was added to the residue. The mixture was extracted with dichloromethane (100 ml). The organic layer was washed with brine and dried over magnesium sulfate. Removal of the solvent under reduced pressure gave a dark oil, which was subjected to column chromatography on silica gel (eluent chloroform/ethanol 19/1) to afford a pale brown oil (24.3 g, 93.5%). MS m/z: 260 (M+). 1H NMR (CDCl3) : 2.12 (3H, s, COCH3), 3.74 (4H, d, J = 3.1, 2xNCH2), 3.83 (3H, s, OCH3), 5.11 (2H, dd, J = 1.8, 10.3 Hz, 2x=CHAHB), 5.15 (2H, dd, J = 1.8, 17.2 Hz, 2x= CHAHB), 5.81 (2H, ddt, J = 3.1, 10.3, 17.3 Hz, 2x-CH=), 6.77 (1H, d, J = 9.2 Hz, ArH), 7.06–7.10 (2H, m, 2xArH), 7.29 (1H, br, CONH).
Synthesis of 2-[(2-bromo-4,6-dinitrophenyl)azo]-5-(diallylamino)-4-methoxyacetanilide (Disperse Blue 373)
The azo coupling reaction of 2-bromo-4,6-dinitrobenzenediazonium sulfate [prepared from 2-bromo-4,6-dinitroaniline (26.2 g, 0.1 mol), sodium nitrite (7 g, 0.1 mol) and concentrated sulfuric acid (40 ml)] with 3-(diallylamino)-4-methoxyacetanilide (26 g, 0.1 mol) was carried out as described above to yield a green powder (45.3 g, 85%). m.p. 175–176°C. FAB-MS m/z: 532 (M+, 79Br), 534 (M+ + 2, 81Br). 1H NMR (CDCl3) : 2.28 (3H, s, COCH3), 3.86 (3H, s, OCH3), 4.14 (4H, d, J = 5.4 Hz, 2xNCH2), 5.25 (2H, dd, J = 1.3, 17.1 Hz, 2xCHAHB), 5.29 (2H, dd, J = 1.3, 10.1 Hz, 2xCHAHB), 5.94 (2H, ddt, J = 5.4, 10.1, 17.1 Hz, 2x=CH-), 7.28 (1H, s, ArH), 8.17 (1H, s, ArH), 8.28 (1H, d, J = 2.4 Hz, ArH), 8.64 (1H, d, J = 2.4 Hz, ArH), 9.03 (1H, br, CONH).
Synthesis of 2-[2-(acetylamino)-4-(diallylamino)-5-methoxyphenyl]-6-amino-4-bromo-2H-benzotriazole (non-ClPBTA-8)
Iron powder (5.5 g, 10 mmol) and magnesium chloride·6H2O (12 g, 60 mmol) were added to a solution of Disperse Blue 373 (5.3 g, 10 mmol) in 200 ml of 50% aqueous acetone. The mixture was stirred at 80°C for 1 h. Insoluble materials were filtered off and washed with acetone. The filtrate was concentrated to 1/3 of its original volume under reduced pressure and extracted with dichloromethane. The organic solution was washed with brine and dried over magnesium sulfate. Removal of the solvent under reduced pressure yielded a dark oil, which was subjected to column chromatography on silica gel (eluent 2% methanol in chloroform) and Sephadex LH-20 (eluent methanol) to yield a yellow powder (357 mg, 7.6%). m.p. 130–131°C. FAB-MS m/z: 470 (M+, 79Br), 472 (M+ + 2, 81Br). 1H NMR (CDCl3) : 2.26 (3H, s, COCH3), 3.86 (4H, d, J = 6.0 Hz, 2xNCH2), 3.95 (3H, s, OCH3), 4.04 (2H, brs, NH2), 5.17 (2H, dd, J = 1.7, 10.0 Hz, 2x=CHAHB), 5.22 (2H, dd, J = 1.7, 16.9 Hz, 2x=CHAHB), 5.81–5.96 (2H, m, 2x-CH=), 6.83 (1H, d, J = 1.7 Hz, ArH), 7.11 (1H, d, J = 1.7 Hz, ArH), 7.74 (1H, s, ArH), 8.30 (1H, s, ArH), 11.11 (1H, br, CONH). 13C NMR (CDCl3) : 25.40, 54.10 (2C), 55.93, 95.21, 105.75, 110.44, 113.10, 117.74 (2C), 121.37, 123.57, 124.54, 134.54 (2C), 138.77, 140.74, 144.68, 146.51, 147.85, 168.46.
Synthesis of 2-[2-(acetylamino)-4-(diallylamino)-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-8)
An aqueous solution of sodium hypochlorite (1 ml, available chlorine >1%) was added dropwise to a stirred solution of non-ClPBTA-8 (130 mg, 0.3 mmol) in dichloromethane (30 ml) at room temperature. After stirring for 5 min, the organic layer was separated, washed with brine and dried over magnesium sulfate. The solvent was removed under reduced pressure to yield a pale yellow solid, which was subjected to medium pressure liquid chromatography on a silica gel column (eluent 2% methanol in chloroform) and then on an ODS silica gel column (eluent 70% methanol) to yield a yellow powder (105 mg, 75%). m.p. 173–175°C. UV (MeOH) max (): 220 (49 800), 266 (29 900), 391 (30 100). FAB-MS m/z: 504 (M+, 79Br, 35Cl), 506 (M+ + 2, 81Br, 35Cl and 79Br, 37Cl), 508 (M+ + 4, 81Br, 37Cl). 1H NMR (CDCl3) : 2.26 (3H, s, COCH3), 3.87 (4H, d, J = 6.5 Hz, 2xNCH2), 3.98 (3H, s, OCH3), 4.36 (2H, brs, NH2), 5.18 (2H, dd, J = 1.8, 10.4 Hz, 2x=CHAHB), 5.23 (2H, dd, J = 1.8, 17.3 Hz, 2x=CHAHB), 5.88 (2H, ddt, J = 6.5, 10.4, 17.3 Hz, 2x-CH=), 7.22 (1H, s, ArH), 7.79 (1H, s, ArH), 8.30 (1H, s, ArH), 11.09 (1H, br, CONH). 13C NMR (CDCl3) : 24.49, 54.11 (2C), 56.06, 99.64, 105.76, 108.15, 112.96, 117.85 (2C), 121.08, 122.79, 124.77, 134.52 (2C), 138.45, 141.17, 142.38, 142.45, 147.83, 168.50.
Mutagenicity assay
All test samples were dissolved in 100 µl of 50% dimethyl sulfoxide and mutagenicity was assayed by the preincubation method (Yahagi et al., 1977) in the presence or absence of S9 mix. Salmonella typhimurium strains TA98 (Maron and Ames, 1983), TA100 (Maron and Ames, 1983), YG1024 (Watanabe et al., 1990) and YG1029 (Watanabe et al., 1990) were used. The S9 mix contained 10 µl of S9 in a total volume of 500 µl. S9 was prepared from the livers of male Sprague–Dawley rats which had been treated with phenobarbital and ß-naphthoflavone. Mutagenic activities of samples were calculated from linear portions of the dose–response curves, which were obtained with four or five doses and duplicate plates, in two independent experiments.
Detection of PBTA-7 and PBTA-8 in river water
Samples were collected at sites downstream from textile dyeing factories along the Kitsune, Mawatari and Asuwa Rivers in Fukui prefecture between August 1999 and August 2000 and at sites below municipal sewage plants that treated effluent from textile dyeing factories on the Nishitakase, Katsura and Uji Rivers in Kyoto prefecture in February 1999 and August 2000. The sampling sites are shown in Figure 1. Many textile dyeing factories are located near the sampling sites. At each sampling site, 15 g blue rayon (5 g/bag) was hung in the river for 24 h after a minimum of several days with no rain. The blue rayon was then washed with distilled water several times. The materials that had adsorbed to the blue rayon were extracted as described previously (Sakamoto and Hayatsu, 1990), with a minor modification. The adsorbed substances were extracted by two rounds of shaking in 80 ml methanol/g blue rayon, instead of methanol/ammonia water (50:1 v/v), for 30 min. The extracts were combined and evaporated to dryness. The residue was dissolved in 1 ml of 75% methanol. Half of the sample solution was applied to a semi-preparative YMC-Pack ODS-AM 324 column (5 µm particle size, 10x300 mm; YMC Co. Ltd, Kyoto, Japan) for HPLC and then eluted with the following gradient system at a flow rate of 1.6 ml/min: 0–25 min, 75% methanol; 25–40 min, linear gradient of 75–80% methanol; 40–50 min, linear gradient of 80–90% methanol; 50–90 min, 90% methanol. This process was repeated twice.
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) denote sampling sites and sewage plants, respectively.Fractions corresponding to PBTA-7, with retention times of 54–56 min on the YMC-Pack ODS-AM 324 column, were combined and evaporated to dryness. The residue was dissolved in 0.5 ml of 50% acetonitrile and injected into a LUNA 5µ Phenyl Hexyl column (5 µm particle size, 4.6x250 mm; Phenomenex, CA) with a mobile phase of 50% acetonitrile at a flow rate of 1 ml/min. The fractions corresponding to PBTA-7 were eluted at retention times of 21–23 min. These fractions were evaporated to dryness. The residue was dissolved in 0.5 ml of 55% acetonitrile and separated by HPLC using a YMC-Pack ODS-A303 column (5 µm particle size, 4.6x250 mm; YMC Co. Ltd). The materials were eluted with 55% acetonitrile at a flow rate of 1 ml/min. Fractions corresponding to PBTA-7, with retention times of 28–29 min, were evaporated to dryness. The residue was dissolved in 55% acetonitrile and applied to a Cosmosil 5C18 AR-II column (5 µm particle size, 4.6x250 mm; Nacalai Tesque Inc., Kyoto, Japan) with a mobile phase of 55% acetonitrile at a flow rate of 1 ml/min. The peak due to authentic PBTA-7 was detected at 28.2 min.
Fractions corresponding to PBTA-8, with retention times of 64–66 min on the YMC-Pack ODS-AM 324 column, were evaporated to dryness and then dissolved in 400 µl of 50% acetonitrile. The solution was further applied to a LUNA 5µ phenyl hexyl column. A mobile phase of 55% acetonitrile was pumped in at a flow rate of 1 ml/min. Fractions corresponding to PBTA-8 were eluted at retention times of 23–25 min. These fractions were evaporated to dryness. The residue was dissolved in 400 µl of 55% acetonitrile and applied to a YMC-Pack ODS-A303 column with a mobile phase of 55% acetonitrile at a flow rate of 1 ml/min. Fractions corresponding to PBTA-8, with retention times of 45–47 min, were evaporated to dryness. The residue was dissolved in 60% acetonitrile and applied to a Cosmosil 5C18 AR-II column with a mobile phase of 60% acetonitrile at a flow rate of 1 ml/min. The peak due to PBTA-8 was detected at 27.5 min.
All HPLC procedures were carried out at ambient temperature and the eluates were monitored using a Shimadzu SPD-M10AV photodiode array detector.
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Synthesis of PBTA-7 and PBTA-8
Disperse Blue 291 was prepared by the coupling reaction of 2-bromo-4,6-dinitrobenzene diazonium sulfate with 3-(diethylamino)-4-methoxyacetanilide, which was prepared from 3-amino-4-methoxynitrobenzene. As shown in Scheme 1, the reduction of Disperse Blue 291 with iron powder gave a 2-phenylbenzotriazole skeleton product. Its FAB-MS spectrum (M+ and M+ + 2, m/z 446, 448; relative abundance 100:98) indicates a molecular formula of C19H23BrN6O2. In the 1H NMR spectrum, aromatic protons are observed as two meta doublets (J = 1.8 Hz) at 6.90 and 7.15 p.p.m. for the benzotriazole ring protons and two singlets at 7.74 and 8.26 p.p.m. for the 2-phenyl moiety. A triplet (6H) at 1.13 p.p.m. and a quartet (4H) at 3.29 p.p.m. indicate the presence of two ethyl groups. Acetamide NH and primary amine NH2 appear at 11.06 (br) and 3.98 (br) p.p.m., respectively. Two singlets at 2.26 and 3.96 p.p.m. are assigned as the methyls of the acetyl and methoxy groups, respectively. Based on these data and the 13C NMR spectrum, this compound was determined to be 2-[2-(acetylamino)-4-(diethylamino)-5-methoxyphenyl]-6-amino-4-bromo-2H-benzotriazole (non-ClPBTA-7).
The chlorinated product, PBTA-7, was obtained in 72% yield by treatment of non-ClPBTA-7 with sodium hypochlorite. The chemical structure of PBTA-7 is shown in Figure 2. The UV absorption spectrum of the product is almost identical to that of PBTA-1 reported previously (Nukaya et al., 1997). The FAB-MS spectrum (M+, M+ + 2, M+ + 4, m/z 480, 482, 484; relative abundance 74:100:29) shows that a hydrogen is replaced by a chlorine. In the 1H NMR (CDCl3) spectrum (Figure 2), three aromatic protons appear as singlets at 7.22, 7.78 and 8.26 p.p.m., which suggests that the chlorine substitution is at the 7 position on the benzotriazole ring. Almost the same signal pattern due to aromatic protons was observed in the spectrum of PBTA-1 produced by chlorination of non-ClPBTA-1, as described previously (Shiozawa et al., 1998). The 13C NMR spectrum is also consistent with the corresponding structures. Thus, 2-[2-(acetylamino)-4-(diethylamino)-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-7) was shown to be produced from the azo dye Disperse Blue 291.
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PBTA-8 was synthesized in a manner similar to PBTA-7, as shown in Scheme 1. First, 3-(diallylamino)-4-methoxyacetanilide was synthesized by diallylation of 3-amino-4-methoxyacetanilide with allyl iodide in the presence of potassium carbonate. Azo coupling with 2-bromo-4,6-dinitrobenzenediazonium sulfate gave Disperse Blue 373, the 1H NMR spectrum of which is consistent with the structure. Reduction of Disperse Blue 373 with iron powder to a benzotriazole derivative (non-ClPBTA-8) was carried out in a similar manner to that of PBTA-7. Finally, chlorination of non-ClPBTA-8 with sodium hypochlorite proceeded smoothly to give PBTA-8. The chemical structure of PBTA-8 is shown in Figure 2
. Its UV spectrum shows an absorption pattern similar to that of PBTA-7. The 1H NMR spectrum is also very similar to that of PBTA-7, except that the peaks due to a diallylamino group appear at 3.87 (4H, d, J = 6.5 Hz), 5.18 (2H, dd, J = 1.8, 10.4 Hz,), 5.23 (2H, dd, J = 1.8, 17.3 Hz) and 5.88 (2H, ddt, J = 6.5, 10.4, 17.3 Hz) p.p.m. (Figure 1). The 13C NMR spectrum is consistent with the structure of PBTA-8.
Mutagenicity of PBTA-7 and PBTA-8
The mutagenicities of PBTA-7 and PBTA-8 in S.typhimurium test strains are summarized in Table I. Both PBTA-7 and PBTA-8 were clearly mutagenic in the four test strains, i.e. TA98, TA100, YG1024 and YG1029, with but not without S9 mix. With S9 mix, both PBTA-7 and PBTA-8 were more mutagenic in TA98 (PBTA-7, 43 000 revertants/nmol; PBTA-8, 40 700 revertants/nmol) than in TA100. Similar phenomena were observed with strains YG1024 and YG1029 with S9 mix, which are O-acetyltransferase-overproducing derivatives of TA98 and TA100 (Watanabe et al., 1990), respectively. These findings indicate that both PBTA-7 and PBTA-8 exhibit more frameshift than base substitution activity. The mutagenic activities of PBTA-7 and PBTA-8 in YG1024 (PBTA-7, 1 430 000 revertants/nmol; PBTA-8, 2 213 000 revertants/nmol) were 33- and 54-fold higher than those in TA98, respectively, suggesting that O-acetyltransferase is required for the mutagenicity of these two PBTA derivatives.
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Detection of PBTA derivatives in river water
To clarify whether river waters are contaminated with PBTA-7 and PBTA-8, concentrates of waters were collected, using blue rayon, from six rivers that flow through two geographically different areas of textile-related industries in Fukui and Kyoto prefectures. All of the water samples were collected downstream of textile dyeing factories or municipal sewage plants that treated effluent from textile dyeing plants. The blue rayon adsorbates were separated by HPLC on a YMC-Pack ODS-A324 column. The fractions corresponding to authentic PBTA-7 and PBTA-8 were further purified using LUNA 5µ phenyl hexyl and YMC-Pack ODS-A303 columns. Purified fractions were finally applied to an analytical Cosmosil 5C18 AR-II column with a photodiode array detector. Single peaks corresponding to PBTA-7 and PBTA-8 in water samples were detected at retention times of 28–29 and 27–28 min, respectively, on an analytical column. Figures 3 and 4
show typical chromatograms of a water sample in the analytical steps and UV absorption spectra of peak fractions coinciding with the retention times of PBTA-7 and PBTA-8 on the chromatograms, respectively. These UV absorption spectra were identical to those of authentic PBTA-7 and PBTA-8, respectively.
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Table II
summarizes the amount of PBTA-7 and PBTA-8 in the river water samples. The values were corrected for recoveries of the compounds during purification, i.e. PBTA-7, 56% and PBTA-8, 54%. PBTA-7 was detected in eight samples collected from five rivers: the Kitsune, Mawatari, Asuwa, Katsura and Uji. On the other hand, PBTA-8 was detected in seven samples from four rivers: the Kitsune, Asuwa, Katsura and Uji. The highest levels of PBTA-7 and PBTA-8 were observed for the sample collected at site A in the Kitsune River in November 1999 (PBTA-7, 101.4 ng/g blue rayon; PBTA-8, 48.9 ng/g blue rayon). The amounts of PBTA-7 and PBTA-8 in the water samples collected at site E in the Katsura River and site G in the Uji River in August 2000 were also high. However, the levels of these PBTA derivatives in the samples collected at the same sampling sites E and G in February 2000 were 1–14% of those detected in the sample collected in August 2000. The levels of PBTA-7 and PBTA-8 were relatively low in the water samples collected at site F in February and August 2000.
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The mutagenicities of the river water samples toward S.typhimurium YG1024 with S9 mix and the percentage contribution of PBTA-7 and PBTA-8 to the mutagenicity of the concentrates of river waters are also shown in Table II
. All water samples were mutagenic in YG1024 with S9 mix and the potencies of the samples collected at sites E and G in August 2000 and sites A–C were high, with >1 000 000 revertants/g blue rayon. The contribution of PBTA-7 to the mutagenicity of the samples was ~15.7% or less and that of PBTA-8 was similar to that of PBTA-7, i.e. 14.5% or less. |
Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The present study has demonstrated that Disperse Blue 291 and Disperse Blue 373 were converted to mutagenic PBTA-type compounds, PBTA-7 and PBTA-8, respectively, by reduction and subsequent chlorination. The mutagenic potencies of PBTA-7 and PBTA-8 in YG1024 with S9 mix were 1 430 000 revertants/nmol for PBTA-7 and 2 213 000 revertants/nmol for PBTA-8; these values are comparable to those of other PBTA derivatives (1 620 000 revertants/nmol for PBTA-1, 1 616 000 revertants/nmol for PBTA-2, 1 404 000 revertants/nmol for PBTA-3, 3 307 000 revertants/nmol for PBTA-4, 431 000 revertants/nmol for PBTA-5 and 248 000 revertants/nmol for PBTA-6 in YG1024 with S9 mix) (Shiozawa et al., 1998
, 2000; Oguri et al., 1998; Nukaya et al., 2001; Watanabe et al., 2001). Reducing agents such as sodium hydrosulfite are generally used for discharge printing and bleaching in textile dyeing factories. Sodium hypochlorite is commonly utilized for bleaching in textile production and decolorization of wastewater and is also used in sewage plants for disinfection. Both PBTA-7 and PBTA-8 were detected in most of the river water samples collected downstream from textile dyeing factories or sewage plants that treat abundant effluent from textile dyeing factories. Based on these findings, PBTA-7 and PBTA-8 may be formed from the corresponding azo dyes during industrial processes in dyeing factories and treatment at sewage plants. The differences in the levels of PBTA-7 and PBTA-8 in the samples examined are probably related to the differences in the amounts and kinds of dinitrophenylazo-type dyes used in dyeing factories that discharge effluent into the rivers. On the other hand, high levels of other PBTA derivatives, such as PBTA-2 and PBTA-3, have been detected in the water samples collected from the Nishitakase and Katsura rivers at other sampling times (Oguri et al., 1998; Ohe et al., 1999; Shiozawa et al., 2000; Watanabe et al., 2001). Since various kinds of dinitrophenylazo-type dyes are used industrially, other PBTA-type compounds besides PBTA-7 and PBTA-8 may also contribute to the mutagenicity of river water samples detected in this study. In many countries, including Japan, water from rivers into which effluent from sewage plants or wastewater from industrial sources is discharged serves as the drinking water supply or irrigation water for agriculture. The Katsura and Uji Rivers flow into the Yodo River and the Yodo River serves as the main drinking water supply for people living in the Osaka area. Recently, Matsuoka et al. (2001) reported that PBTA-2 strongly induced binucleate cells in polynuclear and karyorrhectic (PN) cells in the Chinese hamster cell lines CHL and V79MZ, which suggests that PBTA-2 induces polyploidy. Moreover, they revealed that PBTA-1 induces micronuclei, PN and mitotic cells as well as aneuploidy in V79-MZ cells. To estimate the risk of PBTA-7 and PBTA-8 to human health and to organisms in the river, more detailed investigations, such as quantification of these chemicals in drinking water and biological assays, including carcinogenicity tests, are necessary.
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Acknowledgments |
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This study was supported by Grants-in-Aid for Cancer Research from the Ministry of Health and Welfare of Japan and the Promotion and Mutual Aid Corporation for Private Schools of Japan and funds under a contract with the Environment Agency of Japan.
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Notes |
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6 To whom correspondence should be addressed. Tel: +81 75 595 4650; Fax: +81 75 595 4769; Email: watanabe{at}mb.kyoto-phu.ac.jp
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References |
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Received on September 17, 2001; accepted on January 11, 2002.
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