Isolation and Chemical− Structural Determination of a Novel Aromatic

PBTA-1 is a newly identified potent mutagen, inducing 1 200 000 revertants of Salmonella typhimurium YG1024 per microgram in the presence of S9 mix...
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OCTOBER 1997 VOLUME 10, NUMBER 10 © Copyright 1997 by the American Chemical Society

Articles Isolation and Chemical-Structural Determination of a Novel Aromatic Amine Mutagen in Water from the Nishitakase River in Kyoto Haruo Nukaya,† Jun Yamashita,† Kuniro Tsuji,† Yoshiyasu Terao,‡ Takeshi Ohe,§ Hiroyuki Sawanishi,⊥ Takao Katsuhara,| Kiyoka Kiyokawa,∇ Masakatsu Tezuka,∇ Atsuko Oguri,# Takashi Sugimura,# and Keiji Wakabayashi*,# School of Pharmaceutical Science and Graduate School of Nutrition and Environmental Sciences, University of Shizuoka, 52-1, Yada, Shizuoka 422, Department of Food and Nutrition Science, Kyoto Women’s University, Kitahiyoshi-cho, Imakumano, Higashiyama-ku, Kyoto 605, Faculty of Pharmaceutical Sciences, Hokuriku University, Kanagawa-machi, Kanazawa 920-11, Central Research Laboratories, TSUMURA & Company, 3586 Yoshiwara, Ami-machi, Inashiki-gun, Ibaraki 300-11, Department of Hygienic Chemistry, College of Pharmacy, Nihon University, 7-1, Narashinodai 7-chome, Funabashi-shi, Chiba 274, and Cancer Prevention Division, National Cancer Center Research Institute, 1-1, Tsukiji 5-chome, Chuo-ku, Tokyo 104, Japan Received May 27, 1997X

Water samples from the Nishitakase River in Kyoto, Japan, especially taken at sites below sewage plants, show significantly high mutagenicity in the Ames test. In the present study, mutagens in the river water were adsorbed to 24 g of blue rayon, extracted, and separated by HPLC on ODS columns. Five mutagenic compounds (I-V) were isolated, and they accounted for 21%, 17%, 11%, 12%, and 6%, respectively, of the total mutagenicity of the blue rayonadsorbed materials. With compound I obtained from adsorbate to 24 g of blue rayon as a marker, a large quantity (1.1 mg) of mutagenic compound I was isolated by Sephadex LH-20 column chromatography and HPLC on ODS columns from material adsorbed to 27 kg of blue cotton. X-ray crystal analysis was carried out with the debrominated derivative of compound I. Based on this X-ray crystallography data and the UV, mass, and 1H-NMR spectra of both the derivative and compound I, the structure of compound I was determined to be 2-[2(acetylamino)-4-[bis(2-methoxyethyl)amino]-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2Hbenzotriazole (PBTA-1). PBTA-1 is a newly identified potent mutagen, inducing 1 200 000 revertants of Salmonella typhimurium YG1024 per microgram in the presence of S9 mix.

Introduction Water is essential to human life. Therefore, any pollution of either tap or environmental water should be * To whom correspondence should be addressed. † School of Pharmaceutical Science, University of Shizuoka. ‡ Graduate School of Nutrition and Environmental Sciences, University of Shizuoka. § Kyoto Women’s University. ⊥ Hokuriku University. | TSUMURA & Co. ∇ Nihon University. # National Cancer Center Research Institute. X Abstract published in Advance ACS Abstracts, September 1, 1997.

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regarded as very serious. Various reports have appeared documenting the presence of mutagens and carcinogens in water sources (1). For example, chloroform was found in the Rhine River in 1971 (2), and haloforms in tap water have been identified in various areas of the world (1). Recently, the potent mutagenic compound MX [3-chloro4-(dichloromethyl)-5-hydroxy-2(5H)-furanone], formed by chlorination of humic acid, was detected in tap water of several countries (3-5). Clearly it is necessary to maintain a lookout for other unidentified mutagenic and carcinogenic compounds present as contaminants in water. © 1997 American Chemical Society

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small percentage of the total mutagenicity of the blue cotton adsorbate. In the present study, we attempted to isolate mutagens in water samples concentrated from the Nishitakase River using blue rayon or blue cotton. We describe here isolation of five mutagens, accounting for 67% of the total mutagenicity, by various column chromatographies. Furthermore, spectral analysis of the structure of one of them revealed it to be a novel mutagen.

Experimental Procedures

Figure 1. Site of water sampling and location of sewage plants in the Nishitakase River of the Yodo River system.

It has been reported that water samples from the Yodo River system, including the Katsura and Nishitakase tributaries, flowing through the prefectures of Kyoto and Osaka in Japan, are mutagenic to Salmonella typhimurium strains TA98 and YG1024 with S9 mix (6-9). The Nishitakase River flows parallel to the Katsura River through Kyoto City, these two rivers both flowing into the Yodo. There are two sewage plants, which treat industrial and domestic waste water, along their banks, the first located upstream on the Nishitakase River and the second between the two rivers, as shown in Figure 1. Water treated at the second plant flows into both rivers. Sakamoto and Hayatsu found that the mutagenicity of water samples was especially high at sites downstream from the second sewage plant (8), being about 200-1000 times higher than that of water of the Sumida and Ara Rivers in Tokyo (9). Thus effluent from the two sewage plants is suspected to be a major source of mutagens in the Yodo River. Water from the Yodo River is taken and used in the water supply for more than 13 million people living in the Osaka area. It is thus very important that the chemical structures are clarified so that their effects on humans can be evaluated. It is generally difficult to analyze water pollutants because they are present in only minute concentrations. Therefore, approaches to concentrate the pollutants from river water are very important. Frequently used methods include extraction with organic solvents (10, 11), a variety of XAD resins (6, 7), and Sep-pak cartridges (12). In addition, blue cotton and blue rayon, which consist of cotton and rayon covalently bound to the blue pigment copper phthalocyaninetrisulfonate, can specifically adsorb multicyclic planar compounds, as reported by Hayatsu (13). These adsorbents enable easy monitoring of the mutagenicity of compounds such as heterocyclic amines and polycyclic aromatic hydrocarbons in water. Indeed, mutagens in water samples from the Yodo River system were efficiently concentrated using blue cotton or blue rayon. Polycyclic aromatic compounds, such as anthraquinone and quinoline derivatives, have been reported to exist in the mutagenic adsorbate to blue cotton from the Katsura River water (14). However, these polycyclic aromatic compounds accounted for only a very

Materials. Blue rayon was obtained from Funakoshi Co. Ltd., Tokyo, Japan, and blue cotton was prepared according to the method reported previously (15). HPLC grade acetonitrile, methanol, and 5% palladium on charcoal (Pd/C) were purchased from Wako Pure Chemical Industries Co. Ltd., Osaka, Japan. All other chemicals used were of analytical grade. Isolation of Mutagens from River Water. Six net bags of blue rayon (4 g/bag) were attached to a floating board and hung in the river for 24 h using the method described previously (8). Sampling was performed at a site downstream of two sewage plants along the Nishitakase River, as shown in Figure 1. The six portions of blue rayon were then combined and washed with 1 L of water 10 times. Adsorbed materials were then extracted by shaking the blue rayon in 1 L of methanol/ ammonia water (50:1, v/v) for 3 h three times (16, 17). The combined extracts were evaporated to dryness, and the residue originated from the 24 g of blue rayon was dissolved in methanol. One-third of the solution was applied to a semipreparative TSK gel ODS-120A column (10 µm particle size, 7.8 × 300 mm; Tosoh Corp., Tokyo) for HPLC (Tosoh) and then eluted with the following gradient system of acetonitrile in 25 mM phosphate buffer (pH 2.0): 0-40 min, linear gradient of 30-80%; 40-90 min, 80%, at a flow rate of 2 mL/min. An aliquot of each 2 min fraction was tested for mutagenicity. Fractions with retention times of 26-34 and 36-42 min demonstrated positive results. Isolation of mutagens in those with retention times of 3642 min was accomplished as follows. The evaporated residues were dissolved in 800 µL of 50% acetonitrile in 25 mM phosphate buffer (pH 2.0) and injected into a YMC-Pack ODS-A 303 column (5 µm particle size, 4.6 × 250 mm; YMC Co. Ltd., Kyoto, Japan) with a mobile phase of 50% acetonitrile in 25 mM phosphate buffer (pH 2.0) at a flow rate of 1 mL/min. Two mutagenic fractions with retention times of 12-16 and 18-20 min were separately purified on a CAPCELL PAK C18 ODS column (5 µm particle size, 4.6 × 250 mm; Shiseido Co. Ltd., Tokyo). By eluting the materials with 40% acetonitrile in 25 mM Tris-HCl buffer (pH 8.5) at a flow rate of 1 mL/min, two mutagenic compounds (compounds I and II) were isolated as follows: compound I from the fractions with retention times of 12-16 min and compound II from the fractions with retention times of 18-20 min. The purity of mutagenic compounds I and II was confirmed on the second YMC-Pack ODS-A 303 column with a mobile phase of 50% acetonitrile in 25 mM phosphate buffer (pH 2.0) at a flow rate of 1 mL/min. Next, mutagens in the fractions with retention times of 2634 min on the semipreparative TSK gel ODS-120A column described above were isolated under the same HPLC conditions, except for percentages of acetonitrile in eluents, as used for the isolation of compounds I and II. When the mutagenic fractions at retention times of 26-34 min were combined, applied on YMC-Pack ODS-A 303 column, and eluted with 35% acetonitrile in 25 mM phosphate buffer (pH 2.0), three mutagenic fractions were obtained at retention times of 20-22, 25-28, and 48-51 min. These three mutagenic fractions were further purified on a CAPCELL PAK C18 ODS column with a mobile phase of 30% acetonitrile in 25 mM Tris-HCl buffer (pH 8.5) for the fractions with retention times of 20-22 min and 35% acetonitrile in 25 mM Tris-HCl buffer (pH 8.5) for the two mutagenic fractions with retention times of 25-28 and 48-51 min, and three mutagenic compounds (compounds III-V) were obtained, respectively. The purity of those mutagenic compounds (III-V)

Determination of a New Mutagen in River Water was also confirmed on a second YMC-Pack ODS-A 303 column with 35% acetonitrile in 25 mM phosphate buffer (pH 2.0) as the eluent. The above processes were repeated two times more, and the five mutagens (compounds I-V) were combined for each. All HPLC procedures were carried out at ambient temperature, and the eluates were monitored for absorbance at 260 nm. Preparation of a Large Quantity of Compound I. The amount of mutagenic compound I required for structural determination was isolated from a river water sample collected with 27 kg (3 kg × 9 times) of blue cotton, using the original compound I collected with 24 g of blue rayon as a standard marker, by the following procedure. A total of 3 kg of blue cotton was divided equally into 22 net bags and hung for 24 h in the Nishitakase River at the same site as previously. The blue cotton samples were then combined and washed twice with 40 L of distilled water. After dehydration in a spin-dryer, the adsorbed materials were extracted by stirring once in 18 L of methanol/ammonia water (50:1, v/v) for 3 h and three times more in 12 L of the same solution. The extract was then evaporated to dryness, and the residue (2.6 g) was dissolved in 20 mL of methanol, filtered through a glass filter, and applied to a Sephadex LH-20 column (50 × 877 mm; Pharmacia, Uppsala, Sweden). The materials were eluted with methanol, and fractions of 17 mL each were collected. The factions at elution volumes of 1520-1775 mL, which were found to contain compound I, were combined and evaporated. The residue (89 mg) was dissolved in 2 mL of methanol and applied again to a Sephadex LH-20 column (13 × 1360 mm) with methanol as a mobile phase, and fractions of 1.5 mL each were collected. The fractions containing compound I, which eluted at elution volumes of 136-159 mL, were combined and evaporated. The residue dissolved in 400 µL of methanol was finally purified by HPLC on a semipreparative TSK gel ODS120A column followed by a YMC-Pack ODS-AM 324 column (5 µm particle size, 10 × 300 mm; YMC Co. Ltd., Kyoto). In both cases the mobile phase of 75% methanol was pumped in isocratically at a flow rate of 2 mL/min, and compound I was found in the peak fractions with retention times of 25 and 32 min, respectively. The above processes were repeated nine times to give 1.1 mg of compound I. The presence of a peak corresponding to authentic compound I was confirmed by HPLC on an analytical YMC-Pack ODS-A 303 column with a mobile phase of 50% acetonitrile in 25 mM phosphate buffer (pH 2.0) as described above. Mutagenesis Assay. All test samples were dissolved in 100 µL of 50% dimethyl sulfoxide, and mutagenicities were examined by the preincubation method (18) using S. typhimurium YG1024 in the presence of S9 mix. S. typhimurium YG1024, produced by introducing plasmids containing the acetyltransferase gene from TA1538 into TA98 (19), was kindly provided by Dr. T. Nohmi, National Institute of Health Sciences, Tokyo. The S9 mix contained 30 µL of S9 in a total volume of 500 µL. Spectral Measurement of Compound I. UV absorption spectra were measured with a Tosoh PD-8020 photodiode array detector and a Beckman DU 640 spectrophotometer. 1H-NMR spectra were taken as solutions in chloroform-d with a JEOL JNM-GSX 500 (500 MHz) Fourier transform spectrophotometer. Chemical shifts are shown in ppm using tetramethylsilane as an internal standard. The following abbreviations for signals of 1H-NMR spectra are used: s ) singlet, d ) doublet, t ) triplet, m ) multiplet, br ) broad. High-resolution mass spectra were measured using a JEOL JMS-DX300 mass spectrometer equipped with a direct inlet system. Debromination of Compound I and Its X-ray Crystallographic Analysis. A mixture of 1.0 mg of compound I and 1.0 mg of 5% palladium on charcoal in 2 mL of ethanol was stirred overnight under a hydrogen atmosphere at room temperature. After removal of the catalyst by filtration, the filtrate was concentrated under reduced pressure to yield a crystalline solid which was recrystallized from hexane-ethyl acetate to afford the debrominated derivative as pale yellow prisms (0.70 mg, 82%).

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Figure 2. HPLC profile of mutagens in river water on a semipreparative ODS column. The UV absorbance of the eluate (upper line) was monitored at 260 nm, and the mutagenicity of each 2 min fraction (histogram bars) was tested in S. typhimurium YG1024 with S9 mix.

Figure 3. Purification of mutagenic compounds I and II by HPLC. Two mutagenic fractions from a YMC-Pack ODS-A 303 column with retention times of 12-16 and 18-20 min were purified respectively on a CAPCELL PAK C18 ODS column. Compounds I (A) and II (B) were obtained at retention times of 25 and 32 min, from the former and the latter, respectively. The UV absorbance and mutagenicity are shown by the upper line and lower bars, respectively. Unit-cell parameters and intensity data for X-ray crystallography of the debrominated derivative of compound I were measured on an Enraf Nonius CAD-4 System automatic fourcircle diffractometer with graphite-monochromated Cu KR radiation at room temperature.

Results and Discussion Isolation of Mutagens from River Water. The materials adsorbed to 1 g of blue rayon were found to

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Figure 4. UV absorption spectrum of compound I, measured on the second YMC-Pack ODS-A 303 column with a photodiode array detector. The material was eluted with 50% acetonitrile in 25 mM phosphate buffer (pH 2.0).

induce 3 200 000 revertants of S. typhimurium YG1024 in the presence of S9 mix. The mutagens extracted from blue rayon were separated by HPLC on a semipreparative ODS-120A column using a gradient solvent system. Figure 2 shows the HPLC profile of UV absorbance and mutagenicity. Two major mutagenic fractions with retention times of 26-34 and 36-42 min were observed, and the mutagenicities in these two fractions accounted for 58% and 39%, respectively, of the total mutagenicity of the blue rayon-adsorbed materials. The mutagens in fractions with retention times of 3642 min were purified by HPLC on a YMC-Pack ODS-A

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303 column. By this separation, mutagenicity was mainly recovered in the two fractions with retention times of 12-16 and 18-20 min, and these were further purified on a CAPCELL PAK C18 ODS column. Concerning the former (retention times of 12-16 min), a single UV absorption peak showing mutagenicity was observed at retention times of 23-26 min (Figure 3A). The mutagenicity was confirmed to be due to a single peak on the second YMC-Pack ODS-A 303 column, and the material was designated compound I. Under the same HPLC conditions, another single UV absorption peak showing mutagenicity (compound II) was isolated from factions with retention times of 18-20 min on the first YMC-Pack ODS-A 303 column. Figure 3B shows HPLC elution patterns for compound II on a CAPCELL PAK C18 ODS column. The mutagens in fractions with retention times of 2632 min on a semipreparative ODS-120A column were separated by HPLC successively on YMC-Pack ODS-A 303, CAPCELL PAK C18 ODS, and second YMC-Pack ODS-A 303 columns. These purifications yielded three mutagenic compounds, III-V, that were isolated as single peaks. The recoveries of the mutagenicities of compounds I-V at the purification step on a CAPCELL PAK C18 ODS column were 21% for I, 17% for II, 11% for III, 12% for IV, and 6% for V, of the total mutagenicity of the blue rayon-adsorbed materials.

Figure 5. Electron impact mass spectrum of compound I isolated from river water.

Figure 6.

1H-NMR

spectrum of compound I in chloroform-d.

Determination of a New Mutagen in River Water

The UV absorption spectrum of compound I, obtained on the second YMC-Pack ODS-A 303 column with a photodiode array detector, is shown in Figure 4. The UV absorption maxima were found at 232, 270(sh), and 384 nm. Preparation of a Large Quantity of Compound I. With compound I isolated from adsorbate to 24 g of blue rayon as a marker, a large quantity of compound I was isolated by Sephadex LH-20 column chromatography and HPLC on semipreparative ODS columns from material adsorbed to 27 kg of blue cotton. Using this procedure, 1.1 mg of compound I was obtained. The UV spectrum was identical to that of compound I isolated from the initial adsorbate to 24 g of blue rayon. The mass spectrum of compound I exhibited a molecular ion peak at m/z 540 and two isotopic ion peaks at m/z 542 and 544 (Figure 5). Subsequent high-resolution mass spectrometry indicated the molecular formula to be C21H26BrClN6O4 (540.0870, calcd 540.0887). Figure 6 shows the 1H-NMR spectrum of compound I in chloroformd, indicating the presence of 26 protons in the molecule, which is in agreement with the data from the highresolution mass spectrometry. Two signals at 4.35 (2H) and 11.01 (1H) ppm were found to be exchangeable by D2O treatment, indicating the presence of an amine NH2 and an amide NH, respectively. Three singlets at 2.26 (3H), 3.34 (6H), and 3.95 (3H) ppm suggested the presence of an acetyl methyl and three O-methyl groups. The band (8H) centered at 3.56 ppm showed a typical A2B2 coupling pattern assignable to eight protons of two ethylene groups, and three singlets due to aromatic protons were observed at 7.22, 7.77, and 8.27 ppm. Structural Analysis of the Debrominated Derivative of Compound I. An appropriate crystal for analysis of X-ray crystallography was not obtained from compound I, and its debrominated derivative was therefore prepared. Debromination was confirmed using highresolution mass spectrometry, which indicated that the molecular formula was C21H27ClN6O4 (462.1780, calcd 462.1782). The UV absorption spectrum of the debrominated derivative in methanol showed absorption maxima at 233, 258(sh), and 380 nm. A yellow crystal of the derivative was grown in hexane-ethyl acetate solution. X-ray crystallographic analysis of the derivative crystal, 0.4 × 0.3 × 0.2 mm in size, showed that the crystal had cell dimensions of a ) 13.857(1) Å, b ) 15.661(2) Å, c ) 12.141(2) Å, R ) 107.910(8)°, β ) 108.381(7)°, and γ ) 106.681(6)° and the triclinic space group P1 h with four molecules in a unit cell. The structure was determined by the direct method program MULTAN and was refined by the full-matrix least-squares method. The final R value for the structure was 0.081, including anisotropic temperature factors for the non-hydrogen atoms and an isotropic one for the hydrogen atoms. A perspective view of the whole molecule is shown in Figure 7. According to the structure proposed by X-ray analysis, each peak of the 1H-NMR spectrum of the debrominated derivative was assignable as follows. Three singlets at 2.26 (3H), 3.34 (6H), and 3.94 (3H) ppm were due to the methyl protons of acetylamino, aliphatic methoxy, and aromatic methoxy groups, respectively. Eight ethylene protons were observed as a higher order A2B2 pattern at 3.52-3.59 ppm. Two peaks at 4.39 and 11.09 ppm, which disappeared on addition of a small amount of D2O, were assigned to an amino NH2 and an amide NH, respectively. Four aromatic protons appeared at 6.99 (d, J )

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Figure 7. Chemical structure and perspective drawing of the debrominated derivative of compound I. No hydrogen bonds are shown in the drawing. 1H-NMR spectrum data for the aromatic protons of the debrominated derivative are also given.

9.2 Hz), 7.66 (d, J ) 9.2 Hz), 7.72 (s), and 8.27 (s) ppm. Two doublets at 6.99 and 7.66 ppm were assigned to the protons at the 6 and 7 positions on the benzotriazole ring. The former was due to the proton ortho to the amino group which shields strongly an ortho proton and the latter due to the proton adjacent to the triazole ring. Assignment of the singlet at 7.72 ppm as the ortho proton of the 2-phenyl group was supported by a nuclear Overhauser effect (NOE) to the OCH3 protons (3.94 ppm). The singlet at 8.27 ppm was, accordingly, assigned to the meta proton of the 2-phenyl group. From these observations, the chemical structure of the debrominated derivative of compound I was determined to be 2-[2-(acetylamino)-4-[bis(2-methoxyethyl)amino]-5-methoxyphenyl]-5amino-4-chloro-2H-benzotriazole. Determination of the Structure of Compound I. The structure of compound I was finally determined on the basis of its 1H-NMR spectrum. The higher field pattern than 5 ppm and the broad peak at near 11 ppm were quite similar to those of the debrominated derivative, suggesting the same substituents at the same positions as in the debrominated derivative, except for the bromine moiety. The other three singlets at 7.22, 7.77, and 8.27 ppm were assigned to aromatic protons. The observation of no ortho doublet due to aromatic protons provided unambiguous evidence that the bromine exists on the benzotriazole ring. In addition, only a singlet at 7.22 ppm of compound I changed to two ortho doublets at 6.99 and 7.66 ppm in the spectrum of the compound resulting from debromination. Namely, the singlet at 7.22 ppm of compound I was assigned as the proton ortho to the amino group. It shifted slightly downfield compared with the corresponding 6-H (6.99 ppm) of the debrominated derivative due to a weakly deshielding effect of the ortho bromine. Thus, the structure of compound I was determined to be 2-[2(acetylamino)-4-[bis(2-methoxyethyl)amino]-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-1). This mutagen is a newly identified compound, and its structure is shown in Figure 8.

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(4)

(5) (6)

(7)

(8)

Figure 8. Structure of compound I. Chemical shifts (ppm) of three aromatic protons in the 1H-NMR spectrum of compound I are also indicated. (9)

PBTA-1 induced 1 200 000 revertants/µg of S. typhimurium YG1024, a strain originating from TA98 with high O-acetyltransferase activity, in the presence but not in the absence of S9 mix. This mutagenicity is comparable to that of the heterocyclic amine 2-amino-6-methyldipyrido[1,2-a:3′,2′-d]imidazole (Glu-P-1). PBTA-1 has specific moieties such as bromo, chloro, acetylamino, bis(2-methoxyethyl)amino, and triazole groups. It is very plausible that these groups are derived from dyes, since there are several dye factories in Kyoto City. Because PBTA-1 is not listed as a compound used in industrial processes, this mutagenic compound is most likely produced by treatment of waste water from dye factories at the sewage plant. Of course, the possibility that PBTA-1 is contained in some industrial materials as a contaminant can not be ruled out. A search for the source of the PBTA-1 is now underway in our laboratory, along with analyses of the structures of the other mutagenic compounds II-V.

Acknowledgment. This study was supported by Grants-in-Aid for Cancer Research from the Ministry of Health and Welfare of Japan and funds under a contract with the Environment Agency of Japan.

(10)

(11) (12)

(13)

(14)

(15)

(16)

(17)

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