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carcinogenic heterocyclic amines, MeIQx, Trp-P-1, Trp-P-2 and PhIP, contributing highly to genotoxicity of river water
Mutation Research 393 Ž1997. 73–79
Quantification of mutagenicrcarcinogenic heterocyclic amines, MeIQx, Trp-P-1, Trp-P-2 and PhIP, contributing highly to genotoxicity of river water Takeshi Ohe
)
Department of Food and Nutrition Science, Kyoto Women’s UniÕersity, Kitahiyoshi-cho, Imakumano, Higashiyama-ku, Kyoto 605, Japan Received 29 January 1997; revised 7 April 1997; accepted 8 April 1997
Abstract Four mutagenicrcarcinogenic heterocyclic amines ŽHCAs., 2-amino-3,8-dimethylimidazow4,5-f xquinoxaline ŽMeIQx., 3-amino-1,4-dimethyl-5H-pyridow4,3-b xindole ŽTrp-P-1., 3-amino-1-methyl-5H-pyridow4,3-b xindoleŽTrp-P-2. and 2-amino1-methyl-6-phenylimidazow4,5-b xpyridine ŽPhIP., in organic extracts obtained by blue rayon hanging method from the Yodo River water were quantified. Blue rayon extracts obtained were separated in two stages of fractionation by reversed-phase high performance liquid chromatography ŽHPLC., and the quantification of corresponding fractions was performed by HPLC with an electrochemical detector for MeIQx and a fluorometric detector for Trp-P-1, Trp-P-2 and PhIP. The geometrical mean values of MeIQx, Trp-P-1, Trp-P-2 and PhIP in extracts collected at 11 locations from the Yodo River systems were 4.8, 26.9, 37.3, and 11.9 ngrg blue rayon equivalent, respectively. The total amounts of four HCAs accounted for mean 24% of the genotoxicity of blue rayon extracts evaluated by the umu test using an O-acetyltransferase-overproducing strain NM2009. q 1997 Elsevier Science B.V. Keywords: River water; Heterocyclic amine; Trp-P-1; Trp-P-2; MeIQx; PhIP; Genotoxicity; umu Test; Blue rayon
1. Introduction The widespread occurrence of mutagenic and carcinogenic substances in river water derived from industrial waste, pesticides and naturally occurring substances is a serious problem, especially for sources of drinking water supply. There have been many
Abbreviations: HPLC, High-performance liquid chromatography; MeIQx, 2-amino-3,8-dimethylimidazow4,5-f xquinoxaline; Trp-P-1, 3-amino-1,4-dimethyl-5H-pyridow4,3-b xindole; Trp-P-2, 3-amino-1-methyl-5 H-pyridow4,3-b xindole; PhIP, 2-amino-1methyl-6-phenylimidazow4,5-b xpyridine; HCA, heterocyclic amine ) Corresponding author. Fax: q81 Ž75. 531-7216.
studies on the mutagenicity of river waters in various countries w1–7x. It is also known that organic substances from the Yodo River, which is the source of drinking water for cities located downstream, showed the highest mutagenic activity among river waters reported until now, and the major source of the mutagenicity was the discharge from wastewater treatment plants w8–11x. Also, the occurrence of indirect-acting mutagenic aminoarenes in the river, shown by the Ames test w12x and umu test w13x using an O-acetyltransferase-over-producing strain, was suggested. However, confirmation or quantification of mutagens in the Yodo River systems still remains very limited, except for some PAHs w14x and 1-nitro-
1383-5718r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 1 3 8 3 - 5 7 1 8 Ž 9 7 . 0 0 0 8 7 - 9
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T. Ohe r Mutation Research 393 (1997) 73–79
pyrene w13x. On the other hand, Trp-P-2 was detected in influents and effluents of municipal wastewater treatment plants or nightsoil treatment plants w15–17x. Mutagenicrcarcinogenic heterocyclic amines such as MeIQx, Trp-P-1, Trp-P-2 and PhIP were also found in urine from humans eating normal diet and after ingestion of a cooked meat meal w18–20x. These findings led to the quantification of heterocyclic amines in the Yodo River water, into which effluents from wastewater treatment plants or nightsoil treatment plants were discharged. This paper demonstrates quantification of four HCAs, MeIQx, Trp-P-1, Trp-P-2 and PhIP, in organic extracts obtained by the blue rayon hanging method, which is selective for collection of HCAs w10,21x, from the Yodo River systems, Japan, and evaluation of their contribution to the total genotoxicity.
Tokyo. All other chemicals and reagents were of analytical grade. 2.2. Collection and preparation of organic extracts from riÕer water Organic substances were collected at 11 sampling locations of the Yodo River systems, shown in Fig. 1, by the blue rayon hanging method w10x at a depth of 20–30 cm on 10 December, 1995. For collecting organic substances, 25 g of blue rayon in a plastic net was hung in the river water for 24 h. Blue rayon recovered from the river water was treated as described by Sakamoto and Hayatsu w10x. Blue rayon extracts obtained here were assayed for quantification of HCAs and genotoxicity in the umu test. 2.3. Purification of organic extract
2. Materials and methods 2.1. Materials M eIQx, Trp-P-1 P CH 3 COOH, Trp-P-2 P CH 3 COOH, and PhIP P HCl were obtained from Wako Pure Chemical, Osaka. HPLC grade methanol and acetonitrile were also from Wako. Blue rayon was purchased from Funakoshi Pharmaceutical Co.,
Reversed-phase HPLC analysis was performed using a Shiseido Nanospace SI-1 chromatograph ŽShiseido, Tokyo., equipped with a spectra detector ŽIrica Ý983, Kyoto.. Each extract was applied to a reversed phase PREP-ODS column Ž5 mm particle size, 10 = 250 mm, GL Science. monitored at 260 nm. The mobile phase was a gradient Ž30–80%r40 min. of methanol in 50 mM ammonium acetate ŽpH 6.0. at a flow rate of 1.6 mlrmin at 408C. The
Fig. 1. Sampling locations in the Yodo River systems.
T. Ohe r Mutation Research 393 (1997) 73–79
fractions corresponding to authentic MeIQx, Trp-P-1, Trp-P-2 and PhIP were separately collected. Then the volumes of these fractions were reduced to 1 ml with a centrifugal evaporator. The aliquot of condensed fractions was further purified using a reversed phase 5C18 AR column Ž4.6 = 150 mm, Nacalai Tesque, Kyoto.. A mobile phase of 10% acetonitrile for MeIQx or 20% acetonitrile for Trp-P1, Trp-P-2 and PhIP in 25 mM H 3 PO4rNa 2 HPO4 ŽpH 2.0. was pumped in at a flow rate of 0.8 mlrmin. The fractions corresponding to each authentic heterocyclic amine were separately collected and evaporated to dryness and then dissolved in 400 ml of 50% methanol again. 2.4. Quantification of HCAs The reduced fractions were finally analyzed on a reversed phase QC Pack C18 column Ž4.6 = 150 mm, Irica, Kyoto.. The elution solvent was 100 mM ammonium acetate ŽpH 6.0. containing 10% acetonitrile for MeIQx, and 25 mM ammonium acetate ŽpH 6.0. containing 20% acetonitrile for Trp-P-1, Trp-P-2 and PhIP at a flow rate of 0.7 mlrmin. MeIQx was detected with an electrochemical detector Ž900 mV, Irica Ý985., and Trp-P-1, Trp-P-2 and PhIP were detected by a fluorometric detector ŽShiseido SI-1, Tokyo. with excitation and emission wavelengths of 265 and 410 nm for Trp-P-1 and Trp-P-2, and 345 and 395 nm for PhIP, respectively.
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Salmonella typhimurium NM2009, kindly provided by Dr. Y. Oda ŽOsaka Prefectural Institute of Public Health.. Four dose levels with duplicate determination per dose were used.
3. Results and discussion The quantification of four HCAs, MeIQx, Trp-P-1, Trp-P-2 and PhIP, in blue rayon extracts was performed modifying the 3-step purification method reported by Manabe et al. w23x. One typical preparative HPLC chromatographic profile on a reversed phase PREP ODS column of blue rayon extract ŽLocation I. in the first purification step is shown in Fig. 2. Three fractions corresponding to MeIQx, Trp-P-1 and Trp-P-2, and PhIP were separately collected and condensed using a centrifugal evaporator. The second purification step of each corresponding fraction was carried out on a reversed phase 5C18 AR column ŽFig. 3A–C.. Each fraction corresponding to authentic heterocyclic amines was finally analyzed on a reversed phase QC Pack C18 column. Each peak corresponding to authentic MeIQx, Trp-P1, Trp-P-2 and PhIP was clearly separated as a single peak in the final analysis condition ŽFig. 3D–F.. The structures of the heterocyclic amines in the peak fractions detected on the final HPLC chromatograms were further confirmed to be MeIQx, Trp-P-1,
2.5. Spectrophotometric analysis UV absorbance spectra and fluorescence spectra of the fractions finally purified with HPLC was recorded with a photodiode-array detector ŽShiseido Nanospace SI-1. and a fluorometric detector ŽShiseido NanospaceSI-1.. 2.6. The umu test assay The genotoxic activities of blue rayon extracts were determined by the umu test in the presence of S9 mix according to the method described previously w13,22x. S9 used in this experiment was prepared from livers of rats pretreated with phenobarbital and 5,6-benzoflavone. The strain used in the experiment was an O-acetyltransferase-overproducing strain
Fig. 2. Preparative HPLC elution pattern of the first purification step of authentic MeIQx, Trp-P-1, Trp-P-2 and PhIP ŽA., and a blue rayon extract from Location I ŽB; injection volume, 2.5 g blue rayon equivalent. on an Inertsil PREP ODS column. Three fractions corresponding to MeIQx, Trp-P-1 and Trp-P-2, and PhIP were separetely collected, and further purified on a 5C18 AR column.
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T. Ohe r Mutation Research 393 (1997) 73–79
Trp-P-2 and PhIP by their UV absorbance spectra and fluorescence spectra. The UV absorption spectra of the peak fractions purified from the blue rayon extract of Location I were identical to those of authentic MeIQx, Trp-P-1, Trp-P-2 and PhIP respectively, as shown in Fig. 4. Moreover, the maximal wavelength of fluorescence spectra of the peak fractions were the same as those of authentic Trp-P-1, Trp-P-2 and PhIP, respectively ŽFig. 5.. Thus, the peaks corresponding to four HCAs on the final purification step by HPLC were identified as MeIQx, Trp-P-1, Trp-P-2 and PhIP. The recoveries of four HCAs during the purification process were estimated by spiking a similar level of authentic compounds Žeach 40 ng of HCAsrg blue rayon equivalent. to that detected in the sample. The result was that recoveries of MeIQx, Trp-P-1, Trp-P-2 and PhIP were 51%, 57%, 51% and 56%, respectively. TABLE 1 shows the amounts of four HCAs in blue rayon extracts collected at 11 locations from the Yodo River systems. The original levels of MeIQx, Trp-P-
1, Trp-P-2 and PhIP were found to be at levels of nd ; 365, nd ; 530, nd ; 840 and nd ; 118 ngrg blue rayon equivalent, respectively. These values were corrected for the recovery of four HCAs during the purification process. These levels were also detected on a Capcell Pak C18 column Ž4.6 = 150 mm, Shiseido, mobile phase: 50 mM phosphate buffer, pH 8.5, containing 10% acetonitrile for MeIQx, and 20% acetonitrile for Trp-P-1, Trp-P-2 and PhIP., and the levels on the QC Pack C18 column and the Capcell Pak C18 column were almost the same. The geometrical means of MeIQx, Trp-P-1,Trp-P-2 and PhIP were 4.8, 26.9, 37.3 and 11.9 ngrg blue rayon equivalent, respectively. These values were more than 3–22-times higher than the levels of benzow axpyrene levels Žnd ; 4.9, the average being 1.7 ngrg blue rayon equivalent, n s 6., a representative polynuclear aromatic hydrocarbon detected in the Yodo River, though the sampling period was not the same ŽApril 1995.. Higher HCAs levels and genotoxicity were found in blue rayon extracts at
Fig. 3. HPLC chromatograms of the second purification step Žtop. and the final quantification step Žbottom. of MeIQx, Trp-P-1, Trp-P-2 and PhIP in a blue rayon extract from Location I. A–C: chromatograms on a 5C18 AR column monitored by their absorbance at 260 nm. Injection volume: 0.42 g blue rayon equivalent. D–F: chromatograms on a QC Pack C18 column. The eluate of MeIQx was monitored by the ECD response ŽD; 900 mV; injection volume, 0.042 g blue rayon equivalent.. Those of Trp-P-1 and Trp-P-2 were monitored by their fluorescence with excitation and emission wavelength of 295 nm and 410 nm ŽE; injection volume, 0.02 g blue rayon equivalnt.. That of PhIP was monitored by the fluorescence with 265 nm and 410 nm ŽF; injection volume, 0.1 g blue rayon equivalent..
T. Ohe r Mutation Research 393 (1997) 73–79
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Fig. 4. UV absorbance spectra of the peak fractions coinciding with the retention time of MeIQx, Trp-P-1, Trp-P-2 and PhIP in the blue rayon extract from Location I Ž — — . and authentic MeIQx, Trp-P-1, Trp-P-2 and PhIP Ž PPP . on a QC Pack ODS column. Samples of the fraction containing each ca. 20 ng of MeIQx, Trp-P-1, Trp-P-2 and PhIP were examined with a photodiode-array detector. Purity index of samples were as follows: MeIQx, 950; Trp-P-1, 960; Trp-P-2, 942; PhIP, 965.
Locations E and J, compared with those in samples at other locations. Locations E, F, I and J are situated only 300–500 m downstream, where effluents from the wastewater treatment plant or the nightsoil treatment plant goes into the Yodo River systems. It was
reported that humans could be exposed to mutagenicrcarcinogenic HCAs continuously in normal daily life w19x. Trp-P-1 and Trp-P-2 are known to be ubiquitous environmental components, which are present in airborne particles and rain water w23x.
Table 1 MeIQx, Trp-P-1, TrpP-2 and PhIP contents and genotoxicity of blue rayon extracts from the Yodo River systems Location
MeIQx
Trp-P-1
Trp-P-2
PhIP
Total of HCA
Žngrg BRE a . B D E F G H I J K L M a
nd nd 365 9 nd nd 32 350 nd nd nd
nd 24 530 48 32 3 203 520 20 43 nd
nd 33 840 50 30 41 196 280 52 40 nd
nd 9 41 55 23 nd 118 84 15 10 nd
b
Žunitsr0.1 g BRE a . nd 66 1776 162 85 44 549 1234 87 93 nd
Blue rayon equivalent. umu test ŽNM2009, qS9 mix.. nd, Not detected Ždetected limit: MeIQx, Trp-P-1 and Trp-P-2, 0.2 ngrg; BRE, PhIP, 0.4 ngrg BRE..
b
b-Galactosidase
308 155 1941 620 445 439 1099 1416 475 375 negative
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T. Ohe r Mutation Research 393 (1997) 73–79
MeIQx, Trp-P-1, Trp-P-2 and PhIP were detected in urine from healthy persons eating normal diet, and MeIQx was detected in the highest concentration among four HCAs w18,19x. On the other hand, Segawa et al. w15x and Ono et al. w16x reported that Trp-P-2 was detected in an influent of municipal wastewater treatment plant, and Ono et al. w17x reported that Trp-P-2 remained after biological treatment in a nightsoil treatment plant. Recently, Mohri et al. w24x reported that Trp-P-2 was detected at a concentration of 8.7 ngrl of the Yodo River water. Therefore, the present result demonstrates that the Yodo River was highly polluted with mutagenicrcarcinogenic HCAs, and suggests that the major source of HCAs in the Yodo River was the discharge from wastewater treatment and nightsoil treatment plants. However, further studies are needed to clarify the behavior of HCAs in the water environment. The value of b-galactosidase activity per 0.1 g blue rayon equivalent was estimated from the
Fig. 5. Fluorescence spectra of each 80–100 ng of authentic Trp-P-1, Trp-P-2 and PhIP Ž — — . and the peak corresponding to Trp-P-1, Trp-P-2 and PhIP in the blue rayon extract of Location I Ž- - -.. Ex, excitation spectra monitored at the emission wavelength of 400 nm; Em, emission spectra monitored at the excitation wavelength of 260 nm.
Fig. 6. Contribution rates of MeIQx, Trp-P-1, Trp-P-2 and PhIP to genotoxicity of blue rayon extracts.
linear-regression analysis of their dose–response curves of each blue rayon extract evaluated by the umu test using an O-acetyltransferase-overproducing strain NM2009 with metabolic activation ŽTable 1.. All the sample extracts except for Location M showed distinct dose-dependent mutagenicity, suggesting that the active materials were indirect-acting mutagenic aminoarenes. A significant positive correlation Ž r s 0.96, p - 0.001; n s 11. was observed between the total amount of HCAs and the genotoxicity. On the basis of specific activity, MeIQx, Trp-P-1, Trp-P-2 and PhIP contributed on average at the rate of 5.5%, 15.6%, 2.5% and 0.5%, respectively, to the total genotoxicity of blue rayon extract at 11 sampling locations. The contribution rate of the sum total of four heterocyclic amines varied between 0% and 68.5%, the average being 24% ŽFig. 6.. Mohri et al. w24x reported that the contribution rate of Trp-P-2 detected in the Yodo River water was less than 10%, in accordance with the present result. In conclusion, the present report showed the existence of mutagenicrcarcinogenic MeIQx, Trp-P-1, Trp-P-2 and PhIP in river water, and the quantified heterocyclic amines accounted for a mean 24% of the total genotoxic activity of blue rayon extracts recovered from the water of the Yodo River system.
Acknowledgements The author would like to thank Dr. Yoshimitsu Oda, Osaka Prefectural Institute of Public Health,
T. Ohe r Mutation Research 393 (1997) 73–79
Japan, for giving NM2009 strain, and Yuko Kasahara and Jyunko Morikawa for their excellent technical assistance in this investigation. This study was supported by a Grant-in-Aid for Cancer Research from the Ministry of Health and Welfare of Japan.
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w11x K. Nakamuro, H. Ueno, Y. Sayato, Evaluation of mutagenicity of municipal river water concentrated using XAD resin column method, Water Sci. Technol. 25 Ž1992. 293–299. w12x Y. Sayato, K. Nakamuro, H. Ueno, R. Goto, Mutagenicity of adsorbates to a copper-phthalocyanine derivative recovered from municipal river water, Mutation Res. 242 Ž1990. 313– 317. w13x T. Ohe, Evaluation of SOS-inducing activity with an O-acetyl transferase-overexpressing strain Salmonella typhimurium NM2009 for municipal river water and the identification of 1-nitropyrene, Water Sci. Technol. 33 Ž1996. 313–320. w14x Y. Sayato, K. Nakamuro, H. Ueno, R. Goto, Identification of polycyclic aromatic hydrocarbons in mutagenic adsorbates to a copper-phthalocyanine derivative recovered from municipal river water, Mutation Res. 300 Ž1993. 207–213. w15x T. Segawa, H. Ueno, K. Nakamuro, Y. Sayato, Behavior of Trp-P-2 during activated sludge process, Jap. J. Toxicol. Environ. Health 39 Ž1993. 132–138. w16x Y. Ono, S. Mohri, I. Somiya, Y. Oda, Detection and evaluation of Trp-P-2 in biologically treated human feces and urine, Environ. Mutagen. Res. Commun. 17 Ž1995. 179–186. w17x Y. Ono, I. Somiya, T. Kawaguchi, Genotoxicity of substances in the nightsoil and its biologically treated water, Water Res. 30 Ž1996. 569–577. w18x H. Ushiyama, K. Wakabayashi, M. Hirose, H. Itoh, T. Sugimura, M. Nagao, Presence of carcinogenic heterocyclic amines in urine of healthy volunteers eating normal diet, but not of inpatients receiving parenteral alimentation, Carcinogenesis 12 Ž1991. 1417–1422. w19x K. Wakabayashi, H. Ushiyama, M. Takahashi, H. Nukaya, S.-B. Kim, M. Hirose, M. Ochiai, T. Sugimura, M. Nagao, Exposure to heterocyclic amines, Environ. Health Persp. 99 Ž1993. 129–133. w20x S. Murray, N.J. Gooderham, A.R. Boobis, D.S. Davies, Detection and measurement of MeIQx in human urine after ingestion of a cooked meat meal, Carcinogenesis 10 Ž1989. 763–765. w21x H. Hayatsu, Cellulose bearing covalently linked copper phthalocyanine trisulfonate as an adsorbent selective for polycyclic compounds and its use in studies of environmental mutagens and carcinogens, J. Chromatogr. 497 Ž1992. 37–56. w22x Y. Oda, H. Yamazaki, M. Watanabe, T. Nohmi, T. Shimada, Development of high sensitive umu test system: rapid detection of genotoxicity of promutagenic aromatic amines by Salmonella typhimurium NM2009 possessing high O-acetyltransferase activity, Mutation Res. 334 Ž1995. 145–156. w23x S. Manabe, E. Uchino, O. Wada, Carcinogenic tryptophan pyrolysis products in airborne particles and rain water, Mutation Res. 226 Ž1989. 215–221. w24x S. Mohri, I. Somiya, Y. Ono, Risk assessment of genotoxic substances related to human diet in the water environment, J. Jap. Soc. Water Environ. 19 Ž1996. 847–854.
Quantification of mutagenicrcarcinogenic heterocyclic amines, MeIQx, Trp-P-1, Trp-P-2 and PhIP, contributing highly to genotoxicity of river water Takeshi Ohe
)
Department of Food and Nutrition Science, Kyoto Women’s UniÕersity, Kitahiyoshi-cho, Imakumano, Higashiyama-ku, Kyoto 605, Japan Received 29 January 1997; revised 7 April 1997; accepted 8 April 1997
Abstract Four mutagenicrcarcinogenic heterocyclic amines ŽHCAs., 2-amino-3,8-dimethylimidazow4,5-f xquinoxaline ŽMeIQx., 3-amino-1,4-dimethyl-5H-pyridow4,3-b xindole ŽTrp-P-1., 3-amino-1-methyl-5H-pyridow4,3-b xindoleŽTrp-P-2. and 2-amino1-methyl-6-phenylimidazow4,5-b xpyridine ŽPhIP., in organic extracts obtained by blue rayon hanging method from the Yodo River water were quantified. Blue rayon extracts obtained were separated in two stages of fractionation by reversed-phase high performance liquid chromatography ŽHPLC., and the quantification of corresponding fractions was performed by HPLC with an electrochemical detector for MeIQx and a fluorometric detector for Trp-P-1, Trp-P-2 and PhIP. The geometrical mean values of MeIQx, Trp-P-1, Trp-P-2 and PhIP in extracts collected at 11 locations from the Yodo River systems were 4.8, 26.9, 37.3, and 11.9 ngrg blue rayon equivalent, respectively. The total amounts of four HCAs accounted for mean 24% of the genotoxicity of blue rayon extracts evaluated by the umu test using an O-acetyltransferase-overproducing strain NM2009. q 1997 Elsevier Science B.V. Keywords: River water; Heterocyclic amine; Trp-P-1; Trp-P-2; MeIQx; PhIP; Genotoxicity; umu Test; Blue rayon
1. Introduction The widespread occurrence of mutagenic and carcinogenic substances in river water derived from industrial waste, pesticides and naturally occurring substances is a serious problem, especially for sources of drinking water supply. There have been many
Abbreviations: HPLC, High-performance liquid chromatography; MeIQx, 2-amino-3,8-dimethylimidazow4,5-f xquinoxaline; Trp-P-1, 3-amino-1,4-dimethyl-5H-pyridow4,3-b xindole; Trp-P-2, 3-amino-1-methyl-5 H-pyridow4,3-b xindole; PhIP, 2-amino-1methyl-6-phenylimidazow4,5-b xpyridine; HCA, heterocyclic amine ) Corresponding author. Fax: q81 Ž75. 531-7216.
studies on the mutagenicity of river waters in various countries w1–7x. It is also known that organic substances from the Yodo River, which is the source of drinking water for cities located downstream, showed the highest mutagenic activity among river waters reported until now, and the major source of the mutagenicity was the discharge from wastewater treatment plants w8–11x. Also, the occurrence of indirect-acting mutagenic aminoarenes in the river, shown by the Ames test w12x and umu test w13x using an O-acetyltransferase-over-producing strain, was suggested. However, confirmation or quantification of mutagens in the Yodo River systems still remains very limited, except for some PAHs w14x and 1-nitro-
1383-5718r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 1 3 8 3 - 5 7 1 8 Ž 9 7 . 0 0 0 8 7 - 9
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T. Ohe r Mutation Research 393 (1997) 73–79
pyrene w13x. On the other hand, Trp-P-2 was detected in influents and effluents of municipal wastewater treatment plants or nightsoil treatment plants w15–17x. Mutagenicrcarcinogenic heterocyclic amines such as MeIQx, Trp-P-1, Trp-P-2 and PhIP were also found in urine from humans eating normal diet and after ingestion of a cooked meat meal w18–20x. These findings led to the quantification of heterocyclic amines in the Yodo River water, into which effluents from wastewater treatment plants or nightsoil treatment plants were discharged. This paper demonstrates quantification of four HCAs, MeIQx, Trp-P-1, Trp-P-2 and PhIP, in organic extracts obtained by the blue rayon hanging method, which is selective for collection of HCAs w10,21x, from the Yodo River systems, Japan, and evaluation of their contribution to the total genotoxicity.
Tokyo. All other chemicals and reagents were of analytical grade. 2.2. Collection and preparation of organic extracts from riÕer water Organic substances were collected at 11 sampling locations of the Yodo River systems, shown in Fig. 1, by the blue rayon hanging method w10x at a depth of 20–30 cm on 10 December, 1995. For collecting organic substances, 25 g of blue rayon in a plastic net was hung in the river water for 24 h. Blue rayon recovered from the river water was treated as described by Sakamoto and Hayatsu w10x. Blue rayon extracts obtained here were assayed for quantification of HCAs and genotoxicity in the umu test. 2.3. Purification of organic extract
2. Materials and methods 2.1. Materials M eIQx, Trp-P-1 P CH 3 COOH, Trp-P-2 P CH 3 COOH, and PhIP P HCl were obtained from Wako Pure Chemical, Osaka. HPLC grade methanol and acetonitrile were also from Wako. Blue rayon was purchased from Funakoshi Pharmaceutical Co.,
Reversed-phase HPLC analysis was performed using a Shiseido Nanospace SI-1 chromatograph ŽShiseido, Tokyo., equipped with a spectra detector ŽIrica Ý983, Kyoto.. Each extract was applied to a reversed phase PREP-ODS column Ž5 mm particle size, 10 = 250 mm, GL Science. monitored at 260 nm. The mobile phase was a gradient Ž30–80%r40 min. of methanol in 50 mM ammonium acetate ŽpH 6.0. at a flow rate of 1.6 mlrmin at 408C. The
Fig. 1. Sampling locations in the Yodo River systems.
T. Ohe r Mutation Research 393 (1997) 73–79
fractions corresponding to authentic MeIQx, Trp-P-1, Trp-P-2 and PhIP were separately collected. Then the volumes of these fractions were reduced to 1 ml with a centrifugal evaporator. The aliquot of condensed fractions was further purified using a reversed phase 5C18 AR column Ž4.6 = 150 mm, Nacalai Tesque, Kyoto.. A mobile phase of 10% acetonitrile for MeIQx or 20% acetonitrile for Trp-P1, Trp-P-2 and PhIP in 25 mM H 3 PO4rNa 2 HPO4 ŽpH 2.0. was pumped in at a flow rate of 0.8 mlrmin. The fractions corresponding to each authentic heterocyclic amine were separately collected and evaporated to dryness and then dissolved in 400 ml of 50% methanol again. 2.4. Quantification of HCAs The reduced fractions were finally analyzed on a reversed phase QC Pack C18 column Ž4.6 = 150 mm, Irica, Kyoto.. The elution solvent was 100 mM ammonium acetate ŽpH 6.0. containing 10% acetonitrile for MeIQx, and 25 mM ammonium acetate ŽpH 6.0. containing 20% acetonitrile for Trp-P-1, Trp-P-2 and PhIP at a flow rate of 0.7 mlrmin. MeIQx was detected with an electrochemical detector Ž900 mV, Irica Ý985., and Trp-P-1, Trp-P-2 and PhIP were detected by a fluorometric detector ŽShiseido SI-1, Tokyo. with excitation and emission wavelengths of 265 and 410 nm for Trp-P-1 and Trp-P-2, and 345 and 395 nm for PhIP, respectively.
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Salmonella typhimurium NM2009, kindly provided by Dr. Y. Oda ŽOsaka Prefectural Institute of Public Health.. Four dose levels with duplicate determination per dose were used.
3. Results and discussion The quantification of four HCAs, MeIQx, Trp-P-1, Trp-P-2 and PhIP, in blue rayon extracts was performed modifying the 3-step purification method reported by Manabe et al. w23x. One typical preparative HPLC chromatographic profile on a reversed phase PREP ODS column of blue rayon extract ŽLocation I. in the first purification step is shown in Fig. 2. Three fractions corresponding to MeIQx, Trp-P-1 and Trp-P-2, and PhIP were separately collected and condensed using a centrifugal evaporator. The second purification step of each corresponding fraction was carried out on a reversed phase 5C18 AR column ŽFig. 3A–C.. Each fraction corresponding to authentic heterocyclic amines was finally analyzed on a reversed phase QC Pack C18 column. Each peak corresponding to authentic MeIQx, Trp-P1, Trp-P-2 and PhIP was clearly separated as a single peak in the final analysis condition ŽFig. 3D–F.. The structures of the heterocyclic amines in the peak fractions detected on the final HPLC chromatograms were further confirmed to be MeIQx, Trp-P-1,
2.5. Spectrophotometric analysis UV absorbance spectra and fluorescence spectra of the fractions finally purified with HPLC was recorded with a photodiode-array detector ŽShiseido Nanospace SI-1. and a fluorometric detector ŽShiseido NanospaceSI-1.. 2.6. The umu test assay The genotoxic activities of blue rayon extracts were determined by the umu test in the presence of S9 mix according to the method described previously w13,22x. S9 used in this experiment was prepared from livers of rats pretreated with phenobarbital and 5,6-benzoflavone. The strain used in the experiment was an O-acetyltransferase-overproducing strain
Fig. 2. Preparative HPLC elution pattern of the first purification step of authentic MeIQx, Trp-P-1, Trp-P-2 and PhIP ŽA., and a blue rayon extract from Location I ŽB; injection volume, 2.5 g blue rayon equivalent. on an Inertsil PREP ODS column. Three fractions corresponding to MeIQx, Trp-P-1 and Trp-P-2, and PhIP were separetely collected, and further purified on a 5C18 AR column.
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Trp-P-2 and PhIP by their UV absorbance spectra and fluorescence spectra. The UV absorption spectra of the peak fractions purified from the blue rayon extract of Location I were identical to those of authentic MeIQx, Trp-P-1, Trp-P-2 and PhIP respectively, as shown in Fig. 4. Moreover, the maximal wavelength of fluorescence spectra of the peak fractions were the same as those of authentic Trp-P-1, Trp-P-2 and PhIP, respectively ŽFig. 5.. Thus, the peaks corresponding to four HCAs on the final purification step by HPLC were identified as MeIQx, Trp-P-1, Trp-P-2 and PhIP. The recoveries of four HCAs during the purification process were estimated by spiking a similar level of authentic compounds Žeach 40 ng of HCAsrg blue rayon equivalent. to that detected in the sample. The result was that recoveries of MeIQx, Trp-P-1, Trp-P-2 and PhIP were 51%, 57%, 51% and 56%, respectively. TABLE 1 shows the amounts of four HCAs in blue rayon extracts collected at 11 locations from the Yodo River systems. The original levels of MeIQx, Trp-P-
1, Trp-P-2 and PhIP were found to be at levels of nd ; 365, nd ; 530, nd ; 840 and nd ; 118 ngrg blue rayon equivalent, respectively. These values were corrected for the recovery of four HCAs during the purification process. These levels were also detected on a Capcell Pak C18 column Ž4.6 = 150 mm, Shiseido, mobile phase: 50 mM phosphate buffer, pH 8.5, containing 10% acetonitrile for MeIQx, and 20% acetonitrile for Trp-P-1, Trp-P-2 and PhIP., and the levels on the QC Pack C18 column and the Capcell Pak C18 column were almost the same. The geometrical means of MeIQx, Trp-P-1,Trp-P-2 and PhIP were 4.8, 26.9, 37.3 and 11.9 ngrg blue rayon equivalent, respectively. These values were more than 3–22-times higher than the levels of benzow axpyrene levels Žnd ; 4.9, the average being 1.7 ngrg blue rayon equivalent, n s 6., a representative polynuclear aromatic hydrocarbon detected in the Yodo River, though the sampling period was not the same ŽApril 1995.. Higher HCAs levels and genotoxicity were found in blue rayon extracts at
Fig. 3. HPLC chromatograms of the second purification step Žtop. and the final quantification step Žbottom. of MeIQx, Trp-P-1, Trp-P-2 and PhIP in a blue rayon extract from Location I. A–C: chromatograms on a 5C18 AR column monitored by their absorbance at 260 nm. Injection volume: 0.42 g blue rayon equivalent. D–F: chromatograms on a QC Pack C18 column. The eluate of MeIQx was monitored by the ECD response ŽD; 900 mV; injection volume, 0.042 g blue rayon equivalent.. Those of Trp-P-1 and Trp-P-2 were monitored by their fluorescence with excitation and emission wavelength of 295 nm and 410 nm ŽE; injection volume, 0.02 g blue rayon equivalnt.. That of PhIP was monitored by the fluorescence with 265 nm and 410 nm ŽF; injection volume, 0.1 g blue rayon equivalent..
T. Ohe r Mutation Research 393 (1997) 73–79
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Fig. 4. UV absorbance spectra of the peak fractions coinciding with the retention time of MeIQx, Trp-P-1, Trp-P-2 and PhIP in the blue rayon extract from Location I Ž — — . and authentic MeIQx, Trp-P-1, Trp-P-2 and PhIP Ž PPP . on a QC Pack ODS column. Samples of the fraction containing each ca. 20 ng of MeIQx, Trp-P-1, Trp-P-2 and PhIP were examined with a photodiode-array detector. Purity index of samples were as follows: MeIQx, 950; Trp-P-1, 960; Trp-P-2, 942; PhIP, 965.
Locations E and J, compared with those in samples at other locations. Locations E, F, I and J are situated only 300–500 m downstream, where effluents from the wastewater treatment plant or the nightsoil treatment plant goes into the Yodo River systems. It was
reported that humans could be exposed to mutagenicrcarcinogenic HCAs continuously in normal daily life w19x. Trp-P-1 and Trp-P-2 are known to be ubiquitous environmental components, which are present in airborne particles and rain water w23x.
Table 1 MeIQx, Trp-P-1, TrpP-2 and PhIP contents and genotoxicity of blue rayon extracts from the Yodo River systems Location
MeIQx
Trp-P-1
Trp-P-2
PhIP
Total of HCA
Žngrg BRE a . B D E F G H I J K L M a
nd nd 365 9 nd nd 32 350 nd nd nd
nd 24 530 48 32 3 203 520 20 43 nd
nd 33 840 50 30 41 196 280 52 40 nd
nd 9 41 55 23 nd 118 84 15 10 nd
b
Žunitsr0.1 g BRE a . nd 66 1776 162 85 44 549 1234 87 93 nd
Blue rayon equivalent. umu test ŽNM2009, qS9 mix.. nd, Not detected Ždetected limit: MeIQx, Trp-P-1 and Trp-P-2, 0.2 ngrg; BRE, PhIP, 0.4 ngrg BRE..
b
b-Galactosidase
308 155 1941 620 445 439 1099 1416 475 375 negative
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MeIQx, Trp-P-1, Trp-P-2 and PhIP were detected in urine from healthy persons eating normal diet, and MeIQx was detected in the highest concentration among four HCAs w18,19x. On the other hand, Segawa et al. w15x and Ono et al. w16x reported that Trp-P-2 was detected in an influent of municipal wastewater treatment plant, and Ono et al. w17x reported that Trp-P-2 remained after biological treatment in a nightsoil treatment plant. Recently, Mohri et al. w24x reported that Trp-P-2 was detected at a concentration of 8.7 ngrl of the Yodo River water. Therefore, the present result demonstrates that the Yodo River was highly polluted with mutagenicrcarcinogenic HCAs, and suggests that the major source of HCAs in the Yodo River was the discharge from wastewater treatment and nightsoil treatment plants. However, further studies are needed to clarify the behavior of HCAs in the water environment. The value of b-galactosidase activity per 0.1 g blue rayon equivalent was estimated from the
Fig. 5. Fluorescence spectra of each 80–100 ng of authentic Trp-P-1, Trp-P-2 and PhIP Ž — — . and the peak corresponding to Trp-P-1, Trp-P-2 and PhIP in the blue rayon extract of Location I Ž- - -.. Ex, excitation spectra monitored at the emission wavelength of 400 nm; Em, emission spectra monitored at the excitation wavelength of 260 nm.
Fig. 6. Contribution rates of MeIQx, Trp-P-1, Trp-P-2 and PhIP to genotoxicity of blue rayon extracts.
linear-regression analysis of their dose–response curves of each blue rayon extract evaluated by the umu test using an O-acetyltransferase-overproducing strain NM2009 with metabolic activation ŽTable 1.. All the sample extracts except for Location M showed distinct dose-dependent mutagenicity, suggesting that the active materials were indirect-acting mutagenic aminoarenes. A significant positive correlation Ž r s 0.96, p - 0.001; n s 11. was observed between the total amount of HCAs and the genotoxicity. On the basis of specific activity, MeIQx, Trp-P-1, Trp-P-2 and PhIP contributed on average at the rate of 5.5%, 15.6%, 2.5% and 0.5%, respectively, to the total genotoxicity of blue rayon extract at 11 sampling locations. The contribution rate of the sum total of four heterocyclic amines varied between 0% and 68.5%, the average being 24% ŽFig. 6.. Mohri et al. w24x reported that the contribution rate of Trp-P-2 detected in the Yodo River water was less than 10%, in accordance with the present result. In conclusion, the present report showed the existence of mutagenicrcarcinogenic MeIQx, Trp-P-1, Trp-P-2 and PhIP in river water, and the quantified heterocyclic amines accounted for a mean 24% of the total genotoxic activity of blue rayon extracts recovered from the water of the Yodo River system.
Acknowledgements The author would like to thank Dr. Yoshimitsu Oda, Osaka Prefectural Institute of Public Health,
T. Ohe r Mutation Research 393 (1997) 73–79
Japan, for giving NM2009 strain, and Yuko Kasahara and Jyunko Morikawa for their excellent technical assistance in this investigation. This study was supported by a Grant-in-Aid for Cancer Research from the Ministry of Health and Welfare of Japan.
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