Chem. Res. Toxicol. 2000, 13, 281-286
281
Mutagenic Activity of Surface Soil and Quantification of 1,3-, 1,6-, and 1,8-Dinitropyrene Isomers in Soil in Japan Tetsushi Watanabe,*,† Sumio Goto,‡ Yutaka Matsumoto,§ Masaharu Asanoma,| Teruhisa Hirayama,† Nobuyuki Sera,⊥ Yoshifumi Takahashi,† Osamu Endo,‡ Shigekatsu Sakai,§ and Keiji Wakabayashi@ Kyoto Pharmaceutical University, 5, Nakauchicho, Misasagi, Yamashina-ku, Kyoto 607-8414, Japan, National Institute of Public Health, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8638, Japan, Hokkaido Research Institute of Environmental Sciences, Nishi-12, Kita-19-jo, Kita-ku, Sapporo 060-0819, Japan, Nagoya City Public Health Research Institute, 1-11 Hagiyama-cho, Mizuho-ku, Nagoya 467-8615, Japan, Fukuoka Institute of Health and Environmental Sciences, 39 Mukaeda, Mukaizano, Dazaifu 818-0135, Japan, and National Cancer Center Research Institute, 1-1 Tsukiji 5-chome, Chuo-ku, Tokyo 104-0045, Japan Received November 11, 1999
To clarify the mutagenic potential of nonagricultural surface soil in Japan, 110 soil samples were collected from five geographically different areas between November 1996 and March 1997, and organic extracts of the soil samples were examined by the Ames/Salmonella assay. Most of the soil extracts showed mutagenicity toward both strains TA98 and TA100 in the presence and/or absence of a mammalian metabolic activation system (S9 mix), suggesting that surface soil is largely contaminated with environmental mutagens. Soil samples collected at Hekinan, Kobe, and Osaka were highly mutagenic toward both strains, and their potencies toward TA98 without S9 mix were extremely high, inducing more than 12 000 revertants per gram of soil. On the other hand, soil samples from Muroran showed strong mutagenicity toward TA100 with S9 mix. Furthermore, 1,3-dinitropyrene (DNP), 1,6-DNP, and 1,8-DNP in soil samples collected at 10 sampling sites in three metropolitan areas were quantified by fluorometric detection of the corresponding diaminopyrene isomers using high-performance liquid chromatography (HPLC). Three DNP isomers were detected in all soil samples, and the amounts of 1,3-, 1,6-, and 1,8-DNP isomers in the soil samples were 12-3270, 14-5587, and 13-6809 pg/g, respectively. The gross amount of three DNP isomers in surface soil collected at Hekinan was more than 10 ng per gram of soil. The highest contribution ratios of DNP isomers to the mutagenicity of soil extracts were observed for the samples collected at Osaka, and the total of the contribution ratios of three DNP isomers was about 50%. These results suggest that surface soil is largely contaminated with mutagenic compounds and that DNP isomers are one class of major mutagenic and carcinogenic compounds contaminating surface soil.
Introduction Thousands of chemical compounds are emitted into air by diesel and gasoline engines (1-4), industrial power plants (5, 6), and municipal incinerators (7) in incomplete combustion processes. Many mutagenic and carcinogenic compounds such as polycyclic aromatic hydrocarbons (PAHs)1 and nitrated PAHs (nitro-PAHs) are included among these components (6-10). Urban air particles were reported to contain extractable organic matter which has mutagenic and carcinogenic activity (11-14). Since most of these atmospheric compounds eventually descend to the ground, the surface of the ground is * To whom correspondence should be addressed. † Kyoto Pharmaceutical University. ‡ Institute of Public Health. § Hokkaido Research Institute of Environmental Sciences. | Nagoya City Public Health Research Institute. ⊥ Fukuoka Institute of Health and Environmental Sciences. @ National Cancer Center Research Institute. 1 Abbreviations: DNP, dinitropyrene; HPLC, high-performance liquid chromatography; PAHs, polycyclic aromatic hydrocarbons; nitroPAHs, nitrated polycyclic aromatic hydrocarbons; B[a]P, benzo[a]pyrene; IARC, International Agency for Research on Cancer.
thought to be contaminated with these genotoxic compounds. Menck et al. (15) reported higher lung cancer mortality rates among males living in certain heavily industrialized areas of Los Angeles County, CA, and that those areas were characterized by elevated concentrations of PAHs of primarily industrial origin in the soil and air. There are several reports on the mutagenicity of soil (16-23). Agricultural soil was found to be mutagenic in the Ames test both with and without mammalian metabolic system (16-18), and Brown et al. (16) suggested that the mutagenic activity was related to past agricultural practices. McDaniels et al. (19) revealed that organic extracts of soil from the sites contaminated with industrial waste were mutagenic, and high levels of PAHs were detected in the soil extracts. Soil samples from roadsides in several cities in Japan have also been reported to be mutagenic toward Salmonella typhimurium TA98 and/or TA100 (20-22). Benzo[a]pyrene (B[a]P), which is a representative PAH mutagenic toward both strains TA98 and TA100, was detected in the soil samples (20-22). Arashidani et al. (22) reported that mutagenic
10.1021/tx990190v CCC: $19.00 © 2000 American Chemical Society Published on Web 03/24/2000
282
Chem. Res. Toxicol., Vol. 13, No. 4, 2000
Watanabe et al.
activity of extracts of soil samples from roadsides in Kyushu and Chugoku Districts was correlated with the amount of B[a]P in the extracts; however, the contribution of B[a]P to the mutagenic activity of the soil extracts was less than 2%. A similar low contribution of B[a]P to the mutagenicity of soil extracts was reported for the samples collected from roadsides in other sampling areas such as Tokyo (20) and Sendai (21). We previously reported that nonagricultural soils collected at six parks in Osaka and neighboring cities, which have some of the highest death rates of lung cancer in Japan (24), showed mutagenicity in the Ames/Salmonella assay and that 1,6and 1,8-dinitropyrene (DNP) isomers were detected in the two soil samples from Osaka (23). Furthermore, these two DNP isomers accounted for about 40% of the mutagenicity of these soil samples. In the study presented here, to clarify the mutagenic potential of surface soil in Japan, a total of 110 nonagricultural soil samples were collected from parks, roadsides, banks, etc., in five areas of Japan in the same period, and the mutagenicity of organic extracts of the soil samples was examined using the Ames/Salmonella assay. Moreover, we quantified three carcinogenic DNP isomers, i.e., 1,3-, 1,6-, and 1,8-DNP, in surface soil samples collected in the three largest metropolitan areas in Japan to clarify whether DNP isomers commonly and continuously contaminate the surface soils. The participation of DNP isomers in the mutagenicity of the soil samples is also discussed in this study.
Experimental Procedures Chemicals. 1,3-DNP (CAS registry number 75321-20-9), 1,6DNP (CAS registry number 42397-64-8), and 1,8-DNP (CAS registry number 42397-65-9) were obtained from Sigma Chemical Co. (St. Louis, MO). HPLC grade acetonitrile and methanol were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). All other chemicals that were used were analytical grade. Sample Collection and Preparation of Organic Extracts of Soil. Sample collection was performed in five areas of Japan, i.e., Hokkaido, Kanto, Chubu, Kinki, and Kyushu (Figure 1), and at least 20 surface soil samples were collected from each area. Sampling sites were selected from roadsides, parks, banks, etc., in residential, commercial, and industrial areas. Soil from the surface of the ground to about 10 cm deep was dug up, if necessary, mixed thoroughly, and collected. Sampling was carried out after a minimum of 7 days of nonrainy weather between November 1996 and May 1998. The soil was spread on Petri dishes, allowed to stand to dryness at room temperature, and screened through a 60-mesh sieve (250 µm) to remove large gravel and trash. The sieved soil (15 g) was extracted twice with methanol (200 mL) with an ultrasonic apparatus (185 W) for 10 min. The extracts were filtered and evaporated to dryness for the mutagenicity test and quantification of DNP isomers. Mutagenicity Assay. Organic extracts of the soil were dissolved in dimethyl sulfoxide and assayed for mutagenicity by the preincubation method (25) in the presence and absence of S9 mix (26). S. typhimurium strains TA98 and TA100 were kindly provided by B. N. Ames (Department of Biochemistry, University of California, Berkeley, CA). The S9 mix contained 50 µL of S9 in a total volume of 500 µL. S9 was prepared from male Sprague-Dawley rat liver which was induced by pretreatment with phenobarbital and β-naphthoflavone (27). The soil extracts and authentic dinitropyrenes were assayed with at least three doses with duplicate plates at each dose. Revertant colonies were counted after incubation for 2 days at 37 °C. When the samples induced 2-fold increases over the average yield of
Figure 1. Map of the surface soil sampling areas in Japan. spontaneous revertants, we judged that they exerted mutagenicity in the tester strain. The slope of the dose-response curve (revertants per gram of soil), which was calculated by leastsquares linear regression from the first linear portion of the dose-response curve, was adopted as the mutagenic potency. Quantification of Dinitropyrenes in Soil. 1,3-, 1,6-, and 1,8-DNP isomers in the soil were quantified as the corresponding diaminopyrenes by the method reported previously (28). The organic extracts of soil were cleaned by three column chromatographic steps using a silica gel open column (63-200 µm particle size, 10 mm × 220 mm) and two reversed-phase HPLC columns, an STR ODS II column (5 µm particle size, 4.6 mm × 250 mm, Shimadzu Techno-Research, Inc., Kyoto, Japan) and a WakosilPAHs column (5 µm particle size, 4.6 mm × 250 mm, Wako Pure Chemical Industries, Ltd.). The fractions corresponding to 1,3-, 1,6-, and 1,8-DNP isomers were dissolved in 50% ethanol and injected into an L-column ODS column (5 µm particle size, 4.6 mm × 150 mm, Chemicals Inspection & Testing Institute, Tokyo, Japan) for HPLC. The elution was carried out with 80% methanol at a flow rate of 0.5 mL/min. The eluate was introduced into a catalyst column (4.0 mm × 50 mm), which was packed with 5 µm alumina coated with platinum and rhodium (Johnson Matthey Inc., Malvern, PA), in series. DNP isomers were reduced to diaminopyrens with the catalyst column at 80 °C, and the effluent from the catalyst column was monitored by a Shimadzu RF-535 fluorescence HPLC monitor with excitation and emission wavelengths of 375 and 450 nm, respectively. Experiments were conducted in triplicate for each soil sample. The contents of DNP isomers in the specimens were estimated from standard curves obtained from various doses of the authentic compounds.
Results and Discussion Figure 1 shows the location of five sampling areas of surface soil. Soil samples were collected at 20-25 sites in each area between November 1996 and March 1997,
Mutagenicity of Surface Soils in Japan
Chem. Res. Toxicol., Vol. 13, No. 4, 2000 283
Table 1. Mutagenicities of Organic Extracts of Surface Soil Collected in Five Areas in Japan mutagenic activity (revertants/g of soil)b
positive rate (%) TA98 sampling area Hokkaido Kanto Chubu Kinki Kyushu Whole
TA100
TA98
TA100
na
without S9 mix
with S9 mix
without S9 mix
with S9 mix
without S9 mix
with S9 mix
without S9 mix
with S9 mix
24 20 25 20 21 110
100 100 100 85 100 97
100 100 100 100 100 100
100 100 100 40 100 89
100 100 100 70 100 95
399 ( 236 362 ( 340 1275 ( 4916 2549 ( 5825 382 ( 160 979 ( 3458
847 ( 793 885 ( 503 923 ( 2137 1052 ( 1729 407 ( 201 825 ( 1325
392 ( 195 297 ( 224 355 ( 718 201 ( 390 328 ( 194 319 ( 409
1869 ( 2039 901 ( 505 591 ( 1327 474 ( 807 563 ( 162 900 ( 1308
a Surface soil was collected between November 1996 and March 1997. b The slope of the dose-response curve (revertants per gram of soil) was calculated by least-squares linear regression from the first linear portion of the dose-response curve. Values were represented as the mean ( SD.
Figure 2. Mutagenic potencies of surface soil toward S. typhimurium TA98 and TA100. Table 2. Five Highest Mutagenic Potencies of Surface Soil in Five Areas in Japan TA98 without S9 mix
TA100 with S9 mix
without S9 mix
with S9 mix
no.
sampling sitea
potencyb (revertants/g)
sampling sitea
potencyb (revertants/g)
sampling sitea
potencyb (revertants/g)
sampling sitea
potencyb (revertants/g)
1 2 3 4 5
Hekinan Kobe Osaka-1 Osaka-2 Nishinomiya
24900 20800 13800 12200 1720
Hekinan Osaka-1 Kobe Osaka-2 Muroran-3
11100 6070 4010 3970 2940
Hekinan Kobe Tokyo Osaka-2 Osaka-1
3710 1310 1060 962 951
Muroran-1 Muroran-2 Hekinan Muroran-3 Muroran-4
8440 6970 6880 3550 3210
a The sampling date of surface soil at each site was as follows: Hekinan, March 5, 1997; Kobe, January 30, 1997; Osaka-1, December 16, 1996; Osaka-2, January 30, 1997; Nishinomiya, January 30, 1997; Tokyo, January 16, 1997; Muroran-1-4, December 5, 1996. b The organic extracts were obtained from 15 g of soil (250 µm) with an ultrasonic extractor. Mutagenic potencies are represented as the number of revertants per gram of soil.
and a total of 110 soil samples were examined for mutagenicity. Table 1 shows the percentage of soil samples which were positive in the Ames/Salmonella assay. Almost all samples exerted mutagenicity toward both strains with and/or without S9 mix, suggesting that surface soils are largely contaminated with mutagenic compounds. Figure 2 shows the mutagenic potency of each soil sample toward TA98 (Figure 2A) and TA100 (Figure 2B) with and without S9 mix. Four soil samples exerted strong mutagenicity toward TA98 without S9 mix, inducing more than 12 000 revertants per gram of soil, and the potency was weakened by the addition of S9 mix. The other soil samples showed the same levels or higher levels of mutagenicity in the presence of S9 mix as in the absence of S9 mix. As shown in Figure 2B, most of the soil samples exerted higher mutagenic potency toward TA100 with S9 mix than without S9 mix. Mean values and the standard deviation of the mutagenic potency of the soil samples at each area and the whole
samples are also shown in Table 1. The mean values of all the samples toward both strains ranged from 319 revertants (for TA100 without S9 mix) to 979 revertants (for TA98 without S9 mix) per gram of soil. These potencies were almost the same as those previously reported for nonagricultural “ordinary soil” (29) and soil from roadsides (20-22). Table 2 lists the top five mutagenicities of surface soil toward TA98 and TA100 with and without S9 mix among all the soil samples that were examined. Soil samples collected at Hekinan and Kobe showed strong mutagenicity toward TA98 without S9 mix, inducing 24 900 and 20 800 revertants per gram of soil, respectively. This activity was g20 times higher than the mean values of mutagenic potencies for all the soil samples shown in Table 1. Soil samples from Osaka-1 and Osaka-2 were also highly mutagenic toward TA98 without S9 mix. Osaka-1 and Osaka-2 are geographically different from each other, and the distance between these sites is 6 km.
284
Chem. Res. Toxicol., Vol. 13, No. 4, 2000
Watanabe et al.
Table 3. Amount of 1,3-, 1,6-, and 1,8-DNP Isomers in Soil and Contribution of DNP Isomers to the Mutagenicities of Organic Extracts of Soil in S. typhimurium TA98 without S9 Mix
sampling site Kanto area Tokyo Sinagawa-ku Higashimurayama Hachioji Chubu area Nagoya Gifu Hekinan-1c Hekinan-2c Kinki area Uji Osaka Sumiyoshi-ku-1d Osaka Sumiyoshi-ku-2d Higashiosaka Kobe
sampling date
mutagenicity (revertants/g of soil)a
February 2, 1998 December 29, 1997 April 1, 1998
319 438 380
January 29, 1998 January 29, 1998 January 15, 1997 February 18, 1998
180 260 34300 46800
May 23, 1998 December 16, 1996 April 19, 1997 April 19, 1997 January 30, 1997
3300 6740 9780 248 10200
amount of DNP (pg/g of soil)b 1,31,61,825 ( 3 17 ( 2 21 ( 2
34 ( 1 14 ( 1 22 ( 1
125 ( 4 17 ( 2 30 ( 1
contribution ratio of DNP (%) 1,3- 1,6- 1,8- total 3 2 2
4 1 2
30 3 6
37 6 10
12 ( 4 16 ( 6 13 ( 2 51 ( 6 82 ( 8 77 ( 5 2437 ( 31 4209 ( 156 4369 ( 469 3270 ( 383 5587 ( 315 6809 ( 50
3 8 3 3
3 12 5 5
6 22 10 11
21 42 18 19
318 ( 51 633 ( 47 863 ( 101 1653 ( 93 1928 ( 71 2360 ( 133 2683 ( 346 3069 ( 383 3646 ( 557 29 ( 10 25 ( 13 61 ( 22 1120 ( 111 1849 ( 120 2573 ( 237
4 10 11 4 4
7 11 12 4 7
20 27 28 19 20
31 48 51 27 31
a The organic extracts were obtained from 15 g of soil (
