Genotoxic activity of extractable organic matter from urban airborne particles in Shanghai, China

Mutation Research 514 (2002) 177–192

Genotoxic activity of extractable organic matter from urban airborne particles in Shanghai, China Xiansi Zhao a,∗ , Zhi Wan a , Gang Chen b , Huigang Zhu c , Shunhui Jiang c , Jiaqing Yao c a

Department of Preventive Medicine, School of Basic Medicine, Tongji University, Shanghai, PR China b Department of Environmental Health, Nan Tong Medical College, Nan Tong, PR China c Department of Environmental Health, Fudan University Medical College, Shanghai, PR China Received 1 February 2001; received in revised form 5 November 2001; accepted 7 November 2001

Abstract The aim of this research is to investigate the impact of air pollution on the population in Shanghai. The genotoxicity of extractable organic matter (EOM) from the air particles was investigated by the means of the Salmonella plate incorporation assay, rat hepatocyte unscheduled DNA repair assay, and mice micronuclei test. The airborne particles were collected in 13 locations during the summer of 1992 and winter of 1993. The crude extracts were fractionated by acid–base partitioning into acid, base and neutral fractions. The neutral fractions were further fractionated by resin–silica gel column chromatography into three subfractions. The induction of revertants with the crude extracts was higher in winter samples than in summer samples. Both indirect-acting and direct-acting mutagenicity were observed. The mutagenicity was detected with TA98, but was not detected with TA100. The mutagenic activity was the greatest in the acid, aromatic and polar fractions from summer samples. The fractions from the winter samples did not show clear differences. There was no substantial location-related variance in the mutagenic potencies of EOM, but substantial location- or time-related variances in the mutagenic potencies of the airborne particles per cubic meter air were found. While rat hepatocyte unscheduled DNA synthesis (UDS) assay revealed genotoxicity for all the samples, there was no big variance in the genotoxicity of the fractions. The mouse micronuclei test showed results similar to the UDS assay. The difference of locality did not have statistical significance. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Urban airborne particles; Fractionation; Mutagenicity; Genotoxicity; Rat hepatocyte UDS; Micronuclei test

1. Introduction Shanghai is an industrial city of China, with many kinds of industries located within the urban area. Although many factories that released serious pollu∗ Corresponding author. Present Address: Cancer Research Center, R903, Boston University School of Medicine, 715 Albany Street, Boston, MA 02118, USA. Tel.: +1-617-327-5854; fax: +1-617-638-5837. E-mail address: [email protected] (X. Zhao).

tants moved out of the urban area, the rapid transportation system as well as crowded constructs, still makes for poor air quality. In the last few years, cancer and respiratory diseases remained the leading causes of mortality in Shanghai. Lung cancer is the most frequent cancer among men (57.0 per 100,000 person per year), and is the third leading cause of mortality among women (18.8 per 100,000 person per year) [1]. It is well documented that tobacco smoking represents its predominant cause [2,3], but there are

1383-5718/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 3 - 5 7 1 8 ( 0 1 ) 0 0 3 3 8 - 2

178

X. Zhao et al. / Mutation Research 514 (2002) 177–192

two questions which remain to be answered. Since only 36% of all cases of cancer and 21% of all deaths are attributed to cigarette smoking [3], the Yuan et al. results [3] suggest that smoking is not the most prevalent cause of lung cancer since many cases of cancer may be caused by other environmental factors. Second, the incidence of lung cancer occurs in a 3:1 ratio between men and women, however, the ratio of smoking men and smoking women in Shanghai, according to our estimation, is at least 7:1. The huge difference of ratios suggested that the factors besides smoking contributed to the causes of lung cancer in women. The impact of urban air pollution on health has been studied at many cities in the world. Many research works discovered the mutagenic activity of air pollutants in some short-term bioassays [4–9] or carcinogenic activity in cell and animal experiments [10–12]. Epidemiological investigations over the last 50 years suggest rather consistently that ambient air pollution may be responsible for increased rates of lung cancer [13–22]. The genotoxic potencies and the potential health risks of urban air pollutants are different between cities because of the variation in air pollution sources. Limited research studies revealed the mutagenic activity of airborne particles in Shanghai in a bacterial experimental system [23], but some epidemiological analyses did not find an association between the incidence of lung cancer and exposure to air pollution [24]. Therefore, it is essential to clarify the nature of urban air pollution in Shanghai and to understand its impact on the health of its inhabitants. Many organic air pollutants are adsorbed on the surface of respirable air particles, and some of the components are known, or suspected, human carcinogens. In order to evaluate the mutagenic potency and carcinogenic potential of the airborne particles in Shanghai, we collected air samples from several locations in Shanghai in January of 1992, and in August of 1993, along with epidemiological data, to investigate the impact of air pollution on the health of the inhabitants of the district. A set of experiments with different levels of checkpoints was applied in this project. Here, we report the results from the short-term bioassays including a gene mutation assay, a DNA damage repair assay, and a chromosomal aberration assay, which identified the potential mutagenic potency of airborne particles in Shanghai.

2. Material and methods 2.1. Sampling and organic extracting The samples of suspended particles were collected with high-volume samplers equipped with 20 cm × 20 cm Pallflex filters. Thirteen sampling sites were selected in all the urban districts of Shanghai (Fig. 1 and Table 1). All the sites collected samples at the same time. Meteorological parameters were measured simultaneously. Filters were pretreated at 105 ◦ C to remove absorbed water and weighed. The sampling procedure spanned ten successive days, and the filter replaced with a new one every 12 h. The filter-loaded particles were placed in plastic bags carefully and kept in −80 ◦ C freezer until extraction. Samples were collected and processed in January (winter samples) and in August (summer samples), respectively. The filters were cut and extracted with dichloromethane (DCM) by means of sonication for 45 min. The crude extracts were filtered into dry, clean flasks and divided into two parts. One part was concentrated at 40 ◦ C until dry and then redissolved in dimethyl sulfoxide (DMSO) at concentrations appropriate for experiments. The other part was fractionated by acid–base partitioning into three fractions: acid, base and neutral [7]. The neutral organic fraction was further fractionated into three subfractions, according to increasing polarity, by means of XAD-2 resin:silica

Table 1 The distribution of sampling sites in Shanghai Codes

District

Location

A B C D

Yangpu Zhabei Zhabei Pudong

E F G H I

Huangpu Jingan Putuo Luwan Xuhui

J K L M

Changnin Jiading Tiaopu Wusong

Central preschool of Lunchang Street 701 Baode Street Qingyun High School Second primary school of W. Laoshan Street Labor Bureau of Shanghai Paijing High School Central Hospital of Putuo district Second preschool of Luban Street Shanghai Traditional Chinese Medical University Huding High School Xijiao Park Chemical Fibre Plant Shanghai First Steel Smeltery

X. Zhao et al. / Mutation Research 514 (2002) 177–192

179

Fig. 1. Distribution of sampling sites for collecting ambient suspended particles in Shanghai.

gel (1:1 mixture) column chromatography. The column was eluted by the order of hexane, benzene and methanol to get aliphatic, aromatic and polar fractions. Each fraction was concentrated in a rotary evaporator. After the solvent had completely evaporated, the mass of fractions was determined and dissolved in DMSO. 2.2. Salmonella/microsome mutagenicity assay Mutagenicity of these extracts was determined by the Salmonella plate incorporation method with bacterial cultures as described by Ames and coworkers [25]. The liver S9 fraction was prepared from male

SD rats pretreated with Aroclor 1254 and kept at −80 ◦ C in small aliquots. S. typhimurium tester strains TA98 and TA100 were used in this assay. The mutagenicity of the crude extracts and of individual fractions was assayed at three doses, ranging from 100 to 400 ␮g per plate for crude extracts, and from 200 to 1000 ␮g per plate for each fraction. Each dose was prepared from the stock solution by adding the appropriate amount of extract into 100 ␮l DMSO. Triplicate plates for each group was used. The chemicals, 2-acetylaminofluorene (2-AF), 2,7-diacetyl-aminofluorene (2,7-AF), and sodium azide (SA), were used as positive controls. The plates were incubated for 48 h at 37 ◦ C, and

180

X. Zhao et al. / Mutation Research 514 (2002) 177–192

then counted electronically with a Biotran II colony counter.

[28]. In addition, 200 normochromatic erythrocytes (NCE) were scored for control purposes.

2.3. Unscheduled DNA synthesis assay

2.5. Statistics

The in vitro unscheduled DNA synthesis (UDS) assay was conducted using primary hepatocytes prepared from 8- to 10-week-old male SD rats using the in situ perfusion technique as described by Swierenga et al. [26]. The freshly prepared cells were suspended in Dulbeco’s Modified Eagle Medium (DMEM) supplemented with 5% bovine serum and 0.01 M hydroxyurea. The number of cells was counted and adjusted to 1 × 106 cells/ml cultural medium. One milliliter of cell suspension was seeded into a labeled tube. Ten micro Curie [3 H]-thymidine ([3 H]-dTrd) and various concentrations of extracts were added and gently mixed. The cell suspensions were placed in shaking water bath at 37 ◦ C for 3 h. Thymidine incorporation was stopped by adding 3 ml cold saline. The cells were collected on membrane filters, washed with cold saline and 6% trichloroacetic acid. The incorporation of [3 H]-dTrd was determined by liquid-scintillation counting in a scintillator (4 g PPO and 80 mg POPOP/l toluene) using a Beckmen Scintillation Counter. The viability of the hepatocytes was determined by the trypan blue test and was more than 90%.

All data were statistical analysis with Student’s ttest. Micronucleus (MN) rates applied arcsine change of their square root before doing statistical analyses.

2.4. Micronucleus assay Male Kun-ming mice, 7–8 weeks of age and weighing 18–25 g were used in the experiment. Mice had free access to standard rodent chow and water. Mice were divided randomly into 41 groups of 5 mice each as follows: (a) solvent control; (b) cytoxin (CTX)-treated positive control; and (c) extract-treated groups. The extracts were dissolved in DMSO and the concentration adjusted to 4, 20 and 100 mg/ml. Double treatment of mice with extracts were done 24 h apart. Mice were injected i.p. with extracts at the volume of 0.1 ml/20 g b.w. The same amount of DMSO was used as a solvent control. CTX was the positive control (80 mg/kg b.w.). Mice were sacrificed by cervical dislocation 6 h after final treatment. After sacrificing the mice, bone-marrow smears were prepared and stained using the May–Grunwald–Diemsa technique [27]. From each mouse 1000 polychromatic erythrocytes (PCE) were screened for micronuclei

3. Results 3.1. Concentration, extractable organic matter and organic matter distribution The concentrations of airborne particles in the air of sampling sites are listed in Table 2. ConcentraTable 2 Concentrations of airborne particles at sampling sites (mg/m3 ) Codes

Summer (August)

Winter (January)

A B C D E F G H I J K L M

0.33 0.16 0.17 0.26 0.23 0.24 0.28 0.23 0.18 0.23 0.095 0.24 0.25

0.43 0.43 0.53 0.44 0.51 0.51 0.51 0.53 0.45 0.47 0.24 0.46 0.43

Table 3 The mass contribution after fractionationa Fraction

Mass contribution (%) Aw

Iw

Ew

Es

Acids Bases

16.7 9.6

18.4 24.2

17.8 4.6

14.9 4.5

Neutral Aliphatic Aromatic Polar

17.5 11.2 45.0

14.1 7.8 35.5

30.1 11.5 35.9

14.4 44.8 21.2

Total

36.6

34.7

48.2

20.1

a

Sample codes: A, I, E; w: winter, s: summer.

X. Zhao et al. / Mutation Research 514 (2002) 177–192

tions among sampling sites were very similar except for sample K. In fact, we selected the site K as the background control in this research project. The concentrations of airborne particles in winter were nearly two times higher than that in summer.

181

Table 3 summarizes the contribution of organic mass in individual fractions and subfractions. The fractions of four samples were weighed and were used for comparing mass contribution. From the results in Table 3, it is clear that following acid–base partition-

Table 4 The result of TA98 reverse mutation caused by the organic extracts of airborne particlesa Sample code

Dose (␮g/ml)

SE + S9 ± S.D.

DMSO A

0 100 200 400 100 200 400 100 200 400 100 200 400 100 200 400 100 200 400 100 200 400 100 200 400 100 200 400 100 200 400 100 200 400 100 200 400 100 200 400 10 100

36 36.7 42.7 76.2 49.7 69 79.7 58.3 100.7 118.3 54.7 74.3 92.7 67.3 72.7 103 45.7 66.7 93.3 47 70.7 72.3 75.7 93 123.7 78.7 101.3 130 54.7 67.7 109 98.3 145.7 167.3 106.3 166.7 202 67.3 115.3 130 1278.3

B

C

D

E

F

G

H

I

J

K

L

M

2-AF 2,7-AF a

±2 ± 0.6 ± 7.6 ± 5.3b ± 7.4 ±1 ± 3.1b ± 7.6 ± 10.5b ± 7.4c ± 6.4 ± 4.2b ± 14.8b ± 9.1 ± 1.5b ± 12.2b ± 6.1 ±6 ± 15.7b ± 3.6 ± 11 ± 0.6b ± 10.7b ± 8.9b ± 16c ± 10b ± 17b ± 7.5c ± 8.4 ±7 ± 41.2c ± 24.2b ± 24.1c ± 12.7c ± 13.3b ± 27.3c ± 32.4c ± 5.5 ± 25.8c ± 17.3c ±3 1.9c

SE − S9 ± S.D. 25.3 64.3 75 92 51.3 81.7 106.7 67.3 118 145.7 52 82 118.7 91 116.3 125 44.7 101 128 87.7 96.7 114 82.3 116.7 148.7 68.7 101.3 149.7 55.7 69 127.7 95 135.3 230.7 68.7 109.7 123 53 62.3 66

5.5 10.3b 4.4b 10.5c 7b 3.8c 12.1c 13.6b 7.2c 9.3c 10.4b 13c 1.5c 7.5c 5.5c 8.5c 4.9 1c 13.2c 8.5c 11c 24.3c 9c 16.3c 5.5c 6.4b 6.1c 16.1c 3.8b 4b 11.6c 13.1c 3.1c 8.6c 6.4b 2.1c 27.6c 11.5b 28b 8.5b

1143 ± 82.1c

SE: extracts of summer samples; WE: extracts of winter samples. The results are the mean of triplicate: ≥ 2 × revDMSO . c The results are the mean of triplicate: ≥ 3 × rev DMSO . b

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

WE + S9 ± S.D. 26 46 66.7 83 47 70 73 80 101.3 114.3 39.7 65.3 83 59.7 86 103.3 46.3 70 114.3 56.7 85 106.7 85.7 142.7 192 53.7 75.3 100.3 57 73 99.7 54.7 94.7 148.3 104 165.7 203 65 93 126.7 916.3

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

2.1 2.6 5.5b 6.1c 7 13.1b 10.4b 7c 3.1c 24.5c 4.7 7.5b 10.6c 4b 2c 7.6c 2.5 7b 16.5c 10.8b 9.5c 6.5c 3.5c 5c 24.6c 6.5b 7.8b 16.3c 7.5b 7.8b 3.5c 6b 6.8c 43.9c 6c 18.3c 25.2c 7b 7.9c 17.7c 60.4c

WE − S9 ± S.D. 23.7 56.3 70.3 93.3 44.3 74 104.3 66.7 105.7 141 63 86.7 118 75.7 116.7 126.3 55 96 128.3 81 103 112.3 70.3 112.3 162 57 81.7 118.7 64 80 146.3 74.3 118.3 193.7 75.3 99.3 127.3 49.3 66.3 103.7

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

2.1 11.4b 7.5b 8.4c 4.2 5.6c 8.1c 5.1b 4.9c 9.8c 10.8b 5.5c 16.7c 10.4c 13.3c 5.5c 3.6b 7c 17.6c 5.3c 5.6c 7.5c 9.5b 12.5c 12.5c 6.2b 15c 19.5c 7.2b 2.6c 48.6c 5.5c 22.5c 17.6c 6.8c 6.8c 2.5c 4.9b 4.5b 9c

1241.7 ± 33.9c

182

X. Zhao et al. / Mutation Research 514 (2002) 177–192

ing, the highest amount of organic mass was found in the neutral fractions, and the lowest amount was found in the base fractions, irrespective of the location or season of sampling. Comparing the extractable organic matter (EOM) of sample E, the recovery from winter

sample was higher than that from summer sample. The contribution of organic components in the neutral fraction varied among the samples. The largest amount of organic mass was found in polar subfractions in winter samples (35–45%). The aromatic fraction, which

Table 5 The result of TA100 reverse mutation caused by the organic extracts of airborne particlesa Sample code

Dose (␮g/ml)

DMSO A

0 100 200 400 100 200 400 100 200 400 100 200 400 100 200 400 100 200 400 100 200 400 100 200 400 100 200 400 100 200 400 100 200 400 100 200 400 100 200 400 1 10

B

C

D

E

F

G

H

I

J

K

L

M

SA 2-AF a

SE + S9 ± S.D. 149.7 159.7 161.7 154 128.7 117.7 143.3 114.7 140.7 140.7 154 163.7 164.3 189 180.3 170.7 148.3 169 170 171.3 174.3 162.7 157 165 188.3 187 176 188 148 151.3 179.7 182.7 194 252.3 131 234 217.3 174 154.3 144.3

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

10.7 14.3 3.2 8.5 7.6 6.8 11.7 10.4 6.4 3.1 6.6 24 4.2 12.2 10.7 2.5 11.7 3.6 2.6 9.5 7.1 5.7 27.5 8.2 3.2 3 8.2 4 9.5 9.1 3.1 42 8.2 21.1 13 10.4 9.5 49.5 25.8 14

SE − S9 ± S.D. 123.3 177.7 190.3 162.3 119.7 116.3 128.3 185 152.3 158.3 138.7 148 160.7 205.3 185 174.7 151.3 156.7 161.3 182.7 167.7 146.3 146 195 186 153.7 164.3 176 118.3 168.7 187 134.7 171.3 194.7 192 196 187 114.3 120 150 1029.3

964.3 ± 20c

SE: extracts of summer samples; WE: extracts of winter samples. The results are the mean of triplicate: ≥ 2 × revDMSO . c The results are the mean of triplicate: ≥ 3 × rev DMSO . b

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

18.3 8.1 6.7 25 13.6 10 7.4 4.6 14.3 2.5 6.7 10.8 9.7 7.5 3.6 5.7 12.5 7.1 7.4 4.7 4.2 14.3 22.1 7.9 6.2 8.1 3.2 4.4 4.2 4.9 5.0 18 36.7 46.5 14.2 37 7.9 13.6 7.9 32.6 48.8c

WE + S9 ± S.D. 121.7 127 151.3 100.3 109.3 154 133.3 130.3 152.7 124.7 109.7 127.7 126 114.3 113.7 125 130 136.3 134.3 121 131.7 143.3 238 190.7 266 123 132 151.7 113.3 123.3 160.7 134.3 122.3 136 123.7 188.7 274.3 139.3 136.3 141

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

12.5 13 9.3 6.1 7 9.6 8.1 9 6.5 29.3 7 13.6 17.1 16.2 12.3 7 4.4 5.7 23.5 14.7 8.4 11.6 16.3 21.2 7.9b 7.2 12.5 7.5 10.1 5.5 14.6 7.4 8.1 7 6.7 5.7 19.6b 14.7 12.5 6.1

646 ± 55c

WE − S9 ± S.D. 103.7 120.7 143.3 121.7 172.3 153 148 108.7 136.7 116 101.7 118.7 114.3 115 124.7 126.3 124 124.7 122.7 126.7 113.7 139.7 127.3 194.7 245.3 126.3 123 125.7 124.3 136.3 139.7 114.3 129 130.3 130.3 183 298.7 124 119 126 1300

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

6 11 5.9 53.4 7.6 14 9 15.9 16.7 15.7 11 2.1 5.5 9 4.9 10.2 7.5 8.4 12.1 11.6 12.1 3.2 9.5 10.6 33.3b 6.2 9.5 6.5 8.7 6 3.4 12.7 2.6 10 3.8 3.0 6.7b 17.5 21.4 10.5 110c

X. Zhao et al. / Mutation Research 514 (2002) 177–192

183

Fig. 2. The MEMD (␮g) of EOMs from airborne particles in Shanghai. MEMD = ((2 × revDMSO ) × 200)/rev200 . revDMSO : revertants in DMSO group; rev200 : revertants in 200 ␮g EOM group.

Fig. 3. The mutagenic potency of the airborne particles per cubic meter of air in Shanghai.

184

X. Zhao et al. / Mutation Research 514 (2002) 177–192

Table 6 The revertants of TA98 after exposure of fractions of EOMs of airborne particles in Shanghaia Sample code

Fraction

DMSO A1

Acids

A2

Bases

A3

Aliphatic

A4

Aromatic

A5

Polar

E1

Acids

E2

Bases

E3

Aliphatic

E4

Aromatic

E5

Polar

I1

Acids

I2

Bases

I3

Aliphatic

I4

Aromatic

I5

Polar

K1

Acids

K2

Bases

K3

Aliphatic

Dose (␮g per plate)

SE + S9 ± S.D.

0 200 500 1000 200 500 1000 200 500 1000 200 500 1000 200 500 1000 200 500 1000 200 500 1000 200 500 1000 200 500 1000 200 500 1000 200 500 1000 200 500 1000 200 500 1000 200 500 1000 200 500 1000 200 500 1000 200 500 1000 200 500 1000

31.7 58.7 95.3 43.3 25.7 40.3 44.3 37.7 46.3 69.3 36 85.7 181.7 30 65 152 31.7 44.7 77.3 30 31.7 39 26 35.3 62.3 48.7 105.7 154.7 27.7 42.7 100.7 30.7 32 89 28.7 27 25.3 36 32 71 41.3 68 150 29.7 44.7 100.3 37.3 44.7 88.7 27 27.7 36 33 52.3 103.3

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

2.5 4.5 17.6c 9.5 4.6 10.7 13.1 11.6 14.2 30.8b 4 29.3b 16.3c 1.7 19.1b 20.3c 7.5 9.9 11b 11.4 2.5 5.6 4.6 5.1 7.1 7.5 33.1c 37.8c 2.3 4 19.1c 6.7 7.5 3.6b 4.7 3.5 4.7 7 12.5 13.7b 3.5 16.5b 10.5c 9.3 4 18.5c 8 6.4 10b 9.6 8.5 5 12.2 4.9 18c

SE − S9 ± S.D. 22.3 49 61.3 28.3 25.3 25.3 28.7 27.7 30 54 31.7 92.3 160.7 19.7 48 126.7 24.3 31.3 58.7 27 27.3 25.7 26.3 21.7 41.7 42.7 77.3 131 27 42.3 107.7 28.7 24 58.7 20.3 23.3 31.7 29.7 33.3 43.3 36 55.7 101 18.7 45 81 24 36 81.7 21.7 28 25 25.3 42.3 61.3

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

2.5 8.7b 9.5b 3.1 5.8 7.6 2.9 4.2 16.6 16.1b 6.5 18.4c 11.2c 4.9 8.7b 22.1c 10 2.5 9b 7.8 4.7 2.3 7.1 3.5 12.7 11.7 24.4c 25.5c 7.8 8 12.9c 5.5 1.7 20.5b 5.9 4.7 2.5 5.9 9.3 11.4 7.8 14.8b 32c 1.2 7b 14.2c 7 15.7 30.2c 6.4 8.7 6.2 4.9 10 4.9b

WE + S9 ± S.D. 24 95 214 279 34.7 45.7 70.7 55 76.7 148.7 44.3 89.3 141.3 33 58.7 145.7 37 81.3 116.3 106.3 226.7 117.7 43.7 89.3 138 46.3 77 94.7 75.3 121.7 189.3 47.7 77.3 136.3 43.7 86.7 131 52 88.7 128.7 37.7 59.7 105.7 25 56.7 104 36.3 73.3 123.3 76.3 98 133.3 65.3 92.3 116.7

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

2 9.2c 47.7c 28.1c 5.7 8.7 2.1b 7.2b 4.5c 3.5c 8.4 6.4c 10.6c 4.6 8b 6.1c 5.6 7.4c 7.5c 11c 89c 23.6c 4.5 3.2c 9.5c 11.6 3c 8.3c 7.1c 13.6c 18.4c 9.1 10c 28.7c 5.5 7.1c 14.7c 2.6b 4.7c 15.3c 6.5 6.1b 8c 5.3 7.1b 8.2c 5.7 8.7c 3.1c 9.7c 10.5c 10.7c 6.8b 3.8c 10.1c

WE − S9 ± S.D. 17 29.3 40.3 41.7 20 27 33.3 23.3 27.7 42.7 19.3 20.7 32 20 21.7 32.3 27.7 32.3 35.7 27.7 38 47.7 21 23.7 32 32.3 42.7 63 28.3 45.3 88.3 22 24.7 49.3 26.7 34.3 55.3 22.3 31 56.3 24.3 30.3 44.7 24.3 38.3 76.7 26 56.7 91.3 45.7 67.3 93.7 29.7 68 92.7

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

2 1.5 8b 7b 1 4.6 3.1 5 1.5 6.8b 1.2 1.5 2.6 1 1.5 7.8 1.5 2.1 5b 4.2 2b 6.1b 2 3.8 2.6 2.1 3.2b 7c 2.5 5.9b 3.8c 2.6 2.5 5.5b 2.5 6.7b 14.2c 1.5 2.6 5.7c 2.5 2.1 9.3b 3.5 5.1b 2.1c 4.4 12.1c 10.3c 8.3b 5.1c 7c 4.5 5c 9.1c

X. Zhao et al. / Mutation Research 514 (2002) 177–192

185

Table 6 (Continued) Sample code

Fraction

Dose (␮g per plate)

SE + s9 ± S.D.

K4

Aromatic

K5

Polar

200 500 1000 200 500 1000

42.3 78 187 39 59 117 772.3 1157.7

2-AF 2,7-AF

± ± ± ± ± ± ± ±

9.9 5.6b 19.1c 7.5 20.2 8.2c 81.1c 110.4c

SE − s9 ± S.D. 44.3 68 134.7 33.7 42.3 92.7

± ± ± ± ± ±

7.4 9.2c 13.5c 11.5 16.2 18c

WE + s9 ± S.D. 57 88.7 195 61 83 135 442.3 1037.7

± ± ± ± ± ± ± ±

6.6b 7.6c 50.3c 5.3b 8c 28.7c 38.8c 56.4c

WE − s9 ± S.D. 25 52.3 64 30.7 46.7 73

± ± ± ± ± ±

2.6 7.2c 7.9c 3.1 3.5b 9c

a

SE: extracts of summer samples; WE: extracts of winter samples. The results are mean of triplicate: ≥ 2 × revDMSO . c The results are mean of triplicate: ≥ 3 × rev DMSO . b

contained the PAHs, accounted for 7.8–11.5% of total EOM. Total recovery of EOMs was about 35–48%. Comparing the amounts from location E, the aromatic fraction showed dramatic change between winter and summer samples (11.5–44.8%). Meanwhile, the total recovery of EOMs in summer samples was much lower than in winter samples (20.1–48.2%).

than summer air samples. Locations C, D, E, F, G, H and L have higher mutagenic potencies. The mutagenic activity of the crude extracts from location K is very strong, but its actual mutagenic potency per cubic meter of air is weak. 3.3. Mutagenic activities of fractions and subfractions

3.2. Mutagenic potency of extractable organic matter Table 4 summarizes the results of mutagenicity assay using the TA98 tester strain. All the crude extracts exhibited a linear dose–response relationship whether S9 was present or not. Except for samples L, and M the mutagenic activities of crude EOMs decreased when S9 is added to reaction solution. TA100 tester strain did not show a dose-depended response for any of the crude extracts (data in Table 5). If the revertants equal to or more than two times of the revertants of solvent control are regarded as the standard of positive response, Fig. 2 shows the mutagenic potencies of EOMs that was expressed as Minimum Effective Mutagenic Dose (MEMD). The direct-acting (without S9) mutagenic potency of the crude extracts is similar between locations and seasons. The indirect-acting mutagenic potency is higher in winter samples than in summer samples, and varied in locations and seasons, the samples C, K, I, and M have relative stronger mutagenic potencies. The concentration of airborne particles also influences its mutagenic potency. The mutagenic potency per cubic meter of air was presented in Fig. 3 which shows that winter air samples have greater mutagenic potency

We selected four samples to fractionate in this experiment. The data presented in Table 6 show the mutagenic activities of fractions and the difference of mutagenic activities among fractions on TA98 tester strain. For summer samples, dose–response activity occurred with all of the fractions except for base fractions, irrespective of whether S9 mix was added to the reaction solution on TA98 or not. For winter samples, adding S9 mix dramatically increased the amount of revertants with the TA98 strain. All fractions from samples I and K show dose-dependent response, irrespective of S9 mix being present or absent in reaction solution. The mutagenic activities of fractions from sample A are mainly indirect-acting activities, and all the five fractions had dose–response reactions. The subfractions of the neutral fraction of sample E showed clear dose-dependent reactions with and without S9 mix; the acid fraction showed indirect-acting mutagenicity, and base fraction showed direct-acting mutagenicity. The results with the TA100 strain did not have a positive response (data not shown). Fig. 4 demonstrate that the acid, polar and aromatic fractions from the summer samples have stronger mutagenicities. The distribution of

186

X. Zhao et al. / Mutation Research 514 (2002) 177–192

X. Zhao et al. / Mutation Research 514 (2002) 177–192

mutagenic potency did not show a big difference among the fractions in winter samples except for sample A in which the direct-acting mutagenicity was very low. Indirecting-acting mutagenicity was mainly apparent in the organic acid fraction, although other fractions also showed relative high mutagenic potency. 3.4. Unscheduled DNA synthesis response in hepatocytes after treatment with EOMs Fig. 5 presents the results of the UDS response in rat hepatocytes after exposure to crude EOMs, which showed dose-dependent responses in the range of EOMs from 25 to 100 ␮g/ml. The minor differences between UDS responses elicited by the different sample were not statistically significant. The samples from winter and from summer elicited similar UDS response level. Because of the limitation of sample amount collected, only fractions of sample E and K were used to determine the mutagenic contribution. The results are summarized in Table 7. For summer samples, the main mutagenic potency was seen in

187

aromatic and polar subfractions (sample E), or in aliphatic, aromatic and polar subfractions (sample K). No dramatic difference was observed between the fractions of winter samples. 3.5. Micronucleus formation in mice treated with crude extracts The crude EOMs of winter samples were used in this experiment. The extracts were diluted to give 4, 10 and 100 mg/ml. The results in Fig. 6 showed that the exposure of Kun-ming mice to the crude EOMs caused statistically significant increase of the number of micronucleated PCE in bone-marrow compared to DMSO-treated control mice (samples C, D, E, K, M, (P < 0.05) and G (P < 0.01) at 4 mg/ml; A, F, H, I, J (P < 0.01) at 20 mg/ml; B (P < 0.01) and L (P < 0.05) at 100 mg/ml). Dose-dependent responses were observed, but the number of micronuclei decreased at the concentration of 100 mg/ml in samples C, I and M. There are no significant differences in the potency of clastogenic activities among locations.

Fig. 5. The UDS response in rat primary hepatocytes in vitro for the exposure of EOMs of airborne particles in Shanghai. Three concentrations for each sample: 25, 50, 100 ␮g/ml from left to right in graphs; nitrogen mustard chloride (25 ␮g/ml) as positive control are 32600 ± 1338.

188

X. Zhao et al. / Mutation Research 514 (2002) 177–192

Table 7 Thymidine incorporation in rat primary hepatocytes treated with the fractions of EOMsa Sample code

Fractions

DMSO E

Acids

Bases

Aliphatic

Aromatic

Polar

K

Acids

Bases

Aliphatic

Aromatic

Polar

3-MCA

Dose (␮g/ml)

SEcpm ± S.D.

0 10 25 50 10 25 50 10 25 50 10 25 50 10 25 50 10 25 50 10 25 50 10 25 50 10 25 50 10 25 50 10

524 1369 1553 1373 1436 1287 1604 1412 4854 3005 3201 2058 2089 3699 4407 2317 670 742 1025 784 808 883 750 755 963 1250 1422 1921 1589 2349 2833 3792

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

109 156 115 167 435 88 137 110 1140 369 258 169 325 373 320 135 112 138 162 62 93 169 48 30 143 84 360 417 508 19 93 764

q

WEcpm ± S.D.

10.35 12.12 10.38 7.4 6.49 8.57 5.63 17.76 12.19 22.67 15.09 15.33 27.59 31.65 18.4 2.47 2.99 13.04 4.15 4.48 15.63 4.16 4.26 19.55 5.7 6.71 9.5 8.01 12.21 14.5

499 2972 3305 3557 3264 3743 3768 4889 4710 4060 1817 1852 4035 1224 1748 1914 2155 2327 3722 2244 2077 3137 2580 2294 1923 1941 2538 2837 1392 2268 2658 23971

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

87.76 215.08 156.93 508.54 154.45 445.15 403.18 312.93 776.28 789.54 228 155.47 212.71 141.08 85.14 27.74 210.51 463.92 543.65 157.48 366.64 430.4 103.18 490.09 283.45 308.31 180.63 154.7 144.88 177.36 176.02 1319.01

q 20.83 22.76 24.09 22.14 24.67 24.81 18.84 18.28 16.32 16.98 17.35 34.5 14.29 22.03 24.23 11.68 12.5 18.71 13.89 12.84 18.64 15.2 13.57 11.46 16.28 21.07 23.24 13.72 23.17 26.78

a

SEcpm: cpm value of the extractions of summer samples; WEcpm: cpm value of the extracts of winter samples; 3-MCA: 3-methylchloroanthane.

4. Discussion Shanghai is the biggest city in China. Located at the side of the Buo sea, it has the characteristic of sea-style weather, with frequent changes in airflow. Shanghai has many kinds of industrial plants (electronic, pharmacy, smelt, chemical industry, etc.) that are distributed in many urban districts. Most industries contribute to pollution due to their lack of modern control techniques. Also, the growth of automotive transportation since the later part of the 1980s has made diesel emissions a main source of air pollution in Shanghai. The morbidity and mortality of lung cancer has been the leading causes in Shanghai

the last few years [1,29,30]. Epidemiological research revealed a tentative correlation between the morbidity of lung cancer and air pollution [24]. In this study, we examined the air particles in Shanghai for genotoxic activity. The bioassay-directed chemical fraction helped to identify and quantify the type of mutagenic compounds in the complex mixture. As we already know, the organic extracts from ambient air particles have direct-acting and indirectacting mutagenicity in bacterial mutagenicity assays [4,5,7]. Both types of the mutagenicity were also observed in the crude extracts of our samples. The mutagenicity was seen in strain TA98, but not in TA100, which suggested that the EOM of airborne

X. Zhao et al. / Mutation Research 514 (2002) 177–192

189

Fig. 6. The dose-dependent response of micronuclei in mice exposed to the EOMs of airborne particles in Shanghai. Positive (cytoxin): 29.22 ± 0.51%.

particles in Shanghai cause frame shift mutations. The indirect-acting (+s9) mutagenic potency was stronger in both summer and winter samples from localities L and M, which are the sites of a chemical fibre plant and a steel smelter company, respectively. The s9 mix decreased the mutagenicity of all samples from the other 11 localities, except for winter sample from location H. Similar results were obtained in our previous research work [23]. However, we can not explain why s9 mix decreases the mutagenicity of crude EOM, but increases the mutagenicity of the fractions of EOM. The samples from urban sites did not show large differences in mutagenic potency, which tell us: (a) the frequently airflow in Shanghai makes air pollutants diffuse quickly and evenly; and/or (b) diesel emission may be the main pollution source, especially in the summer. Locality K was selected as a clean control in our project because of its clean environment (i.e. no industrial factories), therefore, the higher mutagenic potency of its airborne particles gave us a great surprise. Because the site K is close to Hongqiao international airport, the emissions from aircraft might be a main pollution source in this area. Further investigation is underway to determine the impact of aircraft pollution on residents in this area.

To identify the most biologically active components, the bioassay-directed fractionation procedure was used. The mutagenic potencies were most elevated in acid, aromatic and polar compounds fractions in all the tested summer samples, but the results in winter samples very between locations. For the winter samples it is hard to say which fraction had higher mutagenic potency. The contribution of the mutagenic activity of each fraction clearly corresponds to the mass contribution in summer sample (locality E) but did not have correlation in winter samples. All of these results can be explained by the change of concentrations, pollution sources and meteorological conditions between summer and winter. Indeed, the wintertime air pollutants should be more complex because there are more pollution sources in winter. The aromatic fraction contained PAHs with both direct and indirect mutagenic effects. The mutagenic potencies, as well as the contributions of aromatic fraction to overall mutagenicity in TA98, were relatively big in summer samples, but did not have obvious differences in winter samples although there were large variations in mass contribution. This is because traffic emissions were the main pollution source in

190

X. Zhao et al. / Mutation Research 514 (2002) 177–192

summer. The particles from incomplete diesel combustion contain PAHs and show indirect-acting and direct-acting mutagenicity with TA98 [6]. Our results provide useful data in assessing the risk of air pollution in urban of Shanghai. Also, there is a need to pay attention to other mutagenic pollutants besides PAHs, such as organic acid fraction and polar compounds. In fact, the importance of the organic acid fraction for mutagenicity was previously documented [7–9], but some nitro compounds, such as nitroaromatic compounds, are the only specific compounds that have been identified to account for mutagenicity [31,32]. The UDS assay has been used extensively for the in vitro detection of DNA damage caused by genotoxin exposure. However, previous studies have not used this technique to detect genotoxins in ambient air samples. We used liquid scintillation counting (LSC) to determine the DNA repair in rat hepatocyte primary cultures, which revealed that all the EOMs induced UDS. The cpm values showed a dose-dependent increase, but showed no difference by locality and seasons. The UDS response caused by the fractions of EOMs showed similar result with Salmonella test. The performance of the UDS assay with primary rat hepatocyte cultures has been documented [26,33]. UDS detection was described as an appropriate system for inclusion in carcinogenicity and mutagenicity testing programs, because it measures the repair of DNA damage induced by many classes of chemicals over the entire mammalian genome. Moreover, the induction of UDS in hepatocytes showed an excellent correlation with bacterial mutagenensis in response to many carcinogens and mutagens [34]. It was known that the UDS result can be influenced by many factors [35–37], and the preferred method of measurement is still in debate. UDS is usually measured either by autoradiography, or liquid scintillation. The autoradiographic technique is the most widely used method for measuring DNA repair. It has proved to be a reliable and reproducible technique in various laboratories, but it has the disadvantage that the slide evaluation is time-consuming. Althaus et al. [38] introduced a technique for measuring the incorporation of radioactive [3 H]-dTrd by LSC after isolation of the nuclear DNA. Although it is less suitable due to the problem of differentiation between UDS activity and replicative DNA synthesis and has the disadvantage that cells cannot be analyzed individually, this technique allows

the rapid screening of a large number of chemicals and has been reported to be at least as sensitive as autoradiography [39]. A few laboratories have modified this technique and published data [38,40]. Our laboratory has used this technique in in vitro genotoxic research. The data presented in Fig. 5 revealed a clearly dose-dependent increase in the number of micronucleated PCE in bone marrow of mice exposed for EOMs. Because of the limitation in the amount of sample collected, only the EOMs of winter samples were used. Although the difference of the micronuclei rates is present among the samples, there is no statistical significance. As a classic genotoxic method, the results demonstrate the in vivo genotoxic effect of the airborne particles in Shanghai. The rates of micronuclei decrease at the high concentration of 100 mg/ml in the samples C, M and I, which may be due to delayed cell cycle. It is well documented that the organic extract of diesel and of tobacco smoke can induce high micronuclei formation [41–43], and it was used for risk assessment of people exposed [44,45], but only a few have published data on urban airborne particles [46,47]. The data presented in this paper provide an additional in vivo confirmation of the genotoxic activity of urban airborne particles. In summary, the short-term genotoxic assays, in combination with the fractionating process, confirmed that the EOMs of airborne particles in Shanghai have strong genotoxicity that is not significantly different between locales across the urban area. However, there are big differences between locations and seasons when considering the concentrations of airborne particles. Both the direct- and indirect-acting mutagens are associated with particles. Through the fractionating process, the apportionment of mutagenic potencies was found primarily in the acid, aromatic and polar fractions in summer samples, but a great variation was seen in winter samples. Furthermore, the risk of mutagenicity is much higher in winter.

References [1] F. Jin, S.S. Devesa, W. Zheng, W.J. Blot, J.F. Fraumeni Jr., Y.T. Gao, Cancer incidence trends in urban Shanghai, 1972–1989, Int. J. Cancer 53 (5) (1993) 764–770. [2] Z. Liu, Smoking and lung cancer in China: combined analysis of eight case-control studies, Int. J. Epidemiol. 21 (1992) 197–201.

X. Zhao et al. / Mutation Research 514 (2002) 177–192 [3] J.M. Yuan, R.K. Ross, X.L. Wang, Y.T. Gao, B.E. Henderson, M.C. Yu, Morbidity and mortality in relation to cigarette smoking in Shanghai, China. A prospective mate cohort study, JAMA 275 (1996) 1683–1684. [4] B.S. De Martinis, N.Y. Kado, L.R. De Carvalho, R.A. Gundel, Genotoxicity of fractionated organic material in airborne particles from Sao Paulo, Brazil, Mutat. Res. 446 (1999) 83–94. [5] Y.A. Courtois, S. Min, C. Lachenal, J.M. Jacquot-Deschamps, F. Callais, B. Festy, Genotoxicity of organic extracts from atmospheric particles, Ann. New York Acad. Sci. 534 (1988) 724–740. [6] F. Pott, U. Heinrich, Relative significance of different hydrocarbons for the carcinogenic potency of emissions from various incomplete combustion processes, IARC Sci. Public. 104 (1990) 288–297. [7] J. Lewtas, J. Chuang, M. Nishioka, B. Peterson, Bioassaydirected fractionation of the organic extract of SRM1649 urban air particulate matter, Int. J. Environ. Anal. Chem. 39 (1990) 245–254. [8] M.G. Nishioka, J. Lewtas, Quantification of nitro- and hydroxylated nitro-aromatic/polycyclic aromatic hydrocarbons in selected ambient air daytime winter samples, Atmos. Environ. 26A (1992) 2077–2087. [9] Z. Zhao, L. Zhang, L. Wei, Mutagenicity and antimutagenicity assay of airborne suspended particles in Shijia Zhuang city, Chung Hua Yu Fang I Hsueh Tsa Chih 29 (1995) 213–215. [10] P.M. Cury, A.J. Lichtenfels, M.S. Reymao, G.M. Conceicao, V.L. Capelozzi, P.H. Saldiva, Urban levels of air pollution modifies the progression of urethane-induced lung tumors in mice, Pathol. Res. Pract. 196 (2000) 627–633. [11] F. Pott, W. Stober, Carcinogenicity of airborne combustion products observed in subcutaneous tissue and lungs of laboratory rodents, Environ. Health Perspect. 47 (1983) 293–303. [12] H. Brune, M. Habs, D. Schmahl, The tumor-producing effect of automobile exhaust condensate and fractions thereof. Part II. Animal studies, J. Environ. Pathol. Toxicol. 1 (1978) 737–745. [13] M.S. Goldberg, R.T. Burnett, J.C. Bailar III, J. Brook, Y. Bonvalot, R. Tamblyn, R. Singh, M.F. Valois, R. Vincent, The association between daily mortality and ambient air particle pollution in Montreal, Quebec, Environ. Res. 86 (2001) 26–36. [14] S. Lagorio, F. Forastiere, M. Lipsett, E. Menichini, Air pollution from traffic and the risk of tumors, Ann. Ist. Super. Sanita 36 (2000) 311–329. [15] F. Nyberg, P. Gustavsson, L. Jarup, T. Bellander, N. Berglind, R. Jakobsson, G. Pershagen, Urban air pollution and lung cancer in Stockholm, Epidemiology 11 (2000) 487–495. [16] K. Katsouyanni, G. Pershagen, Ambient air pollution exposure and cancer, Cancer Causes Control 8 (1997) 284–291. [17] A.J. Cohen, C.A. Pope III, F.E. Speizer, Ambient air pollution as a risk factor for lung cancer, Salud Publica Mex. 39 (1997) 346–355. [18] E. Buiatti, M. Geddes, S. Arniani, Epidemiology of lung cancer, Ann. Ist. Super. Sanita 32 (1996) 133–144.

191

[19] H. Li, S. Jin, S. Shi, The trend of mortality of lung cancer and its association with air pollution, Zhonghua Nei Ke Za Zhi 35 (1996) 451–454. [20] A.J. Cohen, C.A. Pope III, Lung cancer and air pollution, Environ. Health Perspect. 103 (Suppl.) (1995) 219–224. [21] G. Pershagen, Air pollution and cancer, IARC Sci. Public. 104 (1990) 240–251. [22] F.E. Speizer, Assessment of the epidemiological data relating lung cancer to air pollution, Environ. Health Perspect. 47 (1983) 33–42. [23] H.G. Zhu, Q.Y. Zhao, S.H. Jiang, The mutagenicty of the extracts of suspended particles in Shanghai, Shanghai Environ. Sci. 10 (1991) 10–13. [24] Y.T. Gao, Risk factors for lung cancer among nonsmokers with emphasis on lifestyle factors, Lung Cancer 14 (Suppl.) (1996) 39–45. [25] D.M. Maron, B.N. Ames, Revised method for the Salmonella mutagenicity test, Mutat. Res. 113 (1983) 173–215. [26] S.H. Swierenga, J.A. Bradlaw, R.L. Brillinger, J.P. Gilman, E.R. Nestmann, R.C. San, Recommended protocols based on a survey of current practice in genotoxicity testing laboratories. Part I. Unscheduled DNA synthesis assay in rat hepatocyte cultures, Mutat. Res. 246 (1991) 235–253. [27] W. Schmid, The micronucleus test, Mutat. Res. 31 (1975) 9–15. [28] J.N. Heddle, M. Hite, B. Kirkhart, K. Mavournin, J. MacGregor, G. Newel, M. Salamone, The induction of micronuclei as a measure of genotoxicity: a report of the US Environmental Protection Agency Gene-Tox program, Mutat. Res. 123 (1983) 61–118. [29] Y.T Gao, Implication of the time trend of cancer incidence in urban Shanghai, China, for cancer control strategy, Cancer Strategies for the New Millennium, WHO, 1998. [30] F. Dai, The changing cancer spectrum and incidence in Shanghai area, China Med. J. 108 (1995) 314. [31] V. Purohit, A.K. Basu, Mutagenicity of nitroaromatic compounds, Chem. Res. Toxicol. 13 (2000) 673–692. [32] V.L. Dellarco, M.J. Prival, Mutagenicity of nitro compounds in Salmonella typhimurium in the presence of flavin mononucleotide in a preincubation assay, Environ. Mol. Mutagen. 13 (1989) 116–127. [33] A.D. Mitchell, D.A. Casciano, M.L. Meltz, D.E. Robinson, R.H. San, G.M. William, E.S. Von Halle, Unscheduled DNA synthesis tests. A report of the US Environmental Protection Agency Gene-Tox Program, Mutat. Res. 123 (1983) 363–410. [34] G.S. Probst, R.E. MeMahon, L.E. Hill, C.Z. Thompson, J.K. Epp, S.B. Neal, Chemically-induced unscheduled DNA synthesis in primary rat hepatocyte cultures: a comparison with bacterial mutagenicity using 218 compounds, Environ. Mutagen. 3 (1981) 11–32. [35] Y. Oshiro, P.S. Balwierz, R.W. Falk, C.E. Piper, Decision criteria for the in vitro rat hepatocyte UDS assay, J. Appl. Toxicol. 7 (1987) 378–385. [36] G.S. Probst, L.E. Hill, Influence of age, sex and strain on the in vitro induction of unschelduled DNA synthesis in rat hepatocyte primary cultures, Cell Bio. Toxicol. 3 (1987) 113–125.

192

X. Zhao et al. / Mutation Research 514 (2002) 177–192

[37] S. Madle, S.W. Dean, U. Andrae, G. Brambilla, B. Burlinson, D.J. Doolittle, C. Furihata, T. Hertner, C.A. McQueen, H. Mori, Recommendations for the performance of UDS tests in vitro and in vivo, Mutat. Res. 312 (1994) 263–285. [38] F.R. Althaus, S.D. Lawrence, G.L. Sattler, D.G. Longfellow, H.C. Pitot, Chemical quantification of unscheduled DNA synthesis in cultured hepatocytes as an assay for the rapid screening of potential chemical carcinogens, Cancer Res. 42 (1982) 3010–3015. [39] W. Suter, Comparison of the autoradiographic and the liquid scintillation techniques for the assessment of DNA repair synthesis rat hepatocyte primary cultures, Mutat. Res. 181 (1987) 334. [40] A. Martelli, E. Cajelli, M. Ghia, Scintillometric determination of unscheduled DNA synthesis in primary cultures of rat-liver cells. A hydroxylapatite batch assay, Mutat. Res. 143 (1985) 257–261. [41] Y. Odagiri, J.X. Zhang, H. Uchida, K. Kawamura, S. Adachi, K. Takemoto, Predominant induction of kinetochorecontaining micronuclei by extracts of diesel exhaust particulates in cultured human lymphocytes, Environ. Mol. Mutagen. 23 (1994) 45–50. [42] C.J. Smith, T.A. Perfetti, M.A. Mullens, A. Rodgman, D.J. Doolittle, IARC group 2B Carcinogens reported in cigarette mainstream smoke, Food Chem. Toxicol. 38 (2000) 825–848.

[43] E. Massey, M. Aufderheide, W. Koch, H. Lodding, G. Pohlmann, H. Windt, P. Jarck, J.W. Knebel, Micronucleus induction in V79 cells after direct exposure to whole cigarette smoke, Mutagenesis 13 (1998) 145–149. [44] M. Somorovska, E. Szabova, P. Vodicka, J. Tulinska, M. Barancokova, R. Fabry, A. Liskova, Z. Riegerova, H. Petrovska, J. Kubova, K. Rausova, M. Dusinska, A. Collins, Biomonitoring of genotoxic risk in workers in a rubber factory: comparison of the comet assay with cytogenetic methods and immunology, Mutat. Res. 445 (1999) 181–192. [45] C. Bolognesi, F. Merlo, R. Rabboni, F. Valerio, A. Abbondandolo, Cytogenetic biomonitoring in traffic police workers: micronucleus test in peripheral blood lymphocytes, Environ. Mol. Mutagen. 30 (1997) 396–402. [46] S. Baldacci, L. Carrozzi, G. Viegi, C. Giuntini, Assessment of respiratory effect of air pollution: study design on general population samples, J. Environ. Pathol. Toxicol. Oncol. 16 (1997) 77–83. [47] E.T. Guimaraes, M. Domingos, E.S. Alves, N. Caldini, D.J. Lobo, A.J. Lichtenfels, P.H. Saldiva, Detection of the genotoxicity of air pollutants in and around the city of Sao Paulo (Brazil) with the Tradescantia-micronucleus (Trad-MCN) assay, Environ. Exp. Bot. 44 (2000) 1–8.