DNA damage caused by extracts of chlorinated drinking water in human derived liver cells (HepG2)

Toxicology 198 (2004) 351–357

DNA damage caused by extracts of chlorinated drinking water in human derived liver cells (HepG2) Wen-Qing Lu a,∗ , Dan Chen a , Xin-Jiang Wu b , Ai-lin Liu a , Hui Liu a , Jian-Jun Wu a , Volker Mersch-Sundermann b a

b

Department of Occupational and Environmental Health, Tongji Medical College of Huazhong University of Science and Technology, Hangkong Road 13, 430030 Wuhan, PR China Institute of Indoor and Environmental Toxicology, Justus-Liebig-University of Giessen-University Hospital, D-35385 Giessen, Germany

Abstract Dong (D) lake and the Yangtze (Y) river are the main water supplies of the city of Wuhan, PR China. In the present study, the genotoxic effect of chlorinated drinking water (CDW) processed from raw water of D lake and Y river was evaluated in human HepG2 cells using the Comet assay and the micronucleus test. For that, HepG2 cells were exposed to XAD extracts of CDW corresponding to 0.167, 1.67, 16.7 and 167 ml CDW/ml cell culture. All CDW extracts caused a significant and dose-dependent increase of DNA migration in HepG2 cells. The level of DNA damage varied depending on the sampling time (season) and sampling site. The lowest concentration which caused a significant increase of DNA migration was 1.67 ml CDW/ml culture for water samples collected in August. Water samples collected in March showed their lowest observable effect levels in 167 ml and 16.7 ml CDW/ml culture for Y river and D lake, respectively. Additionally, significant increases of micronuclei (MN) frequencies were found in HepG2 cells after CDW treatment. However, in the MN assay the CDW samples collected in March exhibited higher genotoxicity than the August samples. In conclusion, HepG2 cells provide a useful tool for the detection of genotoxic effects of environmental mixtures. © 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Chlorinated drinking water; Genotoxicity; HepG2 cells; Human liver cell line; Comet assay; Micronucleus test

1. Introduction Humans are normally exposed to undefined mixtures of natural and artificial chemicals via food, air and drinking water. To estimate risks, both toxicological analysis of individual agents and of complex mixtures are necessary. ∗ Corresponding author. Tel.: +86-27-8369-2715; fax: +86-27-8369-2701. E-mail address: [email protected] (W.-Q. Lu).

The present study deals with DNA-damage caused by extracts of drinking water processed from raw water of the Dong (D) lake and Yangtze (Y) river using chlorination. Chlorination is the most commonly used method of disinfecting drinking water. Disinfection of drinking water guarantees an effective protection of humans against pathogens. However, chlorination can result in the formation of disinfection by-products, many of which are genotoxic in short-term assays (Douglas et al., 1986; Liimatainen et al., 1988). Using gas chromatography/mass spec-

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trometry (GC/MS) analysis we identified 25 and 28 non-volatile organic compounds in CDW extracts from D lake and the Y river, respectively. Also 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]furanone (MX), a strong mutagen that arise as a result of the chemical reaction between chlorine and organic materials (Kronberg and Vartiainen, 1988), was detected in CDW extracts from D lake and the Y river in concentrations of 0.23 and 0.15 ng/l, respectively. The D lake is one of the main drinking water sources of Wuhan, a city with 8 million residents located in the Hubei province, central China. Since the 1960s D Lake has become eutrophic due to organic pollution caused by agriculture, industries and households. A retrospective cohort study showed that the cancer risk among people provided with water from D Lake was higher than among populations drinking water from the Y river (Li et al., 1992). These results are in accordance with epidemiological studies which showed a modest increase in the risk of colon and bladder cancer among people who had consumed chlorinated drinking water (CDW) (Cantor et al., 1987; Koivusalo et al., 1997; Wilkins et al., 1979). Additionally, it was reported that CDW extracts from D lake cause mutagenic effects in Salmonella typhimurium (Wang et al., 1985), the SOS chromotest with E. coli PQ37 (Li et al., 1995), and in HepG2 cells (Lu et al., 2002). Like D lake the Y river is also an important drinking water source of the city of Wuhan. In recent years, it has also been polluted by industries and households. No data are available on the genotoxicity of CDW processed from raw water of the Y river. A reliable assessment of health risks caused by complex mixtures of chemicals in drinking water is of crucial importance with respect to public health issues. Therefore, during the last decades papers were published using laboratory models with bacteria, mammalian cells and animals to investigate the DNA-damaging potency of CDW. However, as reported earlier (Knasmüller et al., 1998) studies with bacteria or metabolically incompetent mammalian cells possess some shortcomings especially regarding the interpretation of results in the frame of human risk assessment. Additionally, in vivo animal studies are too time-consuming and expensive. Therefore, in the present study HepG2 cells were used to examine the induction of DNA-migration and micronuclei after exposure to CDW extracts from D

lake and the Y river. HepG2 cells are of human origin, similar to human hepatocytes and possess most of the (inducible) enzymes necessary for xenobiotic metabolism. It has been shown that HepG2 cells provide a useful model for the detection of mutagens and combined effects in mutagenesis (reviewed by Knasmüller et al., this issue; Mersch-Sundermann et al., this issue).

2. Methods 2.1. Chemicals and media Dulbecco’s minimal essential medium (DMEM), fetal calf serum, and antibiotics (penicillin, streptomycin) were obtained from Gibco (USA). Cytochalasin B and benzo(a)pyrene (Bap) were purchased from Sigma (USA). Dimethyl sulfoxide (DMSO) was from Sigma (Germany) and XAD-7 resin was from Merck (Germany). 2.2. Water sampling and preparations Chlorinated tap water was supplied from water treatment plants of the Y river and D lake, Wuhan, China. Samples of 200–300 l each were collected in March and August. The water samples were transported to the laboratory immediately, acidified (pH 2) with HCl, and extracted using columns (1.5 × 18) filled with Amberlite resins XAD-7. The XAD extracts were eluted with 60 ml acetic acid and then concentrated by evaporation using a 60 ◦ C water bath. The dried extracts were dissolved in DMSO and diluted to concentrations corresponding to 40 l of CDW/ml DMSO. The XAD extracts were stored at −20 ◦ C until testing. 2.3. Cells and cell culture HepG2 cells were kindly provided by Dr. Firouz Darroudi, University of Leiden, The Netherlands. The human fetal hepatocytes cell line L-02 was purchased from the China Center for Type Culture Collection of Wuhan University. The cells were grown in DMEM supplemented with 15% fetal calf serum and antibiotics at 37 ◦ C and 5% CO2 .

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2.4. Micronucleus assay The in vitro micronucleus assay was carried out according to Natarajan and Darroudi (1991). The HepG2 cells were treated with the CDW extracts at different concentrations corresponding to 0.167, 1.67, 16.7 and 167 ml CDW/ml culture medium. After 24 h incubation (37 ◦ C, 5% CO2 ), the medium containing CDW extracts was replaced by fresh medium containing cytochalasin B (final concentration: 3 ␮g/ml). Twenty-four hours later, the HepG2 cells were harvested by trypsinization, treated with ice cold 0.56% KCl, pelleted by centrifugation (10 min, 800 rpm), resuspended in 0.56% KCl, spread on a slide, dried by air and stained with 2% Giemsa. The experiments were repeated independently three times (n = 3). The cells were observed under the light microscope. Micronuclei (MN) in 1000 binucleated cells (BNCs) with well-preserved cytoplasm were scored for each treatment according to established criteria (Kirsch-Volders et al., 2000). To provide data regarding proliferation kinetics, nucleus dividing index (NDI) of each culture was also calculated at the time of MN scoring according to the formula NDI = (M1 + 2(M2) + 3(M3) + 4(M4))/N, where M1 to M4 represent the number of cells with one to four nuclei and N is the total number of cells scored (Eastmond and Tucker, 1989). Student’s t-test was used to calculate statistical significance. 2.5. Single cell gel electrophoreses (SCGE, Comet assay) The Comet assay was performed according to the method of Singh et al. (1988) with minor modifications. The HepG2 cells were treated with the CDW extracts at different concentrations corresponding to 0.167, 1.67, 16.7 and 167 ml CDW/ml culture medium. L-02 cells were treated only with CDW extracts from D lake (August sample) to compare the sensitivity of the cell line with that of HepG2 cells. After 24 h incubation (37 ◦ C, 5% CO2 ), cells were collected by trypsination. About 100 ␮l of cell suspension in 0.75% low melting agarose (LMPA) was spread onto microscope slides precoated with 1 and 0.5% normal melting agarose (NMPA). Then, the cells were lysed for 1 h at 4 ◦ C in a lysis solution containing 2.5 M NaCl, 100 mM EDTA, 10 mM Tris pH 10,

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1% Triton X-100, and 10% DMSO. After that, the slides were placed in an electrophoresis unit and the DNA was allowed to unwind in fresh alkaline buffer (300 mM NaOH, 1 mM EDTA, pH 13) for 20 min at room temperature and subjected to electrophoresis in the same buffer for 25 min at 25 V and 300 mA. After electrophoresis, the gels were neutralized by rinsing three times with buffer (400 mM Tris pH 7.5), the slides were stained with ethidium bromide (20 ␮g/ml) and covered with cover slips. The experiments were repeated independently three times (n = 3). To prevent additional DNA damage, all the steps described above were conducted under dim light or in the dark. The comets were scored visually from 100 randomly chosen cells by using fluorescence microscope (Olympus, Japan) equipped with an excitation filter of 515–560 nm and a barrier filter of 590 nm. As an indicator of DNA migration, tail length (distance between the edge of the comet head and the end of tail) was analyzed with ocular-micrometer. Based on the approximate percentage of DNA in the tail, DNA damage was classified in five categories according to Anderson et al. (1994): grade 0, no change (<5%); grade 1, low level damage (5–20%); grade 2, medium level damage (21–40%); grade 3, high level damage (41–95%); grade 4, total damage (>95%). The relation of cells with different degree of DNA damage was calculated. Student’s t-test was used to calculate significance.

3. Results Fig. 1 shows the result of the micronucleus assays with HepG2 cells treated with XAD extracts of CDW collected from different water sources (D lake and Y river) in March (M) and August (A). Significant increases of the MN frequency were observed for sample DA at concentrations of 16.7 and 167 ml CDW/ml (P < 0.01). Additionally, an MN increase was seen for samples DM, YM and YA at concentrations of 167 ml CDW/ml medium (P < 0.05) compared to solvent control DMSO. No effect of CDW on NDI was observed, the NDI remained around 1.8 for all CDW concentrations tested. Table 1 shows the mean DNA migration (tail length) and standard deviation in HepG2 and L-02 cells using the Comet assay. All CDW extracts led to an increase of DNA-migration in a dose-dependent manner in

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Fig. 1. Induction of micronuclei in HepG2 using XAD extracts of 0.167–167 ml chlorinated drinking water (CDW)/ml culture medium from Dong (D) lake and the Yangtze (Y) river [DA: D lake (August), DM: D lake (March); YA: Y river (August); YM: Y river (March); DMSO: dimethyl sulfoxide (solvent control), Bap (16 ␮M): benzo(a)pyrene (positive control); data represent the mean value ± standard deviation (n = 3); (*) P < 0.05 compared to DMSO; (**) P < 0.01 compared to DMSO].

both HepG2 and L-02 cells. In HepG2 cells, the lowest concentrations resulting in a significant increase of DNA-migration was 1.67 ml CDW/ml for DA and YA (the August samples). For the March samples

DM and YM, the lowest effective concentration was 16.7 ml CDW/ml and 167 ml CDW/ml for DM and YM, respectively. At the highest concentrations of 167 ml CDW/ml, the August (A) samples exhibited a

Table 1 Effect of CDW treatment on DNA tail length (␮m) in HepG2 and L-02 cells using the alkaline Comet assay Treatment

Concentrations (ml CDW/ml medium) 0.167

HepG2 cells DA DM YA YM DMSO Bap (25 ␮M)

3.08 ± 0.71 8.22 ± 0.65b

L-02 cells DA DMSO Bap (25 ␮M)

2.28 ± 0.25 6.56 ± 0.92b

4.28 4.32 4.41 3.69

± ± ± ±

1.67 0.28 1.16 0.70 0.30

2.58 ± 0.20

5.36 5.11 5.17 4.68

16.7 ± ± ± ±

0.17a 1.22 0.36a 1.01

3.20 ± 0.72

6.33 5.57 5.65 4.74

167 ± ± ± ±

0.60b 1.00a 0.09a 0.78

3.95 ± 0.42b

8.42 6.62 6.65 5.05

± ± ± ±

0.50bc 0.47b 0.2bc 0.61a

5.33 ± 0.17b

CDW: chlorinated drinking water; DA: Dong lake (August), DM: Dong lake (March); YA: Yangtze river (August); YM: Yangtze river (March); DMSO: dimethyl sulfoxide (solvent control), Bap (25 ␮M): positive control; data represent the mean value ± standard deviation (n = 3); (a) P < 0.05 compared to DMSO; (b) P < 0.01 compared to DMSO; (c) P < 0.05 compared DA to DM and compared YA to YM.

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Fig. 2. Increase of DNA damage (in %) indicated by tail length using the alkaline Comet assay with HepG2 and L-02 cells treated with 0.167–167 ml CDW of DA [D lake (August)].

significant increase of DNA-migration in comparison to the March (M) samples (P < 0.05). In L-02 cells, the lowest concentration resulting in significant DNA migration was 16.7 ml/ml for sample DA (Table 1). Fig. 2 compares the increases of DNA-migration caused by DA in HepG2 and L-02 cells. Whereas in HepG2 cells concentrations of 0.167, 1.67, 16.7 and 167 ml CDW/ml led to an increase of DNA-migration of 39, 74, 105.5 and 173.4%, respectively, the in-

crease in L-02 cells was only 13.06, 40.32, 72.96 and 133.61%, respectively. Fig. 3 shows the distribution of the different classes of DNA damage in HepG2 cells treated with CDW at the highest concentration of 167 ml/ml medium. It could be seen that the August samples DA and YA caused a higher percentage of grade 2 and grade 3 DNA damaged cells than the March samples DM and YM. In contrast, the percentage of grade 0 DNA

Fig. 3. Distribution of cells with different grades of DNA damage caused by 167 ml CDW/ml medium in HepG2 cells using the alkaline Comet assay [G0: grade 0, no change (<5%); G1: grade 1, low level damage (5–20%); G2: grade 2, medium level damage (20–40%); G3: grade 3, high level damage (40–95%); DA: Dong lake (August), DM: Dong lake (March); YA: Yangtze river (August); YM: Yangtze river (March); DMSO: dimethyl sulfoxide (solvent control), Bap (25 ␮M): benzo(a)pyrene (positive control); mean values of n = 3 experiments].

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damage caused by CDW from DA and YA was lower than that caused by DM and YM. No grade 4 was observed in any of the experiments.

4. Discussion In the present study we investigated the DNA damaging effects of XAD-7 extracts of CDW from Dong (D) lake and the Yangtze (Y) river in human HepG2 hepatoma cells and L-02 hepatocytes using the micronucleus assay and the alkaline Comet assay, a simple, sensitive, and reliable method for detecting DNA single and double strand breaks and alkali labile sites at a single cell level. The results demonstrated that drinking water produced from polluted raw water using chlorination caused a significant and dose-dependent increase of DNA damage in human cell lines. DNA damage caused by CDW varied depending not only on the sampling time (season) and sampling site but also on the cell lines employed. The results indicated that CDW collected during summertime (August) caused significant higher DNA damage than those collected during the cold season (March). This finding is in agreement with studies published by Shu et al. (2003) who found that organic frame-shift mutagens are the predominant genotoxicants in CDW. According to this study, the order of increase in frame-shift mutagenicity was summer > spring > winter, and higher water temperature could raise mutagenic activities of CDW. DNA damage plays an important role in the development of diseases like hereditary deformities, degenerative diseases and cancer. Many investigators have assayed DNA damage caused by environmental factors under in vitro conditions using various metabolically incompetent indicator organisms, e.g. bacteria, yeasts and mammalian cells. However, most environmental genotoxicants have to be converted endogenously to reactive metabolites by xenobiotic metabolising enzymes. Since most of the commonly used indicator organisms are lacking these enzymes (Shimada and Okuda, 1988) exogenous metabolizing systems containing rat liver microsomal enzymes are usually added to insert mammalian metabolism. Unfortunately, in many case, reactive metabolites (ultimate genotoxicants) are not formed inside the

indicator cells so that their availability at the target molecules (DNA) is questionable. Another approach to monitor DNA damaging properties of chemicals is the use of primary human or animal cells. However, the availability of animals and tissue resections is limited, primary cells undergo only a limited number of cell divisions, and enzyme activities are usually lost during cultivation. To avoid the problems mentioned above we introduced the human HepG2 cell line which expresses many xenobiotic metabolizing enzymes. HepG2 cells are widely used in in vitro studies dealing with the genotoxicity of individual compounds and recently with the identification of synergistic effects in mutagenesis (Natarajan and Darroudi, 1991; Knasmüller et al., 1998; Mersch-Sundermann et al., 2001; Lu et al., 2002). Additionally, the study of DNA damage at the chromosome level is an essential part of genetic toxicology because chromosomal mutation is an important event in carcinogenesis. The mammalian cytokinesis-block micronuclei (MN) assay is a well known cytogenetic technique to quantify DNA damage induced by chemical compounds and complex mixtures (Fenech, 2000). Therefore, in our study we investigated also the micronucleus induction caused by CDW in HepG2 cells. Even though all CDW samples exhibited a dose-dependent genotoxicity, the samples collected in March led to significant higher induction of micronuclei than the August samples. These results are in contrast to the Comet assay results and the outcome of studies dealing with mutagenicity assays indicating higher genotoxicity during summertime (Shu et al., 2002). At the moment we have no explanation for these differences, even considering the compounds present or expected in CDW. Even though the number of CDW samples in this study was low, the results indicated that HepG2 is a useful tool for the detection of genotoxicity of environmental mixtures.

Acknowledgements We thank Firouz Darroudi, Leiden University Medical Center, for providing HepG2 cells. This work was funded by National Natural Science Foundation of China (30170794).

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