Application of human cell transformation assay on assessment of carcinogenic potential of river organic pollutants

Abstract

Approaches for assessing the carcinogenic potential of complex mixtures of environmental pollutants are still under development. In this study, a human cell transformation assay was used to evaluate the carcinogenic potential of organic extracts of water pollutants collected from the Jialu River (S1), one of the main tributaries of the Huaihe River in Henan province, China. The incidence of digestive cancers in the basin has risen dramatically within the past three decades. In addition, we collected water samples from the local wells in two liver cancer patients’ homes. The distance of these two wells is 1 km (S2) and 20 km (S3) from the site of S1. Organic chemicals were extracted using hydrophilic–lipophilic balance solid phase cartridges and the fraction was dissolved in DMSO. Human hepatic immortal cells (HL-7702) were treated with each extract and the cytotoxicity was measured. The cells were treated with each extract and the efficiency of cell transformation was examined periodically. The subcutaneous injection of treated cells in immune-deficient mice was performed to confirm the malignant cell transformation. The latency of malignant transformation for samples S1, S2, and S3 was 14, 14, and 16 weeks, respectively, at lowest concentrations of 1.0, 0.5, and 2.0 microlitres (μL) of extract per millilitre medium (mL), enriched from 10, 50, and 200 mL source water, respectively. Moreover, we analyzed the organic extracts for 16 polycyclic aromatic hydrocarbons (PAHs) using GC-MS analysis and found 13 components appearing in all water samples. Our study indicates that human cell transformation assays can potentially be used for assessing the carcinogenic potential of mixtures of environmental pollutants.

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1. Introduction

Current methods for testing toxicity are mostly conducted using laboratory animals. The prediction of human health effects from animal results, which involves a number of assumptions and extrapolations, remains controversial.¹ The development of alternative methods that are rapid, less expensive, and more relevant to human exposure is extremely urgent in order to meet the requirement of testing huge numbers of emerging chemicals. In addition, there is an increasing demand to address the issue of cumulative exposure to complex pollutant mixtures.

Recent epidemiological studies have revealed an association between water pollution and the high incidence of human digestive cancers in the Huaihe River basin, China, where “cancer villages” were reported.^2,3 The mortality rates for stomach, esophageal, liver, and colorectal cancer in the studied areas were 2–6 times higher than average.² Organic chemical pollutants including PAHs have been detected in the Huaihe River, China.^4–6 The surface water pollution originating from industrial, agricultural, and domestic contamination undoubtedly reduces the groundwater quality.⁷ For a long time, groundwater has been the main source of water for residents living in the Huaihe river basin. However, toxicity assays that assess the health risks of complex mixtures of environmental pollutants have not been established. Thus, it is urgent to develop assays that fulfill the need to quickly screen the carcinogenic potential of organic water pollutants.

Cell-based bioassays that predict health-related biological endpoints may therefore be in keeping with the criteria for risk assessment. The in vitro cell transformation assay (CTA) has been recommended as an alternative method for rodent carcinogenicity testing⁸ due to its accuracy and expeditiousness in the prediction of chemical carcinogens.⁹

In our previous work, we developed in vitro models for human cell transformation and demonstrated their efficiency in detecting both genotoxic and non-genotoxic carcinogens.¹⁰ We established a series of human bronchial epithelial (HBE) cells by expressing oncogene H-Ras (HBER) or c-Myc (HBEM),¹⁰ or by introducing defects in DNA repair genes including excision repair cross-completion 1 (ERCC1), excision repair cross-completion 2 (ERCC2), ataxia telangiectasia mutated (ATM) and mutS homolog 2 (MSH2).¹¹ We demonstrated that HBER and HBEM cells were more sensitive to carcinogen-induced cell transformation with a shortened latency of induction. Thus, we speculate that human cell transformation models are feasible for assessing the carcinogenic potential of water pollutant mixtures.

In this study, organic pollutants were extracted from the surface water of the Jialu River and well water in the basin. The carcinogenic potential was evaluated using an in vitro human cell transformation assay. In addition, thirteen USEPA priority-controlled polycyclic aromatic hydrocarbons (PAHs) were also detected in all water samples, implying a relevance to the carcinogenicity.

2. Materials and methods

2a. Chemicals and reagents

Cell culture medium and glutamine were obtained from GIBCO BRL-Life Technologies (Grand Island, NY). Cytochalasin B was purchased from Sigma-Aldrich (St. Louis, MO). Sixteen PAH (specified on US EPA Method 610 (ref. 12)) standards, including acenaphthene (Acp), acenaphthylene (AcPy), anthracene (Ant), benz[a]anthracene (BaA), benzo[a]pyrene (BaP), chrysene (Chr), dibenz[a,h]anthracene (Daa), fluorene (Flu), fluoranthene (Flua), naphthalene (Nap), phenanthrene (Phe), pyrene (Pyr), benzo[b]fluoranthene (BbF), benzo[k]fluoranthene (BkF), benzo[g,h,i]perylene (Bgp), and indeno[1,2,3-c,d]pyrene (Ind) were purchased from Accu Standard Inc. (Akron, Ohio).

2b. Sample collection and preparation

Water samples from three locations were collected in March 2010, when there was no rainfall at least one week before sampling and during the sampling periods. The organic pollutants were extracted according to the method developed by Ma et al.⁷ The water samples were collected in amber glass bottles and kept on ice while being transported to the laboratory. Then, the water samples were passed through glass fiber filters (GF/C, 1.2 μm, Whatman, Maidstone, UK) using a vacuum system. The pH of the water samples was adjusted to 3.0 with hydrochloric acid. The water samples were concentrated using Oasis HLB cartridges (Waters, USA) at a flow rate of 5–10 mL min⁻¹. The HLB cartridges were pre-conditioned with 10 mL of hexane, 20 mL of acetone, 10 mL of methanol, and 10 mL of ultrapure water consecutively. After extraction, the cartridges were dried under a flow of nitrogen gas. Subsequently, the cartridges were eluted with 20 mL of acetone and dried by nitrogen blowing. The residues were dissolved in DMSO. An experimental blank was prepared by extracting ultrapure water using the same method as described above.

In particular, samples used for the analysis of PAHs were dissolved in 500 μL hexane instead of DMSO. Then, the solvents were subjected to purification using C18 cartridges (Waters, USA) that were pre-conditioned with 25 mL of hexane and eluted with 5 mL hexane, 5 mL of a mixture of hexane and dichloromethane (1 : 1 in volume), and 5 mL of a mixture of hexane and dichloromethane (1 : 4 in volume). The purified extracts were further concentrated to 100 μL and spiked with known amounts of the internal standards prior to instrumental analysis.

2c. Cell lines and cell treatment

Human hepatic immortal cell line HL-7702 was obtained from Shanghai Cell Biology Institute, Chinese Academy of Sciences. Cells were cultivated in RPMI-1640 medium with 10% (v/v) heat-inactivated fetal calf serum at 37 °C in an atmosphere containing 5% CO₂.

For analysis of carcinogenic potential, the cells were treated at least 4 times with organic extracts. After 24-hour exposure, the medium was discarded and the cells were washed twice with PBS and refilled with 2 mL of fresh complete medium for continuous cultivation. Cells were subcultured at 90% confluence, followed by the next round of treatment. It took one week for one round of treatment, and after the 4^th round of treatment cells were subjected to a soft agar assay. Cells which formed colonies in soft agar and grew into tumors in nude mice were considered to be malignantly transformed, and the organic extract treatment was stopped. Treatment of the other groups continued until the 20^th week.

2d. Soft agar assay

Anchorage-independent cell growth was detected using a soft agar assay. 1 × 10⁵ cells per well were seeded in six-well culture plates and treated with water extracts when the cells reached 60% confluence. After 24 h, the medium was discarded, and fresh complete medium was added. Cells were subcultured at 90% confluence, and another round of treatment followed. After the 4^th round of treatment, the cells were trypsinized every two weeks and subjected to a soft agar assay.

The soft agar assay was performed as previously described.¹¹ 6 × 10⁴ HL-7702 cells were suspended in DMEM with 10% FBS in 0.4% agar above a layer of 0.6% agar base. The transformed HBERST cells expressing SV40 large T (LT), the telomerase catalytic subunit (hTERT) and an oncogenic allele H-Ras were used as positive controls. The culture plates were examined 5 days later for signs of cell growth using phase-contrast microscopy. Colonies with a diameter of greater than 100 μm were counted 4 weeks after treatment.

2e. Tumorigenesis study

The BALB nu/nu mice were purchased from the Experimental Animal Center of Guangzhou University of Traditional Chinese Medicine (Guangzhou, P.R. China). A two hundred microliter cell suspension containing 1 × 10⁷ cells was injected subcutaneously into the nude mice, with transformed HBERST cells and DMSO-treated HL-7702 cells as positive controls and negative controls, respectively. Tumors were observed as early as the 5^th day after subcutaneous injection. After 5 weeks, the mice were sacrificed for examination. All the procedures herein were approved by the Animal Care and Use Committee of Sun Yat-sen University.

2f. Cytokinesis-blocked micronucleus (CBMN) assay

HL-7702 cells were seeded on the six-well cell culture plates at a density of 2.5 × 10⁵ cells per well. After 24 hours, the medium was removed and the cells were exposed to 4 doses of extracts and 1/3 IC₅₀ was used as the highest treatment concentration. Mitomycin C (MMC, 0.1 μg mL⁻¹) was used as the positive control and DMSO (0.1%, v/v) as the vehicle control. After treatment, the cells were washed twice with PBS and cytochalasin B (3 μg mL⁻¹) was added. After 36 hours of incubation at 37 °C, the cells were then trypsinized and the slides were coded according to the standard protocol proposed by Fenech,¹³ with minor modifications. The formation of CBMN was viewed and scored under a microscope at 1000× magnification. For each group, 1000 cytokinesis-blocked (CB) cells were examined for the presence of micronuclei, following the criteria described by Fenech.¹⁴ All experiments were performed in triplicate.

2g. Detection of PAHs in water sample extracts

Water sample extracts were probed for sixteen PAHs using a GC-MS (QP2010, SHIMADZU) in electron impact ionization mode (70 eV). Separation was carried out under the following conditions: helium was the carrier gas at a constant flow rate of 1.0 mL min⁻¹ and the temperatures of the injector and ion source were 280 and 200 °C, respectively, using a DB-5 (30 m × 0.25 mm, ID 0.25 μm) capillary column. A 1.0 μL sample was injected in splitless mode. The initial temperature was 50 °C and this was maintained for 3 min, after which it was increased to 300 °C at a rate of 10 °C min⁻¹ and then maintained for 5 min. Selective ion monitoring (SIM) was used to analyze each PAH component according to the corresponding m/z. The GC peaks were identified by accurate assignment of retention time for each standard. For quantification purposes, calibration curves based on a set of six concentration standards (5, 10, 25, 50, 100, and 250 μg L⁻¹) were drawn. The instrumental detection limit was 1 μg L⁻¹.

2h. Statistical analysis

Data are presented as the mean ± standard deviation (SD) for at least three independent experiments. Statistical analyses were performed using SPSS 13.0 (SPSS, Chicago, IL). The differences among different groups were analyzed using one-way analysis of variance. P < 0.05 was considered statistically significant. Specific post-hoc comparisons were used to examine differences among groups.

3. Results

3a. Selection of sampling sites and preparation of organic extracts

The Jialu River is one of the branches of the Huaihe River. It has been contaminated by the direct discharge of industrial and domestic waste water and farmland and livestock contaminants for over 30 years. There are about 1.38 million residents living along the river.^5,15 The study region and sampling sites were selected as shown in Fig. 1. In this region, shallow groundwater is the main source of drinking water consumed by the local residents. The sample S1 was the organic extract from the surface water of the Jialu River. The other two samples S2 and S3 were taken from shallow groundwater sources, which were 1 km (S2, about 10 m in depth) and 20 km (S3, about 10 m in depth) away from the Jialu River. The organic pollutants were extracted as described above and were finally dissolved in a defined volume of DMSO. Specifically, one microliter of DMSO extract was equivalent to 10 mL of original river water, 100 mL of groundwater, and 100 mL of ultrapure water (control), respectively.

Fig. 1 The study area is one of the towns in Henan province (b), located in the middle of China (a). S1 was the surface water collection site at the Jialu River. S2 and S3 were adjacent groundwater sites, which were 1 km and 20 km away from S1 (c). In this district, groundwater serves as the source of drinking water.

3b. Hepatic cell transformation induced by organic extracts of water samples

To investigate the carcinogenic potential of the organic mixtures, an in vitro cell transformation assay was performed using HL-7702 cells. Cell viability was determined by a trypan blue exclusion assay. The highest concentration of each sample used in this experiment has no obvious cytotoxicity (<5%). Three doses for each sample were chosen at 0.11, 0.33 and 1.00 μL mL⁻¹ medium for S1, 0.06, 0.17 and 0.50 μL mL⁻¹ medium for S2 and 0.22, 0.67 and 2.00 μL mL⁻¹ medium for S3. Treatment of 0.1% (v/v) DMSO served as a negative control. HL-7702 cells were treated weekly with organic extract at the concentration described above. After four weeks’ treatment, we examined the anchorage-independent cell growth by a soft agar assay. 14 weeks after treatment, we observed transformed phenotypes in HL-7702 cells at doses of 1.0 μL mL⁻¹ and 0.5 μL mL⁻¹ (Fig. 2) for samples S1 and S2, respectively. 16 weeks after treatment, we found that sample S3 at a dose of 2.0 μL mL⁻¹ showed a phenotype resulting from malignant transformation (Fig. 2). In contrast, we did not observe a cell transformation phenotype in DMSO-treated control cells even after 20 weeks. The transformed cells displayed significant morphological changes including cell enlargement, rotation and overlapping growth, while the non-transformed cells remained long fusiform-shaped and grew in a monolayer fashion (Fig. 3). The malignant cell transformation was confirmed by subcutaneous injection of treated cells in immunodeficient mice. Collectively, although all water samples could induce malignant cell transformation, the concentrations at which malignant cell transformation began to show varied greatly, and this may be relevant to the carcinogenic potential of the mixtures.

Fig. 2 Number of cell colonies in the anchorage-independent growth assay. HL-7702 cells were treated with the extracts of water samples from (a) S1, (b) S2, and (c) S3 at the indicated concentrations, expressed as μL of extract per mL medium. The data were expressed as mean ± SD from three independent experiments. *P < 0.05 is considered as a statistically significant difference compared to the negative control (NC). The indicated highest dose of each group induced 100% tumor formation in nude mice (#).

Fig. 3 The malignant cell transformation induced by river water extracts. The cells were treated with 0.1% DMSO (a, e) or the lowest dose (b, f). A representative image shows colony formation in a soft agar plate 14 weeks after treatment. The cells treated with organic extracts at concentrations of 0.33 (c, g) and 1.00 (d, h) μL mL⁻¹ medium formed obvious colonies.

3c. Genetic toxicity of the organic extracts examined by CBMN assay

To assess the genetic toxicity of organic extracts, we performed a CBMN assay on HL-7702 cells treated with different concentrations of organic extracts. The CBMN method is a simple, sensitive, repeatable and high-accuracy assay to detect genetic damage. Four doses at 0.11, 0.33, 1.00, and 3.00 μL mL⁻¹ medium for S1, 0.06, 0.17, 0.50 and 1.50 μL mL⁻¹ medium for S2 and 0.07, 0.22, 0.67 and 2.00 μL mL⁻¹ medium for S3 were used for administration. DMSO (0.1%, v/v) and MMC (0.1 μg mL⁻¹) served as the negative and positive controls, respectively. After 24 h treatment, a significant increase of micronuclei formation (MNi‰) in cytokinesis-blocked cells (P < 0.05) was observed at 0.33, 0.17 and 0.67 μL mL⁻¹ medium in S1, S2, and S3 (equivalent to 3.3, 16.7 and 66.7 mL of original water per millimeter medium), respectively (Fig. 4). An image of representative micronuclei is shown in Fig. 5. These findings indicate that all water samples displayed DNA damage potential, and the order of genotoxicity of the three different sites (from high to low) was S1 > S2 > S3, which correlates well with the activity of cell transformation.

Fig. 4 Frequencies of MNi in HL-7702 cells treated with extracts of water samples S1 (a), S2 (b) and S3 (c). Treatment of MMC at 0.1 mg mL⁻¹ served as a positive control. The data were expressed as mean ± SD from three independent experiments. *P < 0.05, #P < 0.01 is considered as a statistically significant difference compared to the negative control (0.1% DMSO).

Fig. 5 Micronuclei formed in cells treated with MMC or organic extracts (b), while no micronuclei were observed in normal cells (a). The black arrow indicates micronuclei (1000× magnification).

3d. Detection of PAHs in water samples

To examine whether the organic extracts of the Jialu River and adjacent groundwater were contaminated by polycyclic aromatic hydrocarbons (PAHs), we probed the samples for 16 PAHs listed as priority pollutants for monitoring the quality of water and wastes (USEPA) by GC-MS analysis. As shown in Table 1, 13 out of 16 PAHs appeared in all water samples, among which BaP, BaA, BkF, BbF, and Chr belong to group B2 according to the USEPA IRIS database. The total amounts of PAHs in water samples S1, S2 and S3 were 20.34 ng L⁻¹, 8.87 ng L⁻¹ and 7.75 ng L⁻¹, respectively, which are lower than the safety level of the Standard for Drinking Water Quality (GB5749-2006)¹⁶ executed in China. The BaP concentration in samples S2 and S3 was 0.246 ng L⁻¹ and 0.136 ng L⁻¹, respectively, which is lower than the recommended level (10 ng L⁻¹) according to the Standard GB5749-2006. However, at a low level of exposure, we are unable to exclude the possibility that adverse health effects might develop in residents exposed to combined PAHs.

Table 1 Concentration of PAHs in water samples (ng L⁻¹)^a

Compound	S1	S2	S3
a Concentration levels of PAHs in Jialu River surface water (S1) and adjacent groundwater samples S2 and S3.
Naphthalene (Nap)	4.152	0.709	0.534
Acenaphthylene (AcPy)	0.392	0.098	0.106
Acenaphthylene (Acp)	3.105	1.215	1.160
Fluorene (Flu)	2.470	1.219	1.200
Phenanthrene (Phe)	3.870	2.331	3.632
Anthracene (Ant)	0.476	0.052	0.163
Fluoranthene (Fla)	0.822	0.574	0.730
Pyrene (Pyr)	0.857	0.666	0.662
Benz[a]anthracene (BaA)	0.255	0.122	0.095
Chrysene (Chr)	0.417	0.251	0.245
Benzo[b]fluoranthene (Bbf)	0.145	0.092	0.140
Benzo[k]fluoranthene (Bkf)	0.130	0.075	0.068
Benzo[a]pyrene (BaP)	0.274	0.202	0.111
∑(Polycyclic aromatic hydro-carbons, PAHs)	17.363	7.603	8.848

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4. Discussion

The approach of combining multiple toxicological endpoints with a system-based impact assessment allows us to obtain an insight into time- and dose-dependent molecular perturbations of specific biological pathways.¹⁷ Human cell-based in vitro systems can be applied for testing the biological effects of single toxicants or environmental mixtures, and for the evaluation of environmental quality in time course studies. In this study, organic extracts from the surface water and shallow groundwater of the Jialu River basin were subjected to in vitro bioassays. The results from the human cell transformation assay showed that water extracts from the Jialu River and nearby underground water could transform hepatic HL-7702 cells after 14 or 16 weeks of treatment. Genotoxicity was also observed in a dose-dependent manner. Chemical analysis revealed that 13 priority PAHs were present in all water samples, some of which are human carcinogens. The findings implied a causative role of the organic pollutants in the development of digestive cancers. This study indicated that an in vitro cellular assay can effectively predict the carcinogenic potential of environmental mixtures.

During the past three decades, the Huaihe River has become seriously polluted. The term “cancer villages” has been conferred to the areas with a high incidence of digestive tract tumor.^2,3 Previous studies indicated that organic pollutants such as PAHs were the major contaminants attributable to the adverse health outcome.⁶ In this study, we revealed the presence of 13 PAHs in the complex extract mixtures, albeit at levels lower than the permissible limit adopted by the Standard GB5749-2006.¹⁶ Drinking water pollution exposes people to complex contaminant mixtures, and the combined adverse effects of pollutant mixtures should be taken into consideration in risk assessment. It has been reported that although the concentration of pollutants in the aquatic environment is very low, complex interactions such as synergistic or antagonistic effects may occur.^18,19 Proper models that evaluate the joint effects of environmental mixtures at low levels would be helpful for addressing this issue using an integrated biological and informatics approach.^18,19 Herein, the potential of an in vitro cell transformation assay for risk assessment of pollutant mixture exposure has been shown.

The in vitro cell transformation assay has been proposed as an alternative method for the 2-year rodent cancer bioassay for screening of potential chemical carcinogens. A recent report showed that this method could effectively detect 90% and 95% of the chemicals or compounds listed as group 1 and group 2 carcinogens (IARC), respectively.⁹ Three rodent cell transformation models, SHE, BALB/c 3T3 and C3H10T1/2 have been widely used, and have good accordance with rodent bioassay data. However, these animal cell-based CTAs have limitations such as difficulties in species extrapolation, while human cell-based CTAs are recommended for the prediction of human carcinogenic potential.⁹

Recently, the U.S. National Research Council (NRC) launched a new risk assessment approach based on the “toxicity pathways” obtained through well-designed in vitro human cell assays.²⁰ The mechanism underlying a specific toxic effect could be studied using human cells or cell lines, and key pathways could be obtained and used as cell state biomarkers.¹ Our previous studies have indicated that human CTAs are feasible and effective in testing the carcinogenic potential of chemicals.¹⁰ Using these chemical carcinogen-transformed human cell models, we identified critical regulatory pathways and epigenetic mechanisms involved in malignant cell transformation.^21,22 In this study, we demonstrate that a human CTA assay is able to evaluate the potent carcinogenicity of environmental mixtures. Therefore, in vitro cell transformation could be developed as an alternative method for the assessment of carcinogenicity.

5. Conclusion

In this study, we used an in vitro cell transformation assay to study the carcinogenic potential of organic extracts from polluted water samples. The genotoxic effects of 13 PAH components are associated with the phenotype of human cell transformation. The human cell-based transformation assay exhibits advantages in high-speed, high-sensitivity and accurate prediction of the carcinogenic potential of single pollutants or mixture pollutants. It can be applied for dynamic monitoring of water quality, in particular in a situation where unknown carcinogens are appearing in a water environment.

Acknowledgements

This work was supported by the National High Technology Research and Development Key Program of China (2008AA062504), the National Key Basic Research and Development Program (2010CB912803, 2012CB525003), the National Technology R&D Program in the 12th Five-year Plan of China (2014BAI12B02), the National Nature Science Foundation of China (NSFC) (81072284), the Natural Science Foundation of Guangdong Province (S2012040007713), and the Guangdong Province Universities and Colleges Pearl River Scholar Funded Scheme GDUPS (2010).

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Footnote

† These authors contributed equally to this work.

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