http://informahealthcare.com/txm ISSN: 1537-6516 (print), 1537-6524 (electronic) Toxicol Mech Methods, Early Online: 1–7 ! 2015 Informa Healthcare USA, Inc. DOI: 10.3109/15376516.2014.1003357

RESEARCH ARTICLE

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The development of GADD45 luciferase reporter assays in human cells for assessing the genotoxicity of environmental pollutants Lili Xin1*, Jianshu Wang2*, Yanhu Wu1, and Sifan Guo1 1

School of Public Health, Medical College of Soochow University, Suzhou, Jiangsu, China and 2Suzhou Center for Disease Prevention and Control, Suzhou, Jiangsu, China Abstract

Keywords

Objectives: In order to assess the potential carcinogenic and genotoxic responses induced by environmental pollutants, genotoxicity test systems based on a GADD45 promoter-driven luciferase reporter in human A549 and HepG2 cells were established. Materials and methods: Four different types of environmental toxicants including DNA alkylating agents, precarcinogenic agents, DNA cross-linking agents and non-carcinogenic agents, and three environmental samples collected from a coke oven plant were used to evaluate the test systems. After treated with the tested agents and environmental samples for 12 h, the cell viabilities and luciferase activities of the luciferase reporter cells were determined, respectively. Results: Methyl methanesulfonate, benzo[a]pyrene, formaldehyde and the extractable organic matter (EOM) from coke oven emissions in ambient air generally produced significant induction of relative luciferase activity in a similar dose-dependent manner in A549- and HepG2-luciferase cells. No significant increases in relative luciferase activity were observed in pyrene-treated A549- or HepG2-luciferase cells. Significant increase in relative luciferase activity was already evident after 2.5 mM benzo[a]pyrene, 5 mM formaldehyde, 0.006 mg/L bottom-EOM, 0.10 mg/L side-EOM or 0.06 mg/L top-EOM, where no cytotoxic damage was observed. Compared with the A549-luciferase cells, the tested pollutants produced higher induction of relative luciferase activity in HepG2-luciferase cells. Discussion and conclusion: Therefore, the new genotoxicity test systems can detect different types of genotoxic agents and low concentrations of environmental samples. The luciferase reporter cells, especially the HepG2-luciferase cells, could provide a valuable tool for rapid screening of the genotoxic damage of environmental pollutants and their complex mixtures.

Environmental complex mixtures, extractable organic matter, GADD45 , genotoxicity assessment, luciferase reporter genes

Introduction Increasing concentrations of various environmental chemicals or their combinations are becoming crucial public health problems because of raising rates of cancer and environmentally related diseases. An important aspect of the toxicity of environmental pollutants is their ability to act as carcinogens, either by damaging DNA directly or indirectly by acting through pathways involving activated oxygen species or other reactive agents that can damage DNA. Therefore, the toxicity testing, especially the genotoxicity assessment has become the cornerstone of chemical hazard assessment. To evaluate the genotoxicity, a number of in vivo or in vitro tests including the comet assay and micronucleus test have been

*These authors contributed equally to this work. Address for correspondence: Lili Xin, School of Public Health, Medical College of Soochow University, 199 Renai Road, Suzhou 215123, Jiangsu, China. Tel: 86-512-65880074. Fax: 86-512-65880070. E-mail: [email protected]

History Received 2 October 2014 Revised 17 November 2014 Accepted 14 December 2014 Published online 22 January 2015

developed to screen the potentially mutagenic or carcinogenic agents (Fenech, 2007; Olive & Bana´th, 2006). However, these commonly used assays are time-consuming, laborious and often require expertise for staining and microscopic observation, and consequently are unsuitable for screening of large numbers of environmental pollutants, especially some low concentrations of environmental samples. Hence, a rapid and cost-effective alternative testing system should be explored. As proposed by the US National Research Council in their long-range vision for toxicity testing and risk assessment, more emphasis should be put on developing new approaches based on the molecular modes of action of environmental pollutants (NRC, 2007). In particular, cellular assays using a reporter gene driven by stress-inducible gene promoters have been suggested as valuable tools in toxicity assessment (Simmons et al., 2009). The genotoxic stress, resulting in DNA damage, can cause DNA damage response (DDR) including cell cycle arrest, DNA damage repair and apoptosis via complicated signaltransduction cascades (Lord & Ashworth, 2012). Among the

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various stressful genes involved in DDR, the p53 and BRCA-1 regulated growth arrest, and DNA damage gene a (GADD45 ) is one of the important proteins that participate in cellular response to a wide range of DNA damage agents including the methyl methanesulfonate (MMS) and the carcinogenic benzo[a]pyrene (Akerman et al., 2004; Hollander et al., 1993; Liebermann & Hoffman, 2008; Rosemary Siafakas & Richardson, 2009; Salvador et al., 2013). The expression of GADD45 is reported to be regulated by both p53-dependent and -independent pathways, depending on the types of genotoxic stress (Rosemary Siafakas & Richardson, 2009). Under normal physiological conditions, the half-life of GADD45 gene is very short, usually less than an hour, and the basal level of GADD45a protein is also very low (Sharova et al., 2009). After exposure to various genotoxic stresses, the GADD45 gene will be highly induced by interacting with the activated p53, BRCA1 or NF-kB proteins (Salvador et al., 2013; Tamura et al., 2012; Zhan, 2005; Zhan et al., 1994). The GADD45a protein can also interact with multiple cellular proteins, including p21, core histone protein, Cdc2 protein kinase, indicating that GADD45a may play crucial roles in DDR. Besides, the importance of GADD45a in maintaining the genomic integrity was also reported (Zhan, 2005). Therefore, GADD45 was proposed to be an important stress sensor in response to DNA damage due to its stress responsiveness (Liebermann & Hoffman, 2008; Rosemary Siafakas & Richardson, 2009; Salvador et al., 2013). In this study, two GADD45 promoter-driven luciferase reporter assays based on the human A549 and HepG2 cell lines that express wild-type p53 were developed and validated by a panel of chemical toxicants and environment samples. After treated with MMS, benzo[a]pyrene, formaldehyde, pyrene and extractable organic matters (EOMs) derived from coke oven emissions (COEs) for 12 h, the cell viability and GADD45 promoter activation indicated by luciferase activity were determined by MTT assay and luciferase reporter gene assay, respectively.

Materials and methods Chemicals and materials MMS (99% purity), benzo[a]pyrene (98% purity) and pyrene (99% purity) were obtained from Sigma Chemical Co. (St. Louis, MO). Formaldehyde was purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). Dulbecco’s modified Eagle’s minimal essential medium (DMEM), trypsin, newborn calf serum and geneticin-selective antibiotic (G418) were obtained from GIBCO (Grand Island, NY). The luciferase assay system and pGL4.17 [luc2/Neo] vector were purchased from Promega (Madison, WI). The HepG2 cell line was kindly provided by Dr. Darroudi (Leiden University, the Netherlands). Air sampling and preparation of coke oven-EOMs Detailed information on air sampling, EOM extraction and chemical analysis of polycyclic aromatic hydrocarbons (PAHs) in each COE sample is given in Xin et al. (2014). COEs in the ambient air were collected on the bottom, side and top of the coke oven in a state-run coke oven plant located in Central China in November 2010. The collected samples

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were extracted with Dichloromethane. Quantitative chemical analysis of PAHs in each COE sample was performed by HPLC, and the airborne concentrations of the total PAHs were 30, 100 and 300 mg/m3 for the bottom, side and top-coke oven samples, respectively. For the in vitro experiments, EOM samples were evaporated to dryness and dissolved in DMSO, and the stock solution of the bottom, side and topcoke oven EOM samples contained 18.2, 65.4 and 202 mg PAHs/mL DMSO, respectively. Cell culture The A549, HepG2 and the two developed reporter cell lines were cultured in DMEM culture medium supplemented with 10% newborn calf serum, 2 mM glutamine, 100 units/mL penicillin and 100 mg/mL streptomycin. Cultures were maintained in a humidified atmosphere with 5% CO2 at 37  C, and medium was refreshed every two or three days with sub-culturing. Construction of GADD45a promoter-driven luciferase plasmid The human GADD45 promoter was isolated with primer sequences as follows: Forward primer 50 -CTAACCTCG AGTGCTTTCCACCTACAAGTTGCCA-30 ; reverse primer 50 -CTACAAGATCTATTGCAAACTGCAGGTCGCCCA-30 . The sequence of human GADD45 promoter was inserted into the Xho†–BgLO site of pGL4.17 [luc2/Neo] (Promega) luciferase plasmid. The direction and sequence authenticity of the luciferase reporter plasmid were validated by restriction analysis and direct sequencing. Generation of stable GADD45a promoter-driven luciferase reporter cells The luciferase reporter plasmid was transfected into A549 or HepG2 cells by using the LipofectamineÔ 2000 (Invitrogen, Waltham, MA) according to the manufacturer’s instructions. A549 or HepG2 cells were trypsinized and re-suspended in culture medium to a final concentration of 1.5  105 cells/mL. To each well of a white six-well culture plate, 2 mL suspension was added. After incubating at 37  C for 24 h, a mixture containing 1 mg luciferase reporter plasmid and 2.5 mL lipofectamine was added to each well for the transfection reaction. The transfected cells were incubated with culture medium containing 0.9 mg/mL (for A549 transfected cells) or 0.6 mg/mL G418 (for HepG2 transfected cells) for 14 d. Twenty G418-resistant clones were isolated and screened by measuring their inducible luciferase activities in response to MMS. One positive clone for each cell line, which showed low background and high inducible luciferase activities, was chosen as the luciferase reporter cell line in the subsequent experiments. This cell line is now called the A549-luciferase cells or HepG2-luciferase cells. Treatment of luciferase reporter cells with environmental toxicants Prior to each experiment, the A549- or HepG2-luciferase cells were seeded in triplicate at a density of 2.5  104 per well in 48-well culture plates. After 24 h, the culture medium was

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replaced with fresh medium supplemented with vehicle alone (DMSO) or increasing concentrations of MMS (6.25, 12.5, 25 and 50 mg/mL), benzo[a]pyrene (2.5, 5, 10, 20, 40 and 80 mM), formaldehyde (2.5, 5, 10, 20, 40 and 80 mM), pyrene (3.125, 6.25, 12.5, 25, 50 and 100 mM), bottom-EOM (0.006, 0.03, 0.15, 0.7, 3.8 and 18.2 mg/L), side-EOM (0.02, 0.1, 0.2, 2.6, 13.1 and 65.4 mg/L) and Top-EOM (0.06, 0.3, 1.6, 8.1, 40.4 and 202 mg/L) for 12 h. MMS, benzo[a]pyrene, pyrene and the three coke oven EOMs were dissolved in DMSO, and the concentrations of stock solutions were 50 mg/mL, 80 mM, 100 mM, 18.2, 65.4 and 202 mg/L, respectively. The stock solution of formaldehyde was 80 mM. The final concentrations of DMSO used in all experiments did not exceed 0.1%. Luciferase activity in luciferase reporter cells After the treatments, the A549- or HepG2-luciferase cells were washed with 0.5 M phosphate-buffered saline (pH 7.4). Subsequently, 100 mL lysis buffer was added to each well. Plates were shaken at room temperature for 15 min. Then, the cell lysates were collected and centrifuge at 12 000  g for 15 s. Finally, 50 mL of the cell lysates were added to a 96-well plate, which contains 50 mL luciferase assay reagent per well. The luciferase activities were measured using the SYNERGY 2 microplate reader (Bio-Tek, Winooski, VT). The ratio of the luciferase activities (test sample/control group) is the relative luciferase activity (versus control). Each assay was triplicated and repeated at least twice to corroborate the results. Determination of cell viability The number of viable A549- or HepG2-luciferase cells in culture was detected using the MTT assay. The cell viability was determined by measuring the capacity of the A549 or HepG2-luciferase cells to reduce the tetrazolium salt, MTT, to a blue formazan product (Denizot & Lang, 1986). The cells were seeded in 96-well plates under different treatments in DMEM culture medium with 10% newborn calf serum. Then, the cells were incubated with MTT (500 mg/mL) at 37  C for 4 h and lysed with DMSO. Finally, the absorbance was measured at 570 nm using the SYNERGY 2 microplate reader (Bio-Tek). The untreated cells were the 100% viable control. Statistical analysis Three individual experiments were performed for each dose group, and the results represented means ± SD. Statistical analysis was performed by one-way analysis of variance followed by the least significant difference test or Dunnett’s test. In all tests, differences were considered significant at p50.05. All the data analyses were carried out using the statistical analysis software SPSS 12.0 for windows (SPSS Inc., Chicago, IL).

Results Validation of the luciferase reporter cells The luciferase reporter cells were validated by a classic DNA alkylating agent, MMS. As shown in Figure 1, the low dose of MMS (12.5 mg/mL) led to a significant increase in relative luciferase activity that progressively increased to 1.6  the control level at 50 mg/mL in A549-luciferase cells

Figure 1. Cell viabilities and luciferase activities of the luciferase reporter cells exposed to MMS. Cells were treated with different concentrations of MMS for 12 h. Cell viability was evaluated using the MTT assay (a). Relative luciferase activity was measured using the luciferase assay system (b). DMSO (0.1%) was the vehicle for MMS. Data represent mean ± SD of three individual experiments. *p50.05 compared with untreated control. **p50.01 compared with untreated control.

(p50.01; Figure 1b). While, in HepG2-luciferase cells, the MMS produced a strong dose-dependent induction of relative luciferase activity up to 10  the control level at the highest concentration tested (50 mg/mL; Figure 1b). The MTT assay showed that MMS induced a nearly linear decrease in cell viability over the range of doses tested, with viability decreasing to 62% of the control level at the highest doses in A549-luciferase cells (Figure 1a). Only at the highest concentration tested (50 mg/mL), the viability of HepG2luciferase cells was significantly decreased to 77% of the control group (p50.01; Figure 1a). Induction of the luciferase activity by different environmental toxicants As shown in Figure 2, the low dose of benzo[a]pyrene (2.5 mM) led to a significant increase in relative luciferase activity that progressively increased to 1.7  the control level at 40 mM in A549-luciferase cells (p50.05; Figure 2b). The decrease in relative luciferase activity at 80 mM compared with the maximal level at 40 mM may be attributable to the saturation of PAH-activating enzymes. Even at the highest concentration tested, benzo[a]pyrene induced no cytotoxicity in A549-luciferase cells (Figure 2a). While, in HepG2-luciferase cells, benzo[a]pyrene produced a strong dose-dependent increase in relative luciferase activity up to

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Figure 2. Cell viabilities and luciferase activities of the luciferase reporter cells exposed to the three environmental toxicants. Cells were treated with different concentrations of benzo[a]pyrene, formaldehyde and pyrene for 12 h. Cell viability was evaluated using the MTT assay (a, c and e). The luciferase activity in the luciferase reporter cells was measured and expressed as relative luciferase activity versus control (b, d and f). Chemical concentrations are at the bottom. DMSO (0.1%) was the vehicle for benzo[a]pyrene and pyrene. Data represent mean ± SD of three individual experiments. *p50.05 compared with untreated control. **p50.01 compared with untreated control.

3.2-fold of the control level at 80 mM (Figure 2b), where the cell viability declined to 81% of the control level (Figure 2a). Formaldehyde induced a dose-dependent increase in relative luciferase activity up to 1.5  the control level at the highest concentration tested in A549-luciferase cells (Figure 2d). Significant dose-dependent decreases in cell viability were observed across a suitable range of formaldehyde concentration (10–80 mM; p50.01; Figure 2c). In HepG2-luciferase cells, formaldehyde produced a strong dose-dependent increase in relative luciferase activity peaking at 80 mM. At this dose, the relative luciferase activity was greater than 3.4-fold of the control level (Figure 2d). Only at the highest test doses, the cell viability was significantly declined to 91% of the control level (p50.01; Figure 2c). In Figure 2(f), our results showed that no significant increases in relative luciferase activity were observed in pyrene-treated A549- or HepG2-luciferase cells. The MTT assay showed that significant increases in cell viability were observed in A549-luciferse cells across the suitable range of pyrene tested (6.25–25 mM; p50.05; Figure 2e). In HepG2luciferase cells, only the highest tested dose of pyrene induced a significant decrease in cell viability (p50.05; Figure 2e). Induction of the luciferase activity by coke oven-EOMs In A549-luciferase cells, significant increases in relative luciferase activity were induced by 18.2 mg/L bottom-EOM and 13.1 mg/L side-EOM (p50.05; Figure 3b and d). The topEOM induced low, but detectable dose-dependent increases in relative luciferase activity with a maximum level of 1.4  the control at the highest concentration tested (40.4 mg/L) in A549-luciferase cells (Figure 3f). As shown in Figure 3, compared with the control group, significant dose-dependent increases in relative luciferase activity were observed in

HepG2-luciferase cells treated with different concentrations of the three EOMs (all p50.01). The bottom-EOM induced low, but detectable, increases in relative luciferase activity at concentrations of nanogram per liter (Figure 3b). The sideEOM induced significant dose-dependent increases in relative luciferase activity in HepG2-luciferase cells peaking at 65.4 mg/L (p50.05). At this dose, the relative luciferase activity was greater than 2.0  the control level (Figure 3d). The top-EOM produced a strong dose-dependent induction of relative luciferase activity up to 2.8  the control level at the highest concentration tested (Figure 3f). Using the MTT assay, our results showed that significant decreases in cell viability were observed in both A549- and HepG2-luciferase cells treated with 18.2 mg/L bottom-EOM (p50.05; Figure 3a). For the side-EOM, no significant increases in cell viability were observed even at the highest concentration tested (Figure 3c). Only at the highest concentration of Top-EOM tested, cell viability was significantly decreased to 93% of the control in HepG2-luciferase cells (p50.01; Figure 3e).

Discussion In this study, two novel luciferase reporter assays based on the A549 and HepG2 cell lines are presented that allow rapid screening of environmental pollutants for GADD45 promoter activation, which can be used as a sensitive indicator of the genotoxicity. Using a panel of environmental toxicants and complex mixtures, our results showed that the new genotoxicity test system, based on the GADD45 promoter activation, could efficiently detect different types of genotoxic agents and low concentrations of COEs (nanogram per liter). Our luciferase reporter cells have several advantages over some of the established genotoxicity tests, such as

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Figure 3. Cell viabilities and luciferase activities of the luciferase reporter cells exposed to the three COE-EOMs. Cells were treated with different concentrations of bottom-, side- and top-EOMs for 12 h. Cell viability was evaluated using the MTT assay (a, c and e). The luciferase activity in the luciferase reporter cells was measured and expressed as relative luciferase activity versus control (b, d and f). EOM concentrations are at the bottom. DMSO (0.1%) was the vehicle for EOMs. Data represent mean ± SD of three individual experiments. *p50.05 compared with untreated control. **p50.01 compared with untreated control.

comet assay, micronucleus or other reporter gene assay. The advantages of our luciferase reporter assays are as follows: (1) they are easier to be conducted and are less timeconsuming, (2) they do not require the expertise for staining or microscopic observation, (3) they are allowing for qualitative and quantitative assessment of reporter gene induction in a high through setup (48-well culture plates), (4) our luciferase cells are highly sensitive to assess the genotoxicity of low concentrations of complex mixtures such as EOM from bottom-COE (nanogram per liter). The luciferase signal in the luciferase reporter test system based on the metabolic competent HepG2 cells is obviously more sensitive and stable than that in A549-luciferase cells, which makes the HepG2-luciferase cells to be a better model for detecting the genotoxicity of environmental pollutants. In this study, we have used MMS, a typical DNA alkylating agent, as a positive control to confirm the sensitivity and dose-dependent relationship of GADD45 promoter activation in A549- and HepG2-luciferase cells. Expression of GADD45 is mainly regulated at the transcriptional, posttranscriptional and posttranslational levels (Gao et al., 2009). Under stressful conditions, GADD45 will be rapidly induced and could play critical roles in DNA repair, cell cycle control and apoptosis via interaction with its partner proteins (Liebermann & Hoffman, 2007, 2008; Salvador et al., 2013). Our results showed that the transfected GADD45 promoter-driven luciferase reporter responded to MMS in a dose-dependent manner with the maximum luciferase activity at the highest concentration tested, which were greater than 1.6  and 10  control level in A549- and HepG2-luciferase cells, respectively. In the luciferase reporter cells, the exogenous transfected plasmid containing the GADD45 promoter is driving the luciferase reporter gene. The luciferase response reflects not only the activity of the

exogenous promoter but also the net results of the promoter activity, translation and luciferase accumulation, thus validated the use of our luciferase reporter cells for detection of GADD45 promoter activation. Benzo[a]pyrene is a PAH that has been classified as a human carcinogen (group 1) by the International Agency for Research on Cancer (IARC, 2010). This prototypical procarcinogen can be metabolized by the cytochromes p450 into the ultimate carcinogen, benzo[a]pyrene-diol-epoxide, which will bind covalently to DNA and thus result in the formation of DNA adducts (Hodek et al., 2013; Isabel et al., 2012; Qin & Meng, 2009; Stiborova´ et al., 2014). Formaldehyde, a typical carcinogenic and mutagenic DNA cross-linking agent, can easily pass through the cytoplasmic membrane and cross-link with the nitrogen atoms in DNA, thus causing DNA damage (Luch et al., 2014; Zhang et al., 2012). Pyrene, a PAH with four fused aromatic rings, has also been widely detected in the environment. Though its quinine metabolites are mutagenic and toxic to microorganisms, pyrene is non-genotoxic (Ravelet et al., 2000). In this study, three induction patterns were observed in response to these three tested toxicants. Benzo[a]pyrene induced dose-dependent increases in relative luciferase activity in A549-luciferase cells at non-cytotoxic doses. At cytotoxic doses, benzo[a]pyrene produced a strong dose-dependent induction of relative luciferase activity in HepG2-luciferase cells. Formaldehyde, a DNA cross-linking agent, could induce a strong dose-dependent increase in relative luciferase activity in HepG2-luciferse cells at non-cytotoxic doses. However, though these tested doses were cytotoxic to A549-luciferase cells, formaldehyde can only induced a low, but detectable dose-dependent increase in relative luciferase activity up to 1.5  the control level at 80 mM. Finally, no significant increases in relative luciferase activity were observed in the non-carcinogenic pyrene-treated A549 or

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HepG2-luciferase at the doses tested. Thus, our luciferase reporter cells showed great potential for the detection of different types of genotoxic agents. Three environmental samples collected from a coke oven plant were also tested to validate the application of the luciferase reporter cells in genotoxicity assessment. COEs emitted during the coal pyrolysis for coke production (Cavallo et al., 2008) represent the coal-burning pollution in the air. The COEs have been reported to contain a large variety of toxic compounds including as many as 17 PAHs (Yang et al., 2007). Our results showed that all the three COE-EOMs produced a dose-dependent induction of luciferase activity in HepG2-luciferase cells, and the Top-EOM with higher concentration of PAHs induced higher increases in relative luciferase activity than the other two EOMs. While, in A549luciferase cells, only the top-EOM induced a low, but detectable increases in relative luciferase activity in a doserelated manner. Compared with the A549-luciferase cells, the EOMs produced higher induction of luciferase activity in HepG2-luciferase cells, probably due to the retained activities of xenobiotic-metabolizing enzymes in HepG2 cells. In this study, although a limited number of environmental pollutants were screened, a clear dose-dependent increase in relative luciferase activity was generally observed in both the A549- and HepG2-luciferase cells, except for the noncarcinogenic pyrene. The luciferase activity induced by the tested agents and coke oven EOMs were in accordance with the dose-dependent genotoxic damage detected by comet assay and micronucleus test reported in our previous study (Xin et al., 2012, 2014). Significant increase in relative luciferase activity was already evident after 2.5 mM benzo[a]pyrene, 5 mM formaldehyde, 0.006 mg/L bottomEOM, 0.10 mg/L side-EOM or 0.06 mg/L top-EOM. These concentrations are generally lower than those doses at which the tested environmental toxicants or complex mixtures decreased cell viability and increased genotoxic damage (Xin et al., 2012, 2014). The relative luciferase activity in A549 and HepG2-luciferase cells is generally as sensitive, or more sensitive to the pollutants tested, compared with the cell viability assay, comet assay, micronucleus test and other genotoxicity test system based on a GADD45 promoterdriven GFP reporter (Hastwell et al., 2006; Xin et al., 2012, 2014). In addition, compared with the A549-luciferase cells, the GADD45 promoter in HepG2-luciferase cells is generally more sensitive to the tested pollutants, which makes the HepG2-luciferase cells to be a better model for detecting the genotoxicity of noxious compounds present in air. However, the application of the luciferase reporter cells in genotoxicity assessment needs further investigation.

Conclusions In conclusion, our study showed that the new genotoxicity test system based on transcriptional activation of the GADD45 promoter can detect different types of genotoxic agents and low concentrations of environmental samples. The luciferase reporter cells, especially the HepG2-luciferase cells, could provide a valuable tool for rapid screening of the genotoxic damage of environmental pollutants and their complex mixtures.

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Declaration of interest The authors report no declarations of interest. This work was supported by the Natural Science Foundation of Jiangsu Province (BK20140367), the National Natural Scientific Foundation of China grant (81402705), the Natural Science Foundation of the Jiangsu Higher Education Institutions of China (13KJB330008), the China Postdoctoral Science Foundation (2013M541728) and the Soochow University Student Science Research Foundation (KY2014606B and KY2014608B).

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The development of GADD45α luciferase reporter assays in human cells for assessing the genotoxicity of environmental pollutants.

In order to assess the potential carcinogenic and genotoxic responses induced by environmental pollutants, genotoxicity test systems based on a GADD45...
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