J O U RN A L OF E N V I RO N ME N TA L S CI EN CE S 27 (2 0 1 5 ) 1 08– 1 1 4
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Modulation of the DNA repair system and ATR-p53 mediated apoptosis is relevant for tributyltin-induced genotoxic effects in human hepatoma G2 cells Bowen Li1 , Lingbin Sun1,2 , Jiali Cai2 , Chonggang Wang1 , Mengmeng Wang1 , Huiling Qiu2,⁎, Zhenghong Zuo1,⁎ 1. State Key Laboratory of Cellular Stress Biology, School of Life Sciences, Xiamen University, Xiamen 361102, China. E-mail:
[email protected] 2. Department of Gynaecology, The Affiliated Chenggong Hospital of Xiamen University, Xiamen 361002, China
AR TIC LE I N FO
ABS TR ACT
Article history:
The toxic effects of tributyltin (TBT) have been extensively documented in several types
Received 17 February 2014
of cells, but the molecular mechanisms related to the genotoxic effects of TBT have still
Revised 14 April 2014
not been fully elucidated. Our study showed that exposure of human hepatoma G2 cells
Accepted 12 May 2014
to 1–4 μmol/L TBT for 3 hr caused severe DNA damage in a concentration-dependent
Available online 12 November 2014
manner. Moreover, the expression levels of key DNA damage sensor genes such as the replication factor C, proliferating cell nuclear antigen and poly (ADP-ribose)
Keywords:
polymerase-1 were inhabited in a concentration-dependent manner. We further
Tributyltin
demonstrated that TBT induced cell apoptosis via the p53-mediated pathway, which
DNA damage
was most likely activated by the ataxia telangiectasia mutated and rad-3 related (ATR)
DNA repair
protein kinase. The results also showed that cytochrome c, caspase-3, caspase-8,
Rad-3 related (ATR) protein kinase
caspase-9, and the B-cell lymphoma 2 were involved in this process. Taken together, we
Apoptosis
demonstrated for the first time that the inhibition of the DNA repair system might be more responsible for TBT-induced genotoxic effects in cells. Then the generated DNA damage induced by TBT initiated ATR-p53-mediated apoptosis. © 2014 The Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences. Published by Elsevier B.V.
Introduction Over the past decade, organotin compounds have been used extensively in antifoulants to inhibit the settlement of fouling organisms on vessels and as biocides in various industrial and agricultural settings. Among these compounds, tributyltin (TBT) has received the most attention because of its widespread distribution and almost universal toxicity (Cooke, 2006; Zhang et al., 2011; Zuo et al., 2012; Morales et al., 2013; Komoike and Matsuoka, 2013). Although the use of TBT in paints for vessels is virtually banned by the international treaty at present, TBT was once the major organotin compound used in antifouling paints worldwide and its residue in the aquatic ecosystem is still an important concern (Miki et al., 2011; Castro and Fillmann, 2012;
Sousa et al., 2012). TBT concentrations were the highest reported for gastropod mollusks (up to 662 ng Sn/g dry weight (dw)) in recent studies. Measurable levels of TBT are also found in human blood (Kannan et al., 1999). This study that involved blood analysis of 38 volunteers from Michigan (US) showed a concentration of butyltin ranging from below the detection limit and up to 155 μg/L. While TBT is documented as a neurotoxic, immunotoxic, reproductive and developmental toxin (Zhang et al., 2011, 2013; Cima and Ballarin, 2012; Yu et al., 2013), there has been relatively limited progress on addressing the genotoxicity of TBT. A few studies reveal that TBT exposure can induce DNA damage in both in vitro and in vivo experimental models (Falcioni et al., 2008; Zuo et al., 2012; Morales et al., 2013). Liu et al. (2006) suggest that TBT-induced irreversible DNA breakage is most likely due to
⁎ Corresponding author. E-mail:
[email protected] (Huiling Qiu),
[email protected] (Zhenghong Zuo).
http://dx.doi.org/10.1016/j.jes.2014.05.032 1001-0742/© 2014 The Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences. Published by Elsevier B.V.
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oxidative damage. However, many aspects related to the genotoxic effects of TBT are still puzzling. It is a widely held view that the initial cellular events in a cell following DNA damage are sensing and repairing the damage, and the excessive unfixed damage shift in the intercellular balance to apoptosis (Roos and Kaina, 2006). To fix different kinds/levels of DNA damage, cells employ several specific damage repair pathways, including mainly base excision repair (BER), nucleotide excision repair (NER), homologous recombination (HR), and non-homologous end joining (Bernstein et al., 2002; Roos and Kaina, 2006). The synthesis of repair proteins and the corresponding mRNAs is strictly regulated. Activation by genotoxin-induced DNA damage has been reported for 25 repair genes. Their induction involves multiple players of the DNA damage response such as ataxia telangiectasia mutated (ATM), ATM and Rad3 related (ATR) and PARP1 as well as key transcription factors (Christmann and Kaina, 2013). Therefore, the DNA repair system can be considered to be of the highest relevance to the mechanisms of genotoxins. Our previous study has proved that exposure to TBT induces DNA damage in marine fish livers by altering the transcription levels of the NER genes (Zuo et al., 2012). In the present study, we chose human hepatoma G2 (HepG2) cells as an in vitro model to investigate the genotoxicity of TBT and its mechanisms. HepG2 cells retain many of the characteristics of the hepatocytes such as the activities of phase I and phase II enzymes, and reflect the metabolism of xenobiotics in the human body better than other metabolically incompetent cells (Cai et al., 2009). This study was undertaken to evaluate the extent of DNA damage and the apoptotic related biochemical changes, and to examine the modulations induced by TBT of the key elements in DNA repair and the related apoptosis in HepG2 cells.
respectively (Li et al., 2007), were kindly provided by Prof. Sheng-Cai Lin (School of Life Sciences, Xiamen University, Xiamen, China), and pRL-TK was purchased from Promega (Madison, WI, USA).
1. Materials and methods
1.5. Real time fluorescence quantitative PCR
1.1. Chemicals
Total RNA isolation and cDNA synthesis were performed using the RNAiso Reagent (TaKaRa Biotechnology, Dalian, China) and ReverTra Ace qPCR RT Kit (TOYOBO BioTechnology, Shanghai, China), respectively. Real-time PCR was performed on a Stratagene MX3000P PCR machine using the Sybr green master mix (Stratagene, Canada). 18S rDNA was used as the endogenous assay control. The REST 2008©-version 2 software was used to calculate the relative expression of target genes using the PairWise Fixed Reallocation Randomization Test© (Pfaffl et al., 2002).
Tributyltin chloride was obtained (Fluka AG, Switzerland), with a purity of greater than 97%. Propidium iodide (PI), Hoechst 33342 (HO), methyl thiazolyl tetrazolium, ethidium bromide (EB), dimethyl sulfoxide (DMSO), normal-melting agarose (NMA), and low-melting agarose (LMA) were purchased from Sigma (St. Louis, MO, USA). All tissue culture supplies were purchased from Hyclone (Logan, UT, USA). Polyclonal antibody against ATM, and ATM and ATR were obtained from Calbiochem (San Diego, USA). Polyclonal antibody against DNA-dependent protein kinase catalytic subunit (DNA-PKcs), glyceraldehyde-3-phosphate dehydrogenase and β-actin, monoclonal antibodies against B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), cytochrome c, p53, phosphorylated p53 (Ser15), and enhanced chemiluminescence (ECL) reagent were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Nitrocellulose membrane (0.2 μm pore size) was purchased from Bio-Rad laboratories (Hercules, USA). All other routine laboratory reagents were obtained from commercial sources and were of analytical grade.
1.3. Cell culture and treatment HepG2 cells were cultured in Dulbecco's minimal essential medium supplemented with 10% heat-inactive fetal bovine serum, 100 international unit (IU)/mL penicillin, and 100 μg/mL streptomycin in a humidified atmosphere of 5% CO2 at 37°C. TBT was first dissolved in DMSO at 1, 2 and 4 mmol/L, and then diluted 1000 times with culture medium just before treatment. Control cells were treated with the DMSO (final concentration, 0.1%) alone, which had no detectable effect on the cell viability. After exposure for 3 hr, the cells were washed twice with ice cold phosphate-buffered saline (PBS), harvested with 0.02% EDTA and 0.025% trypsin, rinsed three times in PBS, and then pelleted by centrifugation for the subsequent experiment.
1.4. Single cell gel electrophoresis (the Comet Assay) DNA damage was evaluated by Comet assay according to Cai et al. (2009). After electrophoresis, DNA was visualized using EB staining. In each experiment, only cultures in which the viability of the cells was ≥80% were analyzed. One hundred and fifty randomly selected comet tails from the slides were analyzed using Komet version 3.1 software (Kinetic Imaging Ltd., Liverpool, UK).
1.6. Flow cytometry with the PI/HO Cell death was evaluated using flow cytometry with the Prodium Iodide (PI)/Hoechst (HO). After treatment with TBT, the collected cells were stained with 20 μg/mL PI and 10 μg/mL HO for 15 min, washed twice with PBS, and then analyzed immediately using a fluorescence activated cell sorter. Cell Quest software (BD Biosciences, Franklin Lakes, USA) was used to analyze the data, and 30,000 events were analyzed for each independent experiment.
1.7. Transient transfection and luciferase reporter assay 1.2. Plasmids Plasmids of luciferase reporters containing cyclin-dependent kinase inhibitor 1A (p21) and p53-upregulated modulator of apoptosis (PUMA) promoters, namely p21- and PUMA-Luc
Transient transfections were carried out using FuGENE® HD Transfection Reagent (Roche, Penzberg, Germany) following the manufacturer's instructions. HepG2 cells were transfected in six-well dishes at 70% confluence with p21-Luc and
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PUMA-Luc, and incubated for 24 hr before TBT treatment. All of the experiments for luciferase assay were performed using co-transfection of pRL-TK as an endogenous assay control. After transfection, the cells were treated with 2 μmol/L TBT for 3 hr, and then the luciferase activities of the harvested cells were measured using a luminometer (Wallac 1420 Victor 2 multi label counter system; PerkinElmer, Waltham, MA, USA) and a dual luciferase reporter assay kit (Beyotime, Beijing, China). All experiments were carried out in triplicate and at least five times.
1.8. Western blotting assay Total protein extracted from HepG2 cells (n = 3 per condition) with SDS-sample buffer were applied for Western blotting using anti-actin, anti-P-p53 Ser15, anti-p53, anti-DNA-PKcs, anti-ATM, anti-ATR, anti-Bax, anti-Bcl-2, and anti-Cyt C antibody according to our further research (Cai et al., 2009). The protein band, specifically bound to the primary antibody, was detected using an anti-rabbit IgG-AP-linked antibody.
three key DNA damage sensor genes involved in major DNA repair pathways, namely the replication factor C (RFC), proliferating cell nuclear antigen (PCNA) and poly (ADP-ribose) polymerase-1 (PARP-1), were designed (Table 1S). The expression of these DNA repair related genes in the cells decreased (p < 0.05) in all treated-groups in a concentration-dependent manner compared to the control (Fig. 1).
2.3. Effect of TBT on apoptosis of HepG2 cells After staining of the cell-impermeant dye PI and the cell-permeant dye HO, cells were divided into three groups: cells exhibiting both HO and PI fluorescence (dead or late apoptotic cells), only HO fluorescence (the early apoptotic cells), and neither HO nor PI fluorescence (intact cells). Representative dot plots of PI/HO staining were shown in Fig. 2A. Under control conditions, most cells were intact live cells. After 3 h-incubation with increasing TBT concentrations, intact living cell populations decreased while apoptotic cell populations increased in a TBT concentration-dependent manner (Fig. 2B).
1.9. Analysis of caspase enzymatic activity The activities of caspase-3, caspase-8 and caspase-9 were measured using a Diagnostic Reagent kit (purchased from Nanjing Jiancheng Bioengineering Institute China) following the manufacturer's instructions. Experiments were performed at least three times.
1.10. Statistical analysis All data were presented as the means ± standard deviation. The presence of statistical differences among groups was determined using ANOVA, and the method of least significant difference (Dunnett test) was used to compare the effects between each TBT exposed group and the control. All of the statistical analyses were carried out using the statistical software SPSS 13.0 for Windows. Statistical significance was recognized when p < 0.05.
2. Results 2.1. DNA damage in HepG2 cells Evaluation of DNA damage using Comet Assay provides a direct assessment of the level of DNA modification in individual cells. The extent of DNA damage can be quantified by measuring the displacement of the genetic material between the cell nucleus (“comet head”) and the resulting “tail”. Comet Assay in the present study showed that tail length, tail intensity, and tail moment all increased in a concentration-dependent manner after the cells were treated with TBT (Table 1).
2.2. RFC, PCNA and PARP-1 expressions in HepG2 cells
Real-time quantitative PCR was carried out to examine the expression levels of the candidate genes. The primers for
2.4. TBT induced HepG2 cell apoptosis via the ATR-p53 pathway The tumor suppressor protein p53 plays a major role in the cellular response to DNA damage and other genomic aberrations. The activation of p53 can lead to either cell cycle arrest and DNA repair, or apoptosis. Although treating the HepG2 cells with TBT did not affect the p53 protein level, the phosphorylation (Ser15) level of p53 increased in a concentration-dependent manner (Fig. 3a). In addition, TBT exposure impaired the expression level of DNA-PKcs and ATM in the HepG2 cells. However, exposing the HepG2 cells induced ATR expression (Fig. 3a). To explore the modulation of p53 transcriptional activity following TBT treatment, we investigated key p53-regulated transcription targets, the pro-apoptotic genes PUMA, and anti-apoptotic genes p21, utilizing the specific luciferase reporter plasmid containing the promoter fragment of PUMA or p21. After transfection and incubation, cells were exposed to 2 μmol/L TBT for 3 hr, and a relative luciferase activity assay was performed. TBT exposure did not change p21 transcription, but enhanced PUMA transcription significantly (Fig. 3b).
Table 1 – Change of tail length, tail DNA, and tail moment after the cells were treated with tributyltin (TBT) for 3 hr. TBT (μmol/L)
Tail length (μm)
Tail intensity (%)
0 1 2 4
3.0 29 41 67
0.8 13.7 41.3 78.1
± ± ± ±
0.002 a 4.042 ab 7.000 b 12.067 c
± ± ± ±
0.068 0.950 6.942 2.394
a bc d e
Tail moment 0.02 4.60 22.5 78.0
± ± ± ±
0.002 0.523 5.988 8.312
a a b c
Data was obtained from three independent experiments. The different letters mean significantly different at p < 0.05 at the same column.
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a
A
0.9 b
0.6
c
c 0.3 0
0
1 2 TBT (μmol/L)
4
1.2
a
Relative expression of PARP-1 (% of control)
1.2
Relative expression of PCNA (% of control)
Relative expression of RFC (% of control)
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B
0.9 0.6 b 0.3 0
c 0
d
1 2 TBT (μmol/L)
4
1.2
a
C
0.9 0.6 b
b 0.3
c
0
0
1 2 TBT (μmol/L)
4
Fig. 1 – The relative expression of replication factor C (RFC) (a), proliferating cell nuclear antigen (PCNA) (b) and poly (ADP-ribose) polymerase-1 (PARP-1) (c) in the cells exposed to tributyltin for 3 hr. Gene expression levels were determined as previously described in the Materials and methods section. Each bar indicates the means ± standard deviation (S.D.) of 6 replicate assay experiments. Treatments not sharing a common letter are significantly different at p < 0.05 as assessed by one-way ANOVA and the Dunnett test.
To further investigate TBT-induced apoptosis through ATR-p53 pathway, the Bcl-2 family proteins, cytochrome c and caspase activities responding to TBT exposure were detected. The Bax protein level was augmented in a concentration-dependent manner after treatment with TBT. In contrast, Bcl-2 displayed a significant reduction in all TBT-treated groups as compared with the control (Fig. 3c). We also found that cytochrome c released from the mitochondria showed an obvious elevation in all TBT-treated groups (Fig. 3c). Compared to the control group, the activities of caspase-3, caspase-8 and caspase-9 all increased significantly in a concentration-dependent manner after the cell was treated with TBT (Fig. 3d).
3. Discussion
Organotin compounds accumulate at higher levels in liver than in most other organs (Strand and Jacobsen, 2005). There is an increasing body of evidence linking TBT with elevated risk of DNA damage (Tiano et al., 2001; Gabbianelli et al., 2002; Falcioni et al., 2008). However, studies on the mechanism of TBT genotoxicity are rare compared to reports on its other toxicities (Zuo et al., 2012). The results obtained in the present study indicated that lower concentrations of TBT (1–4 μmol/L) also caused severe DNA strand breaks in the
A 5
0
1
B 3
Q1 Q2
Q1 Q2
Q3 Q4
Q3 Q4
4
2 2 5
3
2
4
5
2
3
4
5
4
4 3
Q1 Q2
Q1 Q2
Q3 Q4
Q3 Q4
2
Percentage of apoptotic cells (%)
Intensity of PI fluorescence PI positive (arbitrary unit in log scale)
4
c 3 b
1
0 2
3 4 5 2 3 4 Intensity of Hoechst 33342 fluorescence Hoechst 33342 positive (arbitrary unit in log scale)
5
b
2 a
0
1
2 TBT (μmol/L)
4
Fig. 2 – Effect of TBT on apoptosis of HepG2 cells. After incubation with 0–4 μmol/L TBT for 3 hr, cells were collected and treated with PI and HO. Samples were analyzed using flow-cytometry. The percentages of apoptotic cells based on the total cell population analyzed were determined. Each bar indicates the means ± standard deviation (S.D.) of 6 replicate assay experiments. Treatments not sharing a common letter are significantly different at p < 0.05 as assessed by one-way ANOVA and the Dunnett test. For each independent experiment, 30,000 events were gated.
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a 0
1
2
4
TBT (μmol/L) P-p53 Ser15
DNA-PKcs
ATM
Relative luciferase activity
p53
25
b
b 20 15 10 a
5 a 0 No plasmid
ATR
p21-Luc
PUMA-Luc
β-Actin
c
0
1
2
4
250
TBT (μmol/L) Bax
Cyt c
GAPDH
200 Enzyme activity (% control)
Bcl-2
c
d
c
b b
b b c
150
b c a
a
Caspase-3
Caspase-8
a
100 50 0
Caspase-9
Fig. 3 – Western blotting analysis of p53, P-p53 Ser15, DNA-PKcs, ataxia telangiectasia mutated and ataxia telangiectasia mutated and rad-3 related (a), Bax, Bcl-2, Cyt c (c) expression in HepG2 cells after TBT exposure. Effects of TBT on p53 transcriptional activity (b), and caspase enzymatic activity (d) in HepG2 cells. Following transfection and incubation, cells were treatment with 2 μmol/L TBT for 3 hr, and then were lysed for dual luciferase assay. All experiments for the luciferase assay were performed by co-transfection with pRL-tk as an internal control (b). After incubation with 0, 1, 2 and 4 μmol/L TBT for 3 hr, cells were assayed for enzymatic activity (d). Each bar indicates the means ± standard deviation (S.D.) of 6 replicate assay experiments. Means of exposures not sharing a common letter are significantly different at p < 0.05 as assessed using one-way ANOVA followed by the Dunnett test.
HepG2 cells. The initial cellular events in a cell following DNA damage are sensing and repairing the damage (Roos and Kaina, 2006). To examine the modulation of DNA repair in response to TBT exposure, three of the DNA damage sensor genes responsible for the major DNA repair pathways were chosen. These chosen sensors play central roles in: (1) BER (PARP-1, RFC/PCNA complex) (Godon et al., 2008); (2) NER (PARP-1, RFC/PCNA complex) (Sancar et al., 2004); and (3) HR (PARP-1) (Hochegger et al., 2006). Our results in the present study showed that the transcription levels of RFC, PCNA and PARP-1 in the cells decreased (p < 0.05) in all treated-groups in a concentration-dependent manner. Although the main mechanisms of exogenous mutagens are direct attack on the DNA (Pavanello et al., 2009), many studies demonstrate that a number of environmental DNA damaging agents act as co-carcinogens through inhibition of DNA repair (Hartwig and Beyersmann, 1989; Hill et al., 2008; Zuo et al., 2012). It is
reported that inhibition of PARP-1 by arsenite interferes with the repair of oxidative DNA damage (Ding et al., 2009). Any deficits in DNA repair capacity will reduce the cellular resistance to spontaneous and exogenous DNA damage, and lead to genome instability and various serious diseases, such as cancer (Helleday, 2008). Our findings demonstrate that micromolar TBT concentrations inhibit the repair of damaged DNA. By this mechanism, TBT may also increase the cellular sensitivity to other exogenous mutagens. However, up to now no direct evidence of TBT mutagenicity and carcinogenicity in humans is available. The tumor suppressor protein p53 provides an important link between DNA damage and apoptosis. p53 can apparently be phosphorylated by ATM, ATR and DNA-PKcs at Ser15, and this phosphorylation impairs the ability of MDM2 to bind p53, promoting both the accumulation and functional activation of p53 in response to DNA damage (Tibbetts et al., 1999). Because
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the phosphorylation of the serine 15 residue of p53 is a very early step in the activation of p53 (Shieh et al., 1997), we assessed the amount of p53 phosphorylated at serine 15 after TBT treatment, using an antibody that specifically recognizes the phosphorylated serine 15 residue of p53. The present study showed that the phosphorylation (Ser15) level of p53 increased in a concentration-dependent manner. However, the mechanisms that link DNA damage to the activation of p53 are also complex. The activation of p53 by ionizing radiation is better understood, and is dependent on the ATM kinase (Canman et al., 1998). However, in our study, only the ATR kinase was activated by TBT exposure. Furthermore, our result proved that TBT exposure induced PUMA transcription. The PUMA gene encodes two BH3 domaincontaining proteins that are located in the mitochondria (Li et al., 2007). In response to transactivation by p53, PUMA proteins are induced, which then form a complex with Bcl-2 or Bcl-XL to induce cytochrome c release and cell apoptosis. In accordance with the induction of the PUMA gene, we found that the Bax expression was induced with a reduction of Bcl-2, the release of cytochrome c, and the activation of caspases. Generally, there are two pathways through which the caspase family proteases can be activated: one is the death signal-induced, death receptor-mediated pathway; the other is the stress-induced, mitochondrion-mediated pathway. Caspase-3, a key factor in apoptosis execution, is the active form of procaspase-3. The downstream substrates of caspase-3 include procaspase-3, procaspase-6, procaspase-9, DNA-PK, PARP and so on (Riedl and Shi, 2004). Taken together, TBT exposure inhibits DNA repair and leads to an increased expression of ATR and the phosphorylation (Ser15) level of p53, thus altering the balance of their downstream pathway components such as Bax/Bcl-2 and PUMA, thereby
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inducing cytochrome c to be released and increasing the activity of cellular caspases, before inducing cell apoptosis (Fig. 4). In conclusion, the present study demonstrated that TBT could produce apoptosis in a concentration-dependent manner in HepG2 cells. The apoptosis appeared to be mediated by inhibition of the DNA repair system. Then, the subsequently generated DNA damage induced by TBT initiated p53-mediated apoptosis, which was most likely activated by ATR, a kinase closely related to ATM.
Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 40606027) and the Project of the Xiamen Science and Technology Program (No. 2013Z20134027). We thank Prof. John Hodgkiss for his assistance with English.
Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.jes.2014.05.032.
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