ARTICLE pubs.acs.org/est
Genotoxicity of Several Polybrominated Diphenyl Ethers (PBDEs) and Hydroxylated PBDEs, and Their Mechanisms of Toxicity Kyunghee Ji,† Kyungho Choi,* John P. Giesy,‡,§,|| Javed Musarrat,|| and Shunichi Takeda^ †
School of Public Health, Seoul National University, Seoul, 151-742, Korea Department of Veterinary Biomedical Sciences and Toxicology Centre, University of Saskatchewan, Saskatoon, Saskatchewan, S7J 5B3, Canada § Department of Zoology, and Center for Integrative Toxicology, Michigan State University, East Lansing, MI, USA Zoology Department, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia ^ Graduate School of Medicine, Kyoto University, Kyoto, 606-8501, Japan
)
‡
bS Supporting Information ABSTRACT: Polybrominated diphenyl ethers (PBDEs) have been extensively utilized as flame retardants, and recently there has been concern about potential adverse effects in humans and wildlife. Their hydroxylated analogs (OH-BDEs) have received increasing attention due to their potential for endocrine and neurological toxicities. However, the potentials and mechanisms of genotoxicity of these brominated compounds have scarcely been investigated. In the present study, genotoxicity of tetra-BDEs, penta BDE, octa-BDE, deca-BDE, and tetra-OH-BDEs were investigated by use of chicken DT40 cell lines including wild-type cells and a panel of mutant cell lines deficient in DNA repair pathways. Tetra-BDEs have greater genotoxic potential than do the other BDEs tested. OH-tetra-BDEs were more genotoxic than tetra-BDEs. DT40 cells, deficient in base excision repair (Polβ/) and translesion DNA synthesis (REV3/) pathways, were hypersensitive to the genotoxic effects of tetra-BDEs and OH-tetra-BDEs. The observation of chromosomal aberrations and gamma-H2AX assay confirmed that the studied brominated compounds caused double strand breaks. Pretreatment with N-acetyl-L-cysteine (NAC) significantly rescued the Polβ/ and REV3/ mutants, which is consistent with the hypothesis that PBDEs and OH-BDEs cause DNA damage mediated through reactive oxygen species (ROS). Some tetra-BDEs and OH-tetra-BDEs caused base damage through ROS leading to replication blockage and subsequent chromosomal breaks.
’ INTRODUCTION Polybrominated diphenyl ethers (PBDEs) have been widely used as flame retardants in electronic equipment, casings for personal computers and television sets, and a variety of other plastic products.1 The number of possible congeners is 209, and their chemical and physical characteristics are similar to those of polychlorinated biphenyls (PCBs),2 which raises concerns about the potential toxicity to humans and wildlife. Since PBDEs do not covalently bind to polymers in plastics, they can be dissipated into the environment from products during use.3 PBDEs, especially the less brominated congeners, are resistant to physical, chemical and biological degradation, and thus persist in the environment.2 The penta- and octa-BDE mixtures were banned by the European Union in 2004,4 and production of those two mixtures was stopped in North America, though voluntarily action by industry.5 Korea is also following the actions of the European Union, and is considering bans.6 Although this ban on penta- and octa-PBDEs will likely result in a decline of residual concentrations in the environment, continued use, degradation, and biotransformation of deca-BDEs will lead to detection of less brominated compounds7 because of debromination of deca-BDE.8 Hydroxylated (OH-) BDEs, which are analogous to PBDEs in structure, are of concern due to their toxicities relative to PBDEs. The OH-BDEs are of particular interest since these compounds r 2011 American Chemical Society
have been shown to lead to a variety of effects on exposed organisms including disruption of thyroid hormone homeostasis, altered estradiol synthesis, and neurotoxic effects.911 While some researchers have reported that OH-BDEs are formed by hydroxylation of synthetic PBDEs12 it has now been shown that some OH-BDEs can be formed naturally in marine organisms and that natural processes are more likely the sources of most OH-BDEs in the environment.13 In spite of widespread occurrence in the environment and growing concerns worldwide, limited information on the toxicity of PBDEs and OH-BDEs is available. It has been reported that exposure to tetra- and penta-BDE congeners affects neurodevelopment of mice,14 and hydroxylation increases the neurotoxic potentials of individual PBDE congeners.9 PBDEs have been found to disrupt thyroid function and alter steroidogenesis in vitro.11,15 It has also been reported that such toxic potentials of OH-BDEs are more potent than those of PBDEs.10,11 However, there is a paucity of data regarding the genotoxicity of PBDEs and OH-BDEs. Commercial PBDE mixtures did not Received: December 26, 2010 Accepted: April 25, 2011 Revised: April 19, 2011 Published: May 05, 2011 5003
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Figure 1. Sensitivity of repair-deficient DT40 mutant cells to PBDEs and OH-BDEs with or without NAC. Values shown are mean ( SD, and “0” indicates solvent control. *p < 0.05, compared with wild-type cells by one-way ANOVA. (A) BDE-47 without NAC, (B) BDE-47 with NAC, (C) BDE-49 without NAC, (D) BDE-49 with NAC, (E) BDE-99 without NAC, (F) BDE-99 with NAC, (G) BDE-138 without NAC, (H) BDE-138 with NAC, (I) 6-OH-BDE-47 without NAC, (J) 6-OH-BDE-47 with NAC, (K) 4-OH-BDE-49 without NAC, (L) 4-OH-BDE-49 with NAC. Mutants exposed to BDE209 with or without NAC did not show significant difference in cell viability compared to wild-type cells.
show mutagenic activity in the Salmonella or Saccharomyces cerevisiae16 assay or in a mouse lymphoma assay.17 Furthermore, no chromosomal aberrations or sister chromatid exchanges were seen in Chinese hamster ovary cells exposed to deca-BDE.17 However, Barsien et al.18 reported induction of micronuclei and other nuclear abnormalities in mussels exposed to BDE-47.
Cytotoxicity and genotoxicity of BDE-47 were observed in human neuroblastoma cells exposed in vitro.2 In addition, An et al.19 reported that 6-OH-BDE-47 could induce inhibition of cell viability, increase of apoptosis rate, cell cycle block, and DNA damages, which might involve the altered oxidative stress response due to the elevated free radicals and impaired antioxidative system. 5004
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Environmental Science & Technology Despite the information available, there is still a gap in knowledge of potential genotoxicity of PBDEs and OH-BDEs, and the molecular mechanism of the toxicity. In the present study we investigated the genotoxicity potential and mechanism of major PBDEs and OH-BDEs using genetically modified chicken DT40 cells.
’ MATERIALS AND METHODS Preparation of Testing Materials. PBDEs (BDE-47, BDE49, BDE-99, BDE-138, and BDE-209) were purchased from Wellington Laboratories (Geulph, ON, Canada). 6-OH-BDE-47 and 4-OH-BDE-49 were synthesized in the Department of Biology and Chemistry of City University of Hong Kong following the methods described in Marsh et al.;20 purities were >98%. PBDEs were concentrated by a gentle stream of nitrogen and dissolved in dimethyl sulfoxide (DMSO), while OH-BDEs were dissolved in acetone for cell bioassay. Cell Types and Culture. A panel of isogenic DT40 mutants, each defective in one of the major DNA damage repair mechanisms, which include base excision repair, nucleotide excision repair, homologous recombination, nonhomologous end-joining, and translesion DNA synthesis were used (See Table S1 in the Supporting Information (SI)). The mutant cell lines were developed elsewhere,2127 and successfully demonstrated as a measure for screening and characterizing genotoxicity of environmental contaminants.28 Cells (1 105) were cultured in 100 mm Petri-dishes with 10 mL RPMI 1640 medium (Welgene, Daegu, Korea) supplemented with 10% fetal bovine serum (FBS; Sigma, St. Louis, MO), 1% chicken serum (Sigma), and 50 μM β-mercaptoethanol (Sigma) at 39.5 C in a humidified atmosphere of 5% CO2 and 95% air. Adenosine-50 -Triphosphate (ATP) Assay. In order to differentiate genotoxicity from cytotoxicity, only doses that resulted in less than 80% decrease of cell viability in wild-type cells were considered. When statistically significant reduction in cell viability relative to the wild-type was observed at these ‘noncytotoxic’ doses, the given chemical was determined to be genotoxic, and the degree of genotoxicity was determined by the differences of fold reduction compared to the viability of wild-type cells. Based on this approach, we detected genotoxicity by simply monitoring for differences in cellular proliferation rates between wild-type cells and the isogenic clones deficient in DNA repair at certain doses. We measured cell viability indirectly by measuring the amount of ATP in cell lysates as previously described in Ji et al.,28 of which details can be found in SI. Effect of a Chemical Scavenger on Cellular Viability. To evaluate the involvement of reactive oxygen species (ROS) in causing DNA damage by PBDEs, DT40 cells were pretreated with 1 mM N-acetyl-L-cysteine (NAC; Sigma), an ROS scavenger, 2 h before PBDEs treatment. Chromosomal Aberration Assay. The chromosomal aberration assay was conducted by use of methods previously described by Ji et al.28 Analysis of chromosome aberrations was limited to the 11 autosomal macrochromosomes and the Z chromosome in Giemsa-stained metaphase cells.29 A total of 50 mitotic cells were evaluated per treatment, and the chromosomal aberrations according to the International System for Human Cytogenetic Nomenclature (ISCN) system were enumerated30 (for details, see SI).
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Immunofluorescence Assay. Visualization of γ-H2AX foci was accomplished with minor modifications, by use of the protocol described by Sonoda et al.31 Approximately 200 cells were evaluated per treatment. Details of this assay can be found in Supporting Information. Statistical Analyses. Results of repeated experiments are expressed as the mean ( standard deviation. Differences among experimental variants were evaluated by ANOVA followed by Dunnett’s test using SPSS 15.0 for Windows (SPSS, Chicago, IL). To compare the frequency of γ-H2AX foci between wildtype and mutant cells, we performed independent sample t tests. P-values less than 0.05 were considered statistically significant.
’ RESULTS AND DISCUSSION Genotoxicity of PBDEs and OH-BDEs. Among the target PBDEs tested, including BDE-47, BDE-49, BDE-99, BDE-138, and BDE-209, tetra-BDEs were more potent initiators of DNA damage than were the more brominated compounds. After exposure to BDE-47, Polβ/ and REV3/ mutant cell lines were significantly affected compared to wild-type cells in a concentration-dependent manner (p < 0.05). However, mutant cells exposed to BDE-99 and BDE-138 began to exhibit significant reduction in cell viability only at concentration of 200 μg/L or greater (Figure 1E and G). Those exposed to BDE-209 did not show significant difference in cell growth (data not shown). These results suggest that the genotoxic potency of tetra-BDEs was greater than those of penta-BDE (BDE-99), octa-BDE (BDE-138), or deca-BDE (BDE-209). Several studies have reported effects that cell viability and cell apoptosis caused by less brominated congeners was greater than that of more brominated congeners. A concentration of 100 μM BDE-47 or BDE-209, resulted in 40% and 50% less cell viability of exposed cells relative to the controls, and the apoptotic rates were 52.6% and 34.6% of that of controls, respectively.32 Similarly, it has been found that the least effective concentration of BDE-47 to inhibit cell proliferation was 41.2 μM,33 while that of BDE-209 was 50 μM.34 These results are consistent with the hypothesis that less brominated congeners are more bioactive than the more brominated PBDE and that the effects are, due in part, to ROS-formation. For instance, with 12.5 μM BDE-47 was reported to increase ROS content 6 h after treatment, while the same concentration of BDE-209 did not significantly increase ROS formation 12 h after treatment.32 Significant increases in intracellular ROS formation probably resulted in oxidative stress and caused lipid damage. Lipid peroxidation could influence membrane fluidity as well as integrity of biomolecules associated with the membrane and cause cell death or apoptosis. These results suggest that less brominated congeners are more potent at producing ROS, which in turn causes greater genotoxicity. OH-tetra-BDEs have greater potential for genotoxicity compared to their nonhydroxylated counterparts. 6-OH-BDE-47 was the most potent congener, and this observation is consistent with previous in vitro and in vivo tests. For instance, the least effective concentration of 6-OH-BDE-47 to induce apoptosis of cells after the 24 h was >5 μM,19 whereas that for BDE-47 was 41.2 μM.2 Similarly, when zebrafish embryos were exposed in vivo to 25 nM 6-OH-BDE-47 several types of developmental defects were observed, while no toxic or teratogenic effects were observed at 5005
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Table 1. Frequency of Chromosomal Aberration in REV3/ DT40 Cells after the Exposure to BDE-47 and 6-OH-BDE-47a cell type wild-type
REV3/
chemicals
NAC
no. (%) of
(μg/L)
(mM)
chromosome aberrations
control control
0 1
1(2) 1(2)
BDE-47 (200)
0
7(14)
BDE-47 (200)
1
3(6)
6-OH-BDE-47 (200)
0
11(22)
6-OH-BDE-47 (200)
1
4(8)
control
0
2(4)
control
1
1(2)
BDE-47 (200) BDE-47 (200)
0 1
11(22) 4(8)
6-OH-BDE-47 (200)
0
15(30)
6-OH-BDE-47 (200)
1
5(10)
Cells were treated with 200 μM PBDEs with or without N-acetyl-Lcysteine (NAC) for 48 h. Fifty mitotic cells were observed per treatment. a
concentrations of BDE-47 as great as 10 μM.35 Therefore, 6-OHBDE-47 was a more potent genotoxin than was BDE-47. There are few studies reporting possible explanations why OH-BDEs are more potent than PBDEs. It has been suggested that OH-BDEs could cause more oxidative stress, which might be associated with the cellular damage and apoptosis than PBDEs. Intracellular ROS concentrations were greater than those observed in background cells, and increased in a concentrationdependent manner when exposed to 6-OH-BDE-47 in the range of 0.12.0 μM.19 Intracellular ROS levels were significantly greater in cells treated with 41.2 μM BDE-47.33 Hydroxylated polychlorinated biphenyls (OH-PCBs) have been reported to be more potent genotoxic agents, and known to generate more ROS than analogous PCBs.36 Since PBDEs are structurally similar to PCBs, it is reasonable to deduce that PBDE exerted its genotoxicity on cells through ROS generation. Mechanism of Genotoxicity of Select PBDEs and OH-BDEs. The sensitivity profile of the DNA repair mutants treated with PBDEs and OH-BDEs exhibited hypersensitivity of cells deficient in base excision repair (Polβ/) and translesion DNA synthesis (REV3/), whereas Rad54/, Ku70/ and XPA/ mutants showed no sensitivity (Figure 1A, C, E, G, I, and K). These observations suggest that PBDEs and OH-BDEs can induce base damage that is repaired by the base excision pathway involving DNA polymerase β. To investigate the cause of reduced cellular survival of Polβ/ and REV3/ clones, chromosomal aberrations in mitotic wild-type and REV3/ cells were also measured. Chromosomal aberrations were barely detectable in nonexposed cells, whereas both chromatid- and chromosome-type breaks were frequently observed in cells exposed to BDE-47 and 6-OH-BDE-47 (Table 1). Formation of double strand break (DSB) triggers extensive phosphorylation of histone H2AX (γ-H2AX), which is detectable using an immuno-cytochemical method.37 Detection of γ-H2AX subnuclear foci is suggested to provide a considerably more sensitive, efficient, and reproducible measurement of the amount of DNA damage compared to other techniques such as pulsed field gel electrophoresis or comet assays.37 In the present study, induction of γ-H2AX was measured after exposure to PBDEs and OH-BDEs to confirm the occurrence of DSBs.
Figure 2. PBDEs and OH-BDEs induced γ-H2AX subnuclear foci formation in wild-type and REV3/ cells. Average number of γ-H2AX foci per cell of the indicated cells. At least 200 cells were analyzed per each sample.
In REV3/ DT40 cells, γ-H2AX focus formation increased significantly after 1 h treatment with BDE-47, BDE-49, 6-OHBDE-47, or 4-OH-BDE-49, compared to that of wild-type cells (Figure 2 and SI Figure S1). These observations also indicate that tetra-PBDEs and tetra-OH-BDEs indeed induce DSBs. It is likely that PBDEs induce base damage that is repaired by base excision repair involving DNA polymerase β. This base damage can cause replication blockage, which is released by REV3, a translesion DNA synthesis polymerase ζ. Furthermore, replication blockage can occasionally result in chromosomal breaks. To gain insight into the nature of the base damage, a study was conducted to determine whether PBDE-induced DNA damage was mediated by ROS. This was done by pretreating cells with an antioxidant, NAC. Treatment with NAC did not affect cellular proliferation. Pretreatment with NAC significantly reversed sensitivity of Polβ/ and REV3/ mutants to PBDEs and OH-BDEs (Figure 1B, D, F, H, J, and L). These results suggest that PBDEs and OH-BDEs induce ROS and thereby generate oxidative base damage, which leads to replication blockages and subsequent chromosomal breaks. Rescue levels observed after pretreatment of NAC to the 6-OH-BDE-47 were greater than when exposed to tetra-PBDEs. These results suggest that the observed genotoxicity of the OH-tetra-BDE can be explained by ROS. The mechanisms by which PBDEs generate ROS have not yet been elucidated. Insight into the mechanisms can be gained from information on PCBs which are structurally similar to PBDEs to some extent. Less-chlorinated PCBs and hydroxylated PCBs are more likely to be oxidized to quinones which are capable of redox cycling, and producing superoxide. Superoxide can be readily converted to hydrogen peroxide and subsequently to highly reactive producing superoxide.36,38,39 The hydroxyl radical can attack DNA and generate additional reactive products, including peroxyl radicals. Further studies are needed to understand precise mechanisms of ROS generation by PBDEs. There is supporting evidence for the ability of PBDEs or OHBDEs to induce oxidative stress. BDE-47 was reported to cause oxidative stress in cells, such as human neutrophil granulocytes,40 hippocampal neurons,33 human neuroblastoma cells,2 human fetal hematopoietic cells,41 and rainbow trout gonadal cells.32 In addition, 6-OH-BDE-47 was also reported to cause oxidative stress response due to the elevated free radicals and impaired antioxidative system in human hepatoma cell lines.19 In the present study, exposure to PBDEs or OH-BDEs resulted in hypersensitivity of DT40 mutant cells, which are deficient in base excision repair and translesion DNA synthesis 5006
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Environmental Science & Technology pathway. The observed hypersensitivity is attributable to the mutagenic potential of PBDEs and OH-BDEs, due to significant increases in the number of chromosomal aberrations and γ-H2AX focus formation in the Polβ/ and REV3/ mutants.
’ ASSOCIATED CONTENT
bS
Supporting Information. Additional information including Figure S1 and Table S1. This material is available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION Corresponding Author
*Phone: 82-2-880-2738; fax: 82-2-745-9104; e-mail: kyungho@ snu.ac.kr.
’ ACKNOWLEDGMENT This research was supported by National Research Foundation of Korea (Project No. 2009-0080808). Prof. Giesy was supported by the Canada Research Chair program and an at large Chair Professorship at the Department of Biology and Chemistry and State Key Laboratory in Marine Pollution, City University of Hong Kong and the Visiting Professor Program of King Saud University. ’ REFERENCES (1) Darnerud, P. O.; Eriksen, G. S.; Johannesson, T.; Larsen, P. B.; Viluksela, M. Polybrominated diphenyl ethers: occurrence, dietary exposure, and toxicology. Environ. Health Perspect. 2001, 109, 49–68. (2) He, W.; He, P.; Wang, A.; Xia, T.; Xu, B.; Chen, X. Effects of BDE-47 on cytotoxicity and genotoxicity in human neuroblastoma cells in vitro. Mutat. Res. 2008, 649, 62–70. (3) de Wit, C. A. An overview of brominated flame retardants in the environment. Chemosphere 2002, 46, 583–624. (4) Restriction of Hazardous Substances Directive, Directive 2002/95/ EC of the European Parliament and of the Council of 27 January 2003 on the Restriction of the Use of Certain Hazardous Substances in Electrical and Electronic Equipment: OJ L37; European Union, 2003; p 19. (5) Canton, R. F.; Scholten, D. E.; Marsh, G.; de Jong, P. C.; van den Berg, M. Inhibition of human placental aromatase activity by hydroxylated polybrominated diphenyl ethers (OH-PBDEs. Toxicol. Appl. Pharmacol. 2008, 227 (1), 68–75. (6) Our stolen future Website; http://www.ourstolenfuture.org/press/ 2003/2003-0810-NYT-cabanspbdes.htm (accessed December 1, 2010). (7) Peters, A. K.; Nijmeijer, S.; Gradin, K.; Backlund, M.; Bergman, Å.; Poellinger, L.; Denison, M. S.; van den Berg, M. Interactions of polybrominated diphenyl ethers with the aryl hydrocarbon receptor pathway. Toxicol. Sci. 2006, 92, 133–142. (8) Soderstrom, G.; Sellstrom, U.; de Wit, C. A.; Tysklind, M. Photolytic debromination of decabromodiphenyl ether (BDE-209). Environ. Sci. Technol. 2004, 38, 127–132. (9) Dingemans, M. M. L.; de Groot, A.; van Kleef, R. G. D. M.; Berman, Å.; van den Berg, M.; Vijverberg, H. P. M.; Westerink, R. H. S. Hydroxylation increases the neurotoxic potential of BDE-47 to affect exocytosis and calcium homeostasis in PC12 cells. Environ. Health Perspect. 2008, 116, 637–643. (10) Hallgren, S.; Darnerud, P. O. Polybrominated diphenyl ethers (PBDEs), polychlorinated biphenyls (PCBs) and chlorinated paraffins (CPs) in rats-testing interactions and mechanisms for thyroid hormone effects. Toxicology 2002, 177 (23), 227–243. (11) Meerts, I. A. T. M.; Letcher, R. J.; Hoving, S.; Marsh, G.; Bergman, Å.; Lemmen, J. G.; van der Burg, B.; Brouwer, A. In vitro Estrogenicity of polybrominated diphenyl ethers, hydroxylated PBDEs,
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Environmental Science & Technology
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dx.doi.org/10.1021/es104344e |Environ. Sci. Technol. 2011, 45, 5003–5008
