UVB-Exposed Chlorinated Bisphenol A Generates Phosphorylated

Aug 26, 2008 - Yuko Ibuki,*,† Yukinori Tani,‡ and Tatsushi Toyooka†. Laboratory of Radiation Biology and Laboratory of EnVironmental Microbiolog...
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UVB-Exposed Chlorinated Bisphenol A Generates Phosphorylated Histone H2AX in Human Skin Cells Yuko Ibuki,*,† Yukinori Tani,‡ and Tatsushi Toyooka† Laboratory of Radiation Biology and Laboratory of EnVironmental Microbiology, Graduate School of Nutritional and EnVironmental Sciences, Institute for EnVironmental Sciences, UniVersity of Shizuoka, 52-1 Yada, Shizuoka-shi 422-8526, Japan ReceiVed April 11, 2008

Bisphenol A (BPA) and chlorinated bisphenol A (ClBPAs) were detected in wastewater from waste paper recycling plants. Previously, we showed that exposure to UV augmented the toxicity of ClBPAs [Mutou (2006) EnViron. Toxicol. Pharmacol. 21, 283-289 and Mutou (2008) Toxicol. in Vitro 22, 864-872]. BPA and ClBPAs are exposed to sunlight in the environment; however, research concerning the change of toxicity during their photodegradation is scarce, especially for ClBPAs. In this study using human keratinocytes and skin fibroblasts, we found that 3,3′-dichlorobisphenol A (3,3′-diClBPA) exposed to UVB induces phosphorylation of histone H2AX, the event considered to be a marker of formation of DNA double strand breaks. The cells treated with the UVB-exposed 3,3′-diClBPA formed clear foci of phosphorylated histone H2AX in the nucleus. Unchlorinated BPA caused no phosphorylation of histone H2AX even when exposed to high doses of UVB (∼200J/cm2). HPLC analysis clarified that several compounds with increased hydrophilicity were produced from 3,3′-diClBPA by UVB irradiation, not from BPA, suggesting the chlorinated chemical structure to be important for the degradation and generation of products related to the phosphorylation of histone H2AX. In separated peaks of 3,3′-diClBPA exposed to UVB, peak fluctuation of 3-hydroxybisphenol A (3-OHBPA) was consistent with the UVB dosedependent appearance of phosphorylated histone H2AX. We suspected that some oxidized BPA involving 3-OHBPA produced by UVB irradiation contributed to the phosphorylation. Considering that the phosphorylation of histone H2AX is required for maintaining the genome’s stability and the repair of DNA, attention to photoproducts from chlorinated compounds is important for the risk evaluation of chemicals.

Bisphenol A (BPA)1 is a synthetic monomer widely used to polymerize polycarbonate plastics and epoxy resins and as a developer in dyes for thermal paper in the paper industry. It was detected in river water at more than 50% of locations investigated in Japan and recently at high concentrations in the effluent from pulping processes for waste paper containing thermal paper and/or other printed paper (1, 2). The bleaching process at waste paper recycling plants using sodium hypochlorite easily chlorinates BPA derived from thermal paper, and the chlorinated derivatives of BPA (ClBPAs) are then released into the wastewater (1–3). Several ClBPAs (3-chlorobisphenol A, 3,3′-dichlorobisphenol A, and 3,3′,5-trichlorobisphenol A) interact more strongly with estrogen receptors than BPA, which is itself known as an endocrine disruptor (2, 4–6), and more significant toxicity was observed in cells treated with 3-chlorobisphenol A and 3,3′-dichlorobisphenol A (3,3′-diClBPA) than with BPA (7).

In addition, the toxicity after photochemical reactions was also significantly different between BPA and ClBPAs (7, 8). The inhibition of cell growth by ClBPAs was augmented by irradiation with UVB or UVC at 100 J/cm2, whereas unchlorinated BPA irradiated with the same dose did not have toxic effects and higher doses were required to induce similar toxicity. Furthermore, 3-hydroxybisphenol A (3-OHBPA) and 3-chloro3′-hydroxybisphenol A were detected in the photoproducts of 3-ClBPA and 3,3′-diClBPA irradiated with UVB or UVC at 100 J/cm2 (7), which might contribute to the augmentation of toxicity. These findings suggested the toxic oxidized BPAs to be formed more easily in ClBPAs than BPA under UV. Many studies about the photochemical reactions of environmental chemicals have been carried out in recent years and showed that some oxidized chemicals were produced. Zhan et al. (9) estimated monohydroxylated BPA and p-hydroquinone as photoproducts of BPA. However, research concerning differences in degradation pattern and photoproducts between unchlorinated and chlorinated BPAs, and the toxicity of their photoproducts, is lacking.

* To whom correspondence should be addressed. Tel/Fax: +81-54-2645799. E-mail: [email protected]. † Laboratory of Radiation Biology. ‡ Laboratory of Aquatic and Soil Environment. 1 Abbreviations: BPA, bisphenol A; BSFGE, biased sinusoidal field gel electrophoresis; ClBPAs, chlorinated bisphenol A; 3,3′-diClBPA, 3,3′dichlorobisphenol A; DSBs, DNA double strand breaks; FCM, flowcytometer; FDA, fluorescein diacetate; γ-H2AX, phosphorylated histone H2AX; 3-OHBPA, 3-hydroxybisphenol A; PI, propidium iodide; PMSF, phenylmethylsulfonyl fluoride; ROS, reactive oxygen species.

In eukaryotes, DNA is packaged into nucleosomes, the core of which is an octameric particle consisting of two each of the class H2A, H2B, H3, and H4 histones. Histone proteins have become a focus of research in many fields because their modification has an important role in the initiation and promotion of cancer (10, 11). H2AX is a minor component of histone H2A, and its phosphorylation has been recently identified as an early event after the formation of DNA double strand breaks

Introduction

10.1021/tx800129n CCC: $40.75  2008 American Chemical Society Published on Web 08/26/2008

UVB-Exposed Cl-BPA Phosphorylates Histone H2AX

(DSBs) (12). Within minutes after the introduction of a DSB, several thousand H2AX near the site of the break are phosphorylated at serine 139, producing foci within the nucleus that are microscopically visible by immunofluorescence staining (13). Although the exact role of the phosphorylation of H2AX is still controversial, it appears to be mainly associated with maintaining the genome’s integrity and protection against tumorigenesis by participating in the repair of DSBs (14–16). The generation of phosphorylated histone H2AX (γ-H2AX) has been wellstudied using general DSB inducers such as ionizing radiation and anticancer drugs, but recently, environmental chemicals such as arsenite (17, 18), methyl methanesulfonate, N-ethyl-Nnitrosourea, benzo[a]pyrene (19), etc. have been found to generate γ-H2AX. We also detected γ-H2AX generated by sunlight-irradiated benzo[a]pyrene, which was due to the production of reactive oxygen species (ROS) (20). Some researchers have reported that hydrogen peroxide generated γ-H2AX (21, 22). Therefore, we suspected that the UV-exposed ClBPAs, in which oxidized products such as 3-OHBPA are formed, might generate γ-H2AX and that the pattern of induction differs between BPA and ClBPAs. In this study, the generation of γ-H2AX by BPA and 3,3′diClBPA exposed to UVB was examined. UVB-exposed 3,3′diClBPA caused significant generation of γ-H2AX in a UVB dose-dependent manner, whereas BPA did not generate γ-H2AX even when exposed to high doses of UVB. HPLC analysis confirmed that 3-OHBPA as one of the photoproducts was formed from 3,3′-diClBPA according to UVB exposure, which was one of causes of the generation of γ-H2AX. This study first found that chlorinated BPA exposed to UVB generates γ-H2AX, considered an index of DSBs, indicating the need to pay attention to chlorinated chemicals whose behavior changes under UV.

Experimental Procedures Chlorinated BPA and UVB Irradiation. BPA (>99%) was purchased from Kanto Chemical Co. Inc. (Japan). 3,3′-diClBPA and 3-OHBPA were synthesized according to previously described procedures (1). They were dissolved in ethanol and kept in the dark at 4 °C as a 100 mM stock solution. BPA and 3,3′-diClBPA (200 µM) solutions were prepared by diluting appropriate volumes of stock solutions with Milli-Q Water (0.2% ethanol) in a glass dish 19 mm in diameter and 11 mm in height. A UVB lamp (HP-30LM; Atto Co., Japan) with a 280-320 nm emission and a maximum peak of 312 nm was used for irradiation. The fluence was simultaneously measured and integrated using a radiometer (ATV-3W; Atto Co., Japan) with a 312 nm detector that was placed at the same distance as the glass dish from the UVB source. The approximate irradiance at the sample level was 1.0 mW/cm2. Cells and Culture Conditions. The immortalized human keratinocyte, HaCaT, was kindly provided by Dr. N. Fusening (German Cancer Research Center, Germany). ASF4-1 fibroblasts established from normal human skin were kindly provided by Dr. K. Kaji (University of Shizuoka, Japan). These cells were cultured in Dulbecco’s modified Eagle medium containing 10% fetal bovine serum. In the experiments, the cells were used at a logarithmic phase of growth. The population doubling level of ASF4-1 cells was 33.5. Survival after Treatment with BPA and 3,3′-diClBPA. HaCaT and ASF4-1 cells were cultured with BPA and 3,3′-diClBPA for 24 h. For the determination of viability, the cells were suspended in PBS containing fluorescein diacetate (FDA) (0.1 µg/mL) and propidium iodide (PI) (2 µg/mL). Cells were incubated for 10 min at 37 °C. Viability was determined using a flowcytometer (FCM) (Epics XL; Coulter, FL). FDA, a substrate of esterases, was hydrolyzed, charged, and then entrapped in living cells. PI can stain

Chem. Res. Toxicol., Vol. 21, No. 9, 2008 1771 the nucleus of dead cells. The percentages of cells stained positive with FDA and negative with PI were calculated to give the rate of survival. Western Blot Analysis for Detection of γ-H2AX. Cells were cultured with BPA, 3,3′-diClBPA, and 3-OHBPA for 4 h and lysed in lysis buffer [50 mM Tris (pH 8.0), 5 mM EDTA, 150 mM NaCl, 0.5% Nonidet P-40, and 1 mM phenylmethylsulfonyl fluoride (PMSF)]. Samples containing 100 µg of whole cell protein were separated by 12.5% SDS-PAGE and blotted onto polyvinylidine fluoride (PVDF) membranes. After they were blocked with 10% nonfat milk, the membranes were incubated with primary antibody against phospho-H2AX (Upstate Biotechnology, VA, 1:1000) overnight at 4 °C or against actin (Santa Cruz Biotechnology Inc., CA, 1:1000) for 1 h at room temperature and then with secondary antibody conjugated with HRP (Jackson Immuno Research Laboratories, PA) for 1 h. Protein expression was visualized with an enhanced chemiluminescence detection kit (GE Healthcare Ltd., United Kingdom). Immunofluorescence Staining for Detection of γ-H2AX. HaCaT cells grown on Lab-Tek chamber slides (Nalge Nunc, IL) were treated with BPA and 3,3′-diClBPA. Treated cells were immediately fixed in 2% paraformaldehyde for 5 min at room temperature and then in 100% methanol for 20 min at -20 °C. Fixed cells were immersed in buffer containing 100 mM Tris-HCl, 50 mM EDTA, and 0.5% Triton X-100 for 20 min at room temperature for better permealization and blocked with 1% bovine serum albumin for 30 min at 37 °C. Cells were washed with PBS and incubated with primary antibody against phospho-H2AX (1: 200) for 2 h and then with secondary antibody conjugated with fluorescein isothiocyanate (FITC) (Jackson Immuno Research Laboratories, PA). To confirm the distribution of foci, the nucleus was stained with PI (20 µg/mL). Images were acquired on a fluorescence microscope (IX70, Olympus Co. Japan). Cells were judged as “positive” for γ-H2AX foci if they displayed five or more discrete dots of brightness. At least 300 cells were counted for each experimental condition. Detection of Double Strand Breaks. DSBs were detected with a biased sinusoidal field gel electrophoresis system (BSFGE) (Atto. Co., Japan) as described in a previous paper (23). In brief, ASF4-1 cells (1 × 106) treated with BPA and 3,3′-diClBPA were solidified in 1% low-melting agarose plugs. As a positive control, the cells were treated with hydrogen peroxide (1 and 10 mM). The solidified agarose plugs were treated with proteinase K (1 mg/mL) for 24 h at 50 °C, with ribonuclease-A (1 mg/mL) for 1 h at 37 °C, and subsequently with PMSF (40 µg/mL) for 30 min at 50 °C. The plugs were placed in a 0.8% agarose gel and electrophoresed in 0.5 × TBE buffer (89 mM Tris-borate and 2 mM EDTA) for 32 h at 20 °C. The gel was visualized by staining with ethidium bromide. HPLC Analysis of UVB-Exposed BPA and 3,3′-diClBPA. UVB-exposed BPA and 3,3′-diClBPA were analyzed using a LC10 series HPLC system (Shimadzu, Japan), connected with a diode array (PDA) detector, monitoring absorbance at 240-350 nm. The separation was carried out on a reverse-phase column (Wakosil 5C18T, 4.6 mm i.d. × 250 mm, 5 µm particles, Wako Chemical Ind., Japan). The column temperature was 30 °C. Mobile phase solvent A consisted of a mixture of methanol/50 mM ammonium formate (pH 3.0) (40:60 by volume). Solvent B was 100% methanol. Linear gradient elution from 100% A to 100% B over 20 min was followed by an isocratic hold for 10 min at 100% B. The flow rate was 1.0 mL/min. Statistics. Values are means ( SD (n ) 3-8). Data were analyzed with a one-way ANOVA followed by Dunnett’s t test for the comparison of multiple samples with a control sample. Statistical significance was reported when p < 0.05 and p < 0.01 and was expressed as one or two asterisks, respectively.

Results Generation of γ-H2AX after Treatment with Chlorinated BPA Exposed to UVB. BPA and 3,3′-diClBPA were exposed to UVB (25, 50, 100, and 200 J/cm2). HaCaT cells were treated

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Figure 1. Generation of γ-H2AX after treatment with BPA and 3,3′-diClBPA exposed to UVB. γ-H2AX was detected by immunofluorescence staining and Western blotting. HaCaT and ASF4-1 cells were treated with BPA and 3,3′-diClBPA (100 µM) exposed to several doses of UVB for 4 h. (A) Images of γ-H2AX foci generated after treatment of HaCaT cells with BPA and 3,3′-diClBPA exposed to UVB (100 J/cm2). Left panel, γ-H2AX detected by immunofluorescence staining; center panel, nuclei detected by PI staining; and right panel, merged images. Upper panel, untreated; middle panel, treated with BPA exposed to UVB; and lower panel, treated with 3,3′-diClBPA exposed to UVB. (B) Expanded images of γ-H2AX foci generated after treatment with 3,3′-diClBPA exposed to UVB. (C) Percentages of γ-H2AX-positive cells. Cells having more than five foci within the nucleus were counted as γ-H2AX-positive. *p < 0.05 and **p < 0.01. (D) Western blot analysis of γ-H2AX generated after treatment of ASF4-1 cells with BPA and 3,3′-diClBPA exposed to UVB. Actin is a standard for the equal loading of proteins for SDS-PAGE, and the values (γ-H2AX/actin) in graphs are expressed as a ratio to the untreated control. These experiments were repeated twice, and similar results were obtained.

with those photoproducts for 4 h, and γ-H2AX was detected by immunofluorescence staining (Figure 1A-C). γ-H2AX is known to produce discrete foci within the nucleus that are microscopically visible by immunofluorescence staining. PI staining shows the location of the nucleus. No foci of γ-H2AX were detected in untreated cells and UVB-exposed BPA-treated cells, whereas many foci of γ-H2AX were observed within the nucleus after the treatment with UVB-exposed 3,3′-diClBPA (Figure 1A). The foci are expressed as expanded images in Figure 1B. Figure 1C shows the percentages of γ-H2AX-positive cells having more than five foci within the nucleus. Percentages increased dependent on the dose of UVB up to 100 J/cm2 in

the 3,3′-diClBPA-treated cells. No γ-H2AX-positive cell was detected among BPA-treated cells even when the dose of UVB increased (∼200 J/cm2). The generation of γ-H2AX by UVBexposed 3,3′-diClBPA was also confirmed by Western blotting in ASF4-1 cells (Figure 1D). γ-H2AX was detected in cells treated with 3,3′-diClBPA exposed to 25-100 J/cm2 of UVB. This was similar in HaCaT cells (data not shown). Relationship between Cytotoxicity and Generation of γ-H2AX by Chlorinated BPA Exposed to UVB. Figure 2 shows the cytotoxicity of UVB-exposed BPA and 3,3′-diClBPA in HaCaT and ASF4-1 cells. BPA showed no cytotoxicity in either cell line, whereas 3,3′-diClBPA exposed to 25-100 J/cm2

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Figure 2. Cytotoxicity of BPA and 3,3′-diClBPA exposed to UVB. HaCaT and ASF4-1 cells were treated with BPA and 3,3′-diClBPA (100 µM) exposed to several doses of UVB. The cells were cultured for 24 h and suspended in PBS containing FDA. They were incubated for 10 min at 37 °C. Viability was determined using a FCM; *p < 0.05.

of UVB was slightly toxic to HaCaT cells and significantly toxic to ASF4-1 cells. The cell death was observed 24 h after the treatment but not 4 h (the generation of γ-H2AX was detected at this time). Furthermore, this toxicity was induced at 100 µM 3,3′-diClBPA and not at the lower concentrations (∼50 µM) (data not shown). On the other hand, 3,3′-diClBPA exposed to 100 J/cm2 of UVB generated γ-H2AX in a concentrationdependent manner (Figure 3). At the concentrations that induced no toxicity, γ-H2AX was significantly generated. Recently, γ-H2AX has become a marker of apoptosis because apoptosis involves DNA fragmentation (24). In this study, γ-H2AX was generated at short time and at low doses of 3,3′-diClBPA, which did not induce cell death, showing that the γ-H2AX was due to DNA damage by 3,3′-diClBPA, not to apoptosis. DSBs after treatment with BPA and 3,3′-diClBPA were detected by BSFGE (Figure 4). In the positive control, hydrogen peroxide induced the fragmentation of DNA. However, no fragmented DNA was detected in BPA- and 3,3′-diClBPAtreated cells. Degradation of BPA and 3,3′-diClBPA after Exposure to UVB. Photoproducts of BPA and 3,3′-diClBPA after exposure to UVB were analyzed by HPLC (Figure 5). UVB did not degradate BPA at up to 200 J/cm2, whereas 3,3′diClBPA produced many photoproducts with greater hydrophilicity than 3,3′-diClBPA itself. We have previously confirmed the existence of 3-OHBPA in photoproducts of 3,3′-diClBPA (7). In Figure 5, we identified 3-OHBPA by comparing its retention time and UV spectrogram to those of synthesized standard 3-OHBPA. The production of 3-OHBPA, the peak of which was seen with 3,3′-diClBPA exposed to 50-100 J/cm2, was consistent with the generation of γ-H2AX in Figure 1. The 3-OHBPA generated the γ-H2AX dose dependently (Figure 6). The photoproduct of 100 µM 3,3′-diClBPA caused the formation of γ-H2AX similar to that by 50 µM 3-OHBPA. In the HPLC analysis as shown in Figure 5, the production rate of 3-OHBPA was about 4% of the initial 3,3′-diClBPA, indicating that other oxidized compounds also correlated with the generation of γ-H2AX.

Discussion γ-H2AX has been studied as part of the cellular response to the formation of DSBs because it is clearly generated immediately after irradiation with γ- and X-rays (12, 13), and the number of γ-H2AX foci/cell and number of DSBs/cell by ionizing radiation were strikingly similar (25). In addition, γ-H2AX plays an important role in the processing and repair of DSBs, which is crucial for the maintenance of the genome’s

Figure 3. 3,3′-diClBPA concentration-dependent generation of γ-H2AX. γ-H2AX was assessed by immunofluorescence staining (A) and Western blotting (B). (A) HaCaT cells were treated with several concentrations of 3,3′-diClBPA exposed to UVB (100 J/cm2) for 4 h. Immunofluorescence staining against γ-H2AX was carried out as in Figure 1A, and cells having more than five foci within the nucleus were counted as γ-H2AX-positive. *p < 0.05 and **p < 0.01. (B) ASF4-1 cells were treated with several concentrations of 3,3′-diClBPA exposed to UVB (100 J/cm2) for 4 h. Western blotting against γ-H2AX was carried out as in Figure 1D. These experiments were repeated twice, and similar results were obtained.

integrity and stability, since H2AX-/- mice exhibited sensitivity to radiation, growth retardation, etc. (14–16). Consequently, high endogeneous levels of γ-H2AX appear in some tumor cells (26). Recently, stressors other than typical DSB inducers such as ionizing radiation and anticancer agents were also reported to generate γ-H2AX foci. Arsenite (17, 18), methyl methanesulfonate, N-ethyl-N-nitrosourea (19), and oxidized benzo[a]pyrene (20) form γ-H2AX foci in a time- and dose-dependent manner. In this study, we clarified that UVB-exposed 3,3′diClBPA-generated γ-H2AX foci. This was attributable to the formation of oxidized BPA such as 3-OHBPA because UVB dose-dependent generation of γ-H2AX was consistent with the

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Figure 4. Induction of DSBs after treatment with BPA and 3,3′diClBPA exposed to UVB. ASF4-1 cells were treated with BPA and 3,3′-diClBPA (100 µM) exposed to several doses of UVB for 4 h. As a positive control, the cells were treated with hydrogen peroxide. They were solidified in 1% low-melting agarose and treated as described in the Experimental Procedures. The gel stacks containing the cells were loaded onto a 0.8% agarose gel, and BSFGE was carried out.

appearance of a 3-OHBPA peak identified by HPLC analysis and synthesized 3-OHBPA-generated γ-H2AX. In the reversedphase HPLC analysis, products more hydrophilic than 3,3′diClBPA itself were detected in a UVB dose-dependent manner, implying that some oxidized products along with 3-OHBPA also contributed to the generation of γ-H2AX. We propose three mechanisms by which γ-H2AX is generated by UVB-exposed 3,3′-diClBPA. (i) Oxidized products of 3,3′diClBPA after UVB irradiation produced ROS, which induced DSBs. We have previously shown that sunlight-exposed benzo[a]pyrene generated γ-H2AX foci, mainly due to ROS produced by oxidized photoproducts of benzo[a]pyrene (20). ROS would cause DSBs not directly but through proximity of two single strand breaks, and the repair of multiple damaged sites within 10-15 bp might contribute to the generation. Hydrogen peroxide has been reported to induce H2AX’s phosphorylation (21, 22). Zhao et al. (22) discussed that phosphorylation of H2AX occurs by metabolically generated oxidants. In MCF-7 cells having the estrogen receptor, BPA caused γ-H2AX (27), possibly via BPA-derived oxidants in the process of metabolism. In the skin cells used in this study, BPA could not generate γ-H2AX. The relationship of γ-H2AX with the metabolism of oxidized products of 3,3′-diClBPA is unclear. (ii) DNA adducts of oxidized products of 3,3′-diClBPA by UVB irradiation formed, and DSBs formed in the processing and repair of the adducts. Benzo[a]pyrene, the metabolic products of which cause DNA covalent adducts, were reported to induce the formation of γ-H2AX foci (19). As oxidized BPA as BPA 3,4-quinone could form adducts with dG in DNA (28–30), the generation of γ-H2AX in the process of repair of DNA adducts with oxidized BPA is expected. (iii) Oxidized products of 3,3′diClBPA inhibited the repair of DNA strand breaks. A nongenotoxic stressor, heat shock-generated γ-H2AX, was speculated to be caused by heat-induced inhibition of the repair of DSBs (31). At present, the exact mechanism is not understood; however, this study indicated that γ-H2AX becomes an indicator of DNA damage by environmental chemicals, not only DSBs but also DNA adducts, oxidative DNA lesions, etc., which eventually or partially produce DSBs.

Figure 5. HPLC analysis of degradation of BPA and 3,3′-diClBPA by UVB irradiation. BPA and 3,3′diClBPA after UVB irradiation were analyzed as described in the Experimental Procedures. The peak of 3-OHBPA identified with a synthesized standard (b). Percentages of 3-OHBPA in total peaks have been calculated, and the graph has been inserted in the chromatograph of 3,3′-diClBPA.

Although the relationship between the generation of γ-H2AX and DSBs is described above, clear DSBs could not be detected using BSFGE after treatment with UVB-exposed 3,3′-diClBPA. In addition, γ-H2AX foci formed even at concentrations of UVB-exposed 3,3′-diClBPA, which have no effect on survival. Our previous studies showed that the phototoxicity of PAHs induced by photosensitizing events could be detected sensitively using γ-H2AX; that is, γ-H2AX was detectable at a low concentration of PAHs, which did not affect cell viability, indicating that γ-H2AX was applicable to the sensitive evaluation of phototoxicity (32). Furthermore, toxicity induced by photomodified benzo[a]pyrene also was detectable at doses, which showed no effect on survival and no DSBs by BSFGE (20). Yu et al. (33) reported that γ-H2AX foci formed very soon after chemical treatment, the damage not detectable by neutral comet assay, suggesting the value of γ-H2AX as a sensitive indicator for DNA damage. Although the route to γ-H2AX might vary, it is clear that γ-H2AX is usable as a sensitive indicator for the toxicity of several chemicals. A more notable finding of this study is that 3,3′-diClBPA was degradated by UVB exposure easier than unchlorinated BPA, resulting in the formation of γ-H2AX foci, whereas UVBexposed BPA did not generate γ-H2AX foci. The photodegradation of halogenated compounds has been characterized (34–37). UV irradiation induced a wavelength-dependent photoreaction in a halogenated chemical structure (34, 35). For

UVB-Exposed Cl-BPA Phosphorylates Histone H2AX

Figure 6. Generation of γ-H2AX after treatment with 3-OHBPA. ASF4-1 cells were treated with several concentrations of 3-OHBPA and 100 µM UVB-exposed 3,3′-diClBPA for 4 h. Western blotting against γ-H2AX was carried out as in Figure 1D. This experiment was repeated twice, and similar results were obtained.

example, dechlorination of polychlorinated biphenyl occurred first on the benzene ring with more chlorines attached and at a higher electric charge distribution of carbon (36, 37). Dechlorination of 3,3′-diClBPA during UVB exposure started at the early stage of irradiation and was completed after irradiation with 50-75 J/cm2 (6). Dechlorination would be important for augmentation of the production of oxidized chemicals such as 3-OHBPA. At up to 200 J/cm2 of UVB, no by-production of BPA was detected in the HPLC analysis, and the formation of γ-H2AX foci and cell death were not induced. Some oxidized products, monohydroxylated BPA and p-hydroquinone, were estimated as photoproducts of BPA (10). Higher doses of UVB would induce a similar induction in BPA as 3,3′-diClBPA exposed to lower doses of UVB. Our data provide for the first time evidence that oxidized BPA produced from chlorinated BPA by exposure to UVB generates γ-H2AX and that the chlorinated chemical structure is important for the production. The generation of γ-H2AX means the induction of DSBs, the worst type of DNA damage. This study highlights the risk of chlorinated compounds after exposure to UV. Acknowledgment. We thank Prof. N. Fusening and Prof. K. Kaji for kindly providing the cell lines and Prof. Y. Terao (University of Shizuoka) for the synthesized 3,3′-diClBPA and 3-OHBPA. This work was supported in part by the Sumitomo Foundation and a Grant-in-Aid for Scientific Research (C) (19510071) from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

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