Environ. Sci. Technol. 2006, 40, 3603-3608
New Method for Testing Phototoxicity of Polycyclic Aromatic Hydrocarbons TATSUSHI TOYOOKA AND YUKO IBUKI* Laboratory of Radiation Biology, Graduate School of Nutritional and Environmental Sciences, Institute for Environmental Sciences, University of Shizuoka, Shizuoka, Japan
Polycyclic aromatic hydrocarbons (PAHs), widespread environmental pollutants, were recently reported to show photomutagenesis. As contaminants in the environment are usually exposed to sunlight, a way to evaluate the phototoxic characteristics of pollutants is required. We have previously found that phosphorylation of histone H2AX (γ-H2AX), which accompanied the induction of DNA double strand breaks (DSBs), was significantly induced by low concentrations of benzo[a]pyrene (10-9-10-7 M) and UVA (0.6 J/cm2) in CHO-K1 cells. Higher concentrations have been required for the detection of DSBs. The aim of the present study is to investigate the applicability of γ-H2AX in a new phototoxicity assay of PAHs. The human keratinocytes, HaCaT, were treated with four model PAHs (naphthalene, phenanthrene, pyrene, and benzo[a]pyrene, 10-11-10-7 M) and/or UVA (5 J/cm2), and the induction of γ-H2AX was assessed. Furthermore, DSBs were directly detected using a biased sinusoidal field gel electrophoresis, and the cell viability was examined as a general assay of phototoxicity. The induction of γ-H2AX was detected in the presence of all the PAHs except naphthalene at concentrations of 10-9-10-7 M, whereas neither DSBs nor cell death could be detected at those concentrations, and higher concentrations were required for the detection. Naphthalene showed no phototoxicity in any of the three different assays. These findings suggest that histone H2AX is a potential molecular target for detecting the phototoxicity of PAHs more sensitively than the detection of cell viability and DSBs.
Introduction Polycyclic aromatic hydrocarbons (PAHs) are widespread mutagenic and carcinogenic environmental pollutants (1). The metabolic products of PAHs, such as diol-epoxides and diones, cause DNA covalent adducts and oxidative DNA lesions (2), resulting in mutagenic and carcinogenic effects. On the other hand, because of their multiple aromatic ring system, PAHs can absorb light energy in the ultraviolet (UV) A range, forming reactive species and causing damage to cellular components (3). It has been reported that concomitant exposure to PAHs and UVA can cause certain kinds of DNA damage, single strand breaks (SSBs), oxidative DNA bases, and DNA covalent adducts (4-8). Furthermore, we * Corresponding author phone and fax: +81-54-264-5799; e-mail; ibuki@u-shizuoka-ken. ac.jp. 10.1021/es060182i CCC: $33.50 Published on Web 05/04/2006
2006 American Chemical Society
have previously shown that coexposure to benzo[a]pyrene and UVA induced DNA double strand breaks (DSBs), one of the most serious lesions in cells (9, 10). DSBs and SSBs are produced by ionizing radiation. Considering that exposure to PAHs plus UVA acts in fashion similar to ionizing radiation in terms of DNA damage, a risk of cancer is expected. Recently, Yan et al. (11) demonstrated that 11 of 16 PAHs listed by the U.S. Environmental Protection Agency as priority pollutants were photomutagenic. Until now, the phototoxicity of environmental pollutants has not been fully examined. Much more attention should be paid to the phototoxicity of environmental pollutants such as PAHs, that is, phototoxicity assays should be carried out when examining the toxicities of pollutants including mutagenic and carcinogenic properties. Evaluations of the phototoxicity of chemicals have been performed on animals. However, for ethical and financial reasons, simple and reproducible in vitro tests are required to estimate the phototoxic potential of the large number of chemicals that are currently being developed (12). At present, a neutral red uptake (NRU) assay with 3T3 cells is generally used via an international validation trial (13). Concomitant exposure to environmental pollutants and sunlight might occur on the skin. Thus using keratinocytes for phototoxicity assays is preferable; however, keratinocytes (both primary culture and permanent cell line) are less sensitive than fibroblasts such as 3T3 cells (13, 14). Furthermore, in actual environments, the contamination from pollutants such as PAHs occurs at very low concentrations in the skin. Therefore, more sensitive and specific methods using keratinocytes are required to assay the phototoxicity of environmental pollutants. We have previously shown that coexposure to benzo[a]pyrene and UVA induced phosphorylation of histone H2AX in Chinese hamster ovary (CHO)-K1 cells and found that the induction was achieved at very low doses of benzo[a]pyrene (10-9-10-7 M) and UVA (0.6 J/cm2), near actual environmental concentrations (15). In eucaryotes, DNA is packaged into nucleosomes, which are composed of about 145 bp of DNA and eight histone proteins. Histone H2AX is a minor component of nucleosome core histone H2A (16). The phosphorylation of histone H2AX (termed γ-H2AX) has been recently identified as an early event after the induction of DSBs. Several thousand molecules of H2AX near the site of the DSB are phosphorylated at serine 139 (17, 18). γ-H2AX produces discrete foci within the nucleus that are microscopically visible by immunofluorescence staining. The detection of a single focus of γ-H2AX within the nucleus is the most sensitive method currently available for identifying DSBs (19). We considered that detection of γ-H2AX would be a useful strategy for screening the phototoxicity of PAHs in the environment. The aim of this study was to investigate the applicability of γ-H2AX in a new phototoxicity assay using human keratinocytes. HaCaT cells were treated with four selected PAHs (naphthalene, phenanthrene, pyrene, and benzo[a]pyrene) and/or UVA exposure, and γ-H2AX induction was assessed. Furthermore, DSBs induced by coexposure to PAHs and UVA were directly detected using a biased sinusoidal field gel electrophoresis, and cell viability after the treatments was examined as an end point of phototoxicity. Based on a comparison among the results of the three different methods of detection, the availability of γ-H2AX for a new phototoxicity assay for human skin was discussed. VOL. 40, NO. 11, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 1. Detection of γ-H2AX after coexposure to PAHs and UVA in HaCaT cells. (A) Detection of γ-H2AX by immunofluorescence staining 2 h after treatment with PAHs (10-11-10-7 M) and/or UVA (5 J/cm2). Images represent γ-H2AX by immunofluorescence staining. Asterisks represent significant differences; *p < 0.05, ***p < 0.001. (B) Images of γ-H2AX generated by coexposure to 10-7 M of pyrene and UVA (5 J/cm2). Left panel, γ-H2AX by immunofluorescence staining; center panel, nuclei by PI staining; right panel, merged images. (C) Detection of γ-H2AX by Western blotting 2 h after treatment with PAHs (10-7 M) and/or UVA (5 J/cm2). Actin is a standard for the equal loading of proteins for SDS-PAGE. In the figure, naphthalene, phenanthrene, pyrene, and benzo[a]pyrene are referred to as Nap, Phe, Pyr, and BaP, respectively.
Experimental Section Chemicals. Naphthalene (CAS 91-20-3), phenanthrene (CAS 85-01-8), pyrene (CAS 129-00-0), and benzo[a]pyrene (CAS 50-32-8) were purchased from Sigma-Aldrich (USA). They were dissolved in dimethyl sulfoxide (DMSO) at a stock concentration of 40 mM and stored at -20 °C. Fluorescein diacetate (FDA) (Wako, Japan) was dissolved in acetone at a concentration of 1 mg/mL and the solution was stored at -20 °C. 3604
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Cells and Cell Culture Conditions. The immortalized human keratinocyte, HaCaT, was kindly provided by Dr. Norbert Fusening (German Cancer Research Center, Germany). Cells were maintained in Dulbeccos’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and 100 units/mL penicillin/ streptomycin at 37 °C in an atmosphere of 5% CO2. For experiments, proper cell numbers compatible with each dish size were seeded for 2 days before treatments.
FIGURE 2. Direct detection of DSBs after coexposure to PAHs and UVA in HaCaT cells. HaCaT cells (A) exposed to PAHs (10-7, 10-6, and 10-5 M) and/or UVA (5 J/cm2) or (B) exposed to naphthalene or phenanthrene (2.5 × 10-5, 5 × 10-5, and 10-4 M) and/or UVA (5 J/cm2) were treated as described in the Experimental Section and loaded for BSFGE. The fluorescent staining in the gel indicates generation of DSB. Exposure to PAHs and UVA. HaCaT cells were treated with various concentrations of PAHs (10-4-10-11 M) for 1 h and subsequently irradiated with several doses of UVA (1-5 J/cm2). The UVA lamp (HP-30LM; Atto, Tokyo, Japan) had a spectral output of 3% in the UVB region (10-5 M, >10-7 M, and >10-7 M, respectively. On the other hand, detection of γ-H2AX was possible at the concentrations ranging from 10-9 to 10-7 M of PAHs. In the case of higher concentrations of PAHs (phenanthrene >10-5 M, pyrene and benzo[a]pyrene >10-7
M) and UVA, the assessment of phototoxicity based on γ-H2AX was not appropriate because of acute phototoxicity as described above. The order of ability to generate γ-H2AX was benzo[a]pyrene > pyrene > phenanthrene. Naphthalene did not have any ability to induce γ-H2AX. This order agreed with that for induced phototoxic intensity assessed based on the viability (22, 23) and growth (24) of several kinds of cells as an index. UVA absorption of PAHs is considered to be an important factor to determine phototoxic effect, that is, benzo[a]pyrene and pyrene strongly absorb UVA, but not naphthalene. Furthermore, it was approximately consistent with the photomutagenic ability of PAHs, as examined with Salmonella typhimurium bacteria strain TA102 (8). The results strongly indicated that γ-H2AX is useful for predicting the phototoxicity of PAHs in keratinocytes because it was successfully detected at very low concentrations that did not affect cell viability. Although γ-H2AX could be used to detect phototoxicity in a relatively sensitive manner, naphthalene, a nonphototoxic compound reported by others (22), did not generate γ-H2AX under UVA irradiation even at high concentrations. This is another important reason that γ-H2AX is applicable to phototoxicity assays. This assay is based on counting of γ-H2AX positive cells, which is semiquantitative. In the future, we will improve the method more quantitatively, e.g. counting of the number of foci in nucleus and determination of fluorescence intensity of each foci. In conclusion, the detection of γ-H2AX can be used to predict phototoxicity at very low concentrations of PAHs using keratinocytes. This method is suitable for the initial screening of phototoxic environmental contamination.
Acknowledgments We sincerely thank Prof. Naoki Matsuda (Nagasaki University) for his suggestions about cell culture techniques. This work was supported in part by a Grant-in-Aid for Scientific Research (C) (16510041) from the Ministry of Education, Culture, Sports, Science and Technology, Japan.
Literature Cited (1) Bostrom, C. E.; Gerde, P.; Hanberg, A.; Jernstrom, B.; Johansson, C.; Kyrklund, T.; Rannug, A.; Tornqvist, M.; Victorin, K.; Westerholm, R. Cancer risk assessment, indicators, and guideVOL. 40, NO. 11, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
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(2) (3)
(4)
(5)
(6)
(7)
(8)
(9)
(10)
(11) (12)
(13)
lines for polycyclic aromatic hydrocarbons in the ambient air. Environ. Health Perspect. 2002, 110 (3), 451-488. Xue, W.; Warshawsky, D. Metabolic activation of polycyclic and heterocyclic aromatic hydrocarbons and DNA damage: a review. Toxicol. Appl. Pharmacol. 2005, 206 (1), 73-93. Arfsten, D. P.; Schaeffer, D. J.; Mulveny, D. C. The effects of near-ultraviolet radiation on the toxic effects of polycyclic aromatic hydrocarbons in animals and plants: a review. Ecotoxicol. Environ. Saf. 1996, 33 (1), 1-24. Dong, S.; Hwang, H. M.; Harrison, C.; Holloway, L.; Shi, X.; Yu, H. UVA light-induced DNA cleavage by selected polycyclic aromatic hydrocarbons. Bull. Environ. Contam. Toxicol. 2000, 64 (4), 467-474. Liu, Z.; Lu, Y.; Rosenstein, B.; Lebwohl, M.; Wei, H. Benzo[a]pyrene enhances the formation of 8-hydroxy-2′-deoxyguanosine by ultraviolet A radiation in calf thymus DNA and human epidermoid carcinoma cells. Biochemistry 1998, 37 (28), 1030710312. Dong, S.; Hwang, H. M.; Shi, X.; Holloway, L.; Yu, H. UVA-Induced DNA single-strand cleavage by 1-hydroxypyrene and formation of covalent adducts between DNA and 1-hydroxypyrene. Chem. Res. Toxicol. 2000, 13 (7), 585-593. Ibuki, Y.; Warashina, T.; Noro, T.; Goto, R. Coexposure to benzo[a]pyrene plus ultraviolet A induces 8-oxo-7,8-dihydro-2′deoxyguanosine formation in human skin fibroblasts: preventive effects of anti-oxidant agents. Environ. Toxicol. Pharmacol. 2002, 12, 37-42. Zhang, X.; Wu, R. S.; Fu, W.; Xu, L.; Lam, P. K. Production of reactive oxygen species and 8-hydroxy-2′deoxyguanosine in KB cells co-exposed to benzo[a]pyrene and UV-A radiation. Chemosphere 2004, 55 (10), 1303-1308. Toyooka, T.; Ibuki, Y.; Koike, M.; Ohashi, N.; Takahashi, S.; Goto, R. Coexposure to benzo[a]pyrene plus UVA induced DNA double strand breaks: visualization of Ku assembly in the nucleus having DNA lesions. Biochem. Biophys. Res. Commun. 2004, 322 (2), 631-636. Toyooka, T.; Ibuki, Y.; Takabayashi, F.; Goto, R. Coexposure to benzo[a]pyrene and UVA induces DNA damage: First proof of double-strand breaks in a cell-free system. Environ. Mol. Mutagen. 2005, 47 (1), 38-47. Yan, J.; Wang, L.; Fu, P. P.; Yu, H. Photomutagenicity of 16 polycyclic aromatic hydrocarbons from the US EPA priority pollutant list. Mutat. Res. 2004, 557 (1), 99-108. Spielmann, H.; Lovell, W. W.; Holzle, E.; Johnson, B. E.; Maurer, T.; Miranda, M. A.; Pape, W. J. W.; Sapora, O. Sladowski, D. In vitro phototoxicity testing, The report and recommendations of ECVAM workshop 2. ATLA 1994, 22, 314-348. Spielmann, H.; Balls, M.; Dupuis, J.; Pape, W. J.; Pechovitch, G.; de Silva, O.; Holzhutter, H. G.; Clothier, R.; Desolle, P.; Gerbercik, F.; Liebsch, M.; Lovell, W. W.; Maurer, T.; Pfannenbecker, U.;
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9
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(14)
(15)
(16)
(17)
(18)
(19)
(20)
(21)
(22)
(23)
(24)
Pot-thast, J. M.; Csato, M.; Sladowski, D.; Steiling, W.; Brantom, P. The International EU/COLIPA In Vitro Phototoxicity Validation Study: results of phase II (blind trial). Part 1: the 3T3 NRU phototoxicity test. Toxicol. in Vitro 1998, 12, 305-327. Maier, K.; Schmitt-Landgraf, R.; Siegemund, B. Development of an in vitro test system with human skin cells for evaluation of phototoxicity. Toxicol. in Vitro 1991, 5, 457-461. Toyooka, T.; Ibuki, Y. Co-exposure to benzo[a]pyrene and UVA induces phosphorylation of histone H2AX. FEBS Lett. 2005, 579 (28), 6338-6342. Redon, C.; Pilch, D.; Rogakou, E.; Sedelnikova, O.; Newrock, K.; Bonner, W. M. Histone H2A variants H2AX and H2AZ. Curr. Opin. Genet. Dev. 2002, 12 (2), 162-169. Rogakou, E. P.; Pilch, D. R.; Orr, A. H.; Ivanova, V. S.; Bonner, W. M. DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139. J. Biol. Chem. 1998, 273 (10), 5858-5868. Rogakou, E. P.; Boon, C.; Redon, C.; Bonner, W. M. Megabase chromatin domains involved in DNA double-strand breaks in vivo. J. Cell Biol. 1999, 146 (5), 905-916. Sedelnikova, O. A.; Rogakou, E. P.; Panyutin, I. G.; Bonner, W. M. Quantitative detection of (125)IdU-induced DNA doublestrand breaks with gamma-H2AX antibody. Radiat. Res. 2002, 158 (4), 486-492. Ibuki, Y.; Goto, R. Antiapoptotic effects induced by different wavelengths of ultraviolet light. Photochem. Photobiol. 2002, 75 (5), 495-502. Wang, Y.; Gao, D.; Atencio, D. P.; Perez, E.; Saladi, R.; Moore, J.; Guevara, D.; Rosenstein, B. S.; Lebwohl, M.; Wei, H. Combined subcarcinogenic benzo[a]pyrene and UVA synergistically caused high tumor incidence and mutations in H-ras gene, but not p53, in SKH-1 hairless mouse skin. Int. J. Cancer 2005, 116, 193-199. Kagan, J.; Kagan, E. D.; Kagan, I. A.; Kagan, P. Do polycyclic aromatic hydrocarbons, acting as photosensitizers, participate in the toxic effects of acid rain? ACS Symp. 1987, 327, 191-204. Schirmer, K.; Chan, A. G.; Greenberg, B. M.; Dixon, D. G.; Bols, N. C. Ability of 16 priority PAHs to be photocytotoxic to a cell line from the rainbow trout gill. Toxicology 1998, 127 (1-3), 143-155. Grote, M.; Schuurmann, G.; Altenburger, R. Modeling photoinduced algal toxicity of polycyclic aromatic hydrocarbons. Environ. Sci. Technol. 2005, 39 (11), 4141-4149.
Received for review January 26, 2006. Revised manuscript received March 28, 2006. Accepted April 5, 2006. ES060182I
