Bisphenol A Promotes Cell Survival Following Oxidative DNA Damage in Mouse Fibroblasts

February Bisphenol A Promotes Cell Survival Following Oxidative DNA Damage in Mouse Fibroblasts Natalie R. Gassman 0 1 Erdem Coskun 0 1 Donna F. Stefanick 0 1 Julie K. Horton 0 1 Pawel Jaruga 0 1 Miral Dizdaroglu 0 1 Samuel H. Wilson 0 1 0 1 Genomic Integrity and Structural Biology Laboratory, NIEHS, National Institutes of Health , 111 T.W. Alexander Drive, Research Triangle Park , NC 27709, United States of America, 2 Biomolecular Measurement Division, National Institute of Standards and Technology , Gaithersburg, MD 20899 , United States of America, 3 Faculty of Pharmacy, Gazi University , Ankara , Turkey 1 Academic Editor: Robert W Sobol, University of South Alabama Mitchell Cancer Institute, UNITED STATES Bisphenol A (BPA) is a biologically active industrial chemical used in production of consumer products. BPA has become a target of intense public scrutiny following concerns about its association with human diseases such as obesity, diabetes, reproductive disorders, and cancer. Recent studies link BPA with the generation of reactive oxygen species, and base excision repair (BER) is responsible for removing oxidatively induced DNA lesions. Yet, the relationship between BPA and BER has yet to be examined. Further, the ubiquitous nature of BPA allows continuous exposure of the human genome concurrent with the normal endogenous and exogenous insults to the genome, and this co-exposure may impact the DNA damage response and repair. To determine the effect of BPA exposure on base excision repair of oxidatively induced DNA damage, cells compromised in double-strand break repair were treated with BPA alone or co-exposed with either potassium bromate (KBrO3) or laser irradiation as oxidative damaging agents. In experiments with KBrO3, co-treatment with BPA partially reversed the KBrO3-induced cytotoxicity observed in these cells, and this was coincident with an increase in guanine base lesions in genomic DNA. The improvement in cell survival and the increase in oxidatively induced DNA base lesions were reminiscent of previous results with alkyl adenine DNA glycosylase-deficient cells, suggesting that BPA may prevent initiation of repair of oxidized base lesions. With laser irradiation-induced DNA damage, treatment with BPA suppressed DNA repair as revealed by several indicators. These results are consistent with the hypothesis that BPA can induce a suppression of oxidized base lesion DNA repair by the base excision repair pathway. - Funding: This research was supported by Research Project Numbers Z01-ES050158 and Z01-ES050159 in the Intramural Research Program of the National Institutes of Health, National Institute of Environmental Health Sciences. NRG is funded by 1K99ES023813-01. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Certain commercial equipment or materials are identified in Bisphenol A (BPA) is found in a variety of consumer products such as adhesives, food and beverage containers, and dental composites and sealants [1]. Concern about BPA exposure is often linked to its estrogenic properties, but the affinity of BPA for cellular estrogen receptors is much lower than that of estradiol [2,3]. Additionally, there are inconsistent data regarding Competing Interests: The authors have declared that no competing interests exist. genotoxicity of BPA [47]. Despite these inconsistencies, BPA exposure has been shown to cause DNA damage independently of its estrogenic properties [2,6,810]. The response of DNA repair pathways to BPA exposure and BPA-induced DNA damage, however, has not been extensively investigated. DNA damaging effects of BPA are thought to occur indirectly through the generation of reactive oxygen species (ROS). ROS create stable base lesions and abasic sites in genomic DNA [1114]. While previous studies had pointed to DNA damaging effects of BPA, the oxidatively induced DNA damage produced by BPA exposure has not been investigated, nor has BPA exposure in combination with other DNA damaging agents, especially other oxidizing agents. The ubiquity of BPA results in exposure concurrent with endogenous and exogenous DNA damaging events, like oxidative stress or environmental toxicants, and together these exposures can increase the DNA damage load of genomic DNA and have implications for genomic stability and human disease development and progression. In the current study, we sought to address the influence of BPA on the oxidative DNA damage response in the model experimental system of cultured mouse fibroblasts. The base excision repair (BER) pathway is the main repair system responsible for removal of modified bases (such as 8-oxo-guanine (8-oxoGua) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua)) formed upon oxidative stress. In the cases of the 8-oxoGua and FapyGua lesions, 8-oxoGua-DNA glycosylase (OGG1) removes the lesions from doublestranded genomic DNA leaving abasic sites. While OGG1 is known as a bifunctional enzyme capable of carrying out both base removal and AP-lyase activity, cleaving the phosphodiester bond of the resulting abasic site by a - or --elimination mechanism, its AP-lyase activity is relatively weak [1517]. Therefore, another enzyme, AP endonuclease 1 (APE1) incises the abasic site, resulting in a single-nucleotide gapped DNA with 3-OH and 5-dRP groups at the gap margins. Subsequently, DNA polymerase (Pol ) loads onto this BER intermediate, removes the 5-dRP group, and then fills the single-nucleotide gap. DNA ligase I, or in some cases the ligase III -XRCC1 complex, then seals the nick in the repair intermediate to complete the pathway. Repair of other oxidized bases, such as 5-hydroxycytosine (5-OH-Cyt), thymine glycol (ThyGly), and 4,6-diamino-5-formamidopyrimidine (FapyAde), are initiated by other DNA glycosylases, e.g., NEIL1 and NTH, and these glycosylases have overlapping substrate specificities including excision of FapyGua by NEIL1 [18,19]. Cells make use of the BER pathway as a first-line defense against oxidized base damage induced by endogenous and exogenous agents, but other DNA repair pathways can back-up a deficiency in BER. To examine an effect of BPA on the response to oxidative stress in mouse fibroblasts, we chose to use a Ku70-deficient cell line. These cells were selected because they are deficient in strand break repair by non-homologous end joining (NHEJ), a back-up repair pathway for BER [20,21]. Thus, NHEJ-deficient cells provide an opportunity for study of BER responses to oxidatively induced DNA damage in the absence of a back-up repair pathway. Cell phenotypes were characterized after treatment with the oxidative stress agent KBrO3, or with the combination of BPA plus KBrO3. Ku70+/+ and Ku70-/- (a gift from Dr. Shigemi Matsuyama, Cleveland, OH) mouse embryonic fibroblasts (MEFs) were grown at 37C in a 10% CO2 incubator in Dulbeccos modified Eagles medium (DMEM) supplemented with glutamine, 10% fetal bovin (...truncated)