Generation Of γ-H2AX Research Articles

Spermatogenesis in mouse testicular organoids with testis-specific architecture, improved germ cell survival and testosterone production

This study presents a biphasic approach to overcome the limitations of current testicular organoid (TO) cultures, including histological heterogeneity, germ cell loss and absence of spermatogenesis. Agarose microwells were utilized to create TOs from prepubertal C57BL/6 J testicular cells. First emphasis was on improving germ cell survival during the initial 2-week reorganization phase by comparingα-MEM + 10% knockout serum replacement (KSR) medium, known to support TO generation in mice, to three optimized media (1-3). Cell densities and culture dynamics were also tested to recreate histological resemblance to testes. After optimizing germ cell survival and cell organization, the effect of growth factors and immunomodulation through CD45+immune cell depletion or dexamethasone (DEX) supplementation were assessed for enhancing spermatogenesis during the subsequent differentiation phase. Testicular cells self-reorganized into organoids resembling the testicular anatomical unit, characterized by one tubule-like structure surrounded by interstitium. Media 1-3 proved superior for organoid growth during the reorganization phase, with TOs in medium 3 exhibiting germ cell numbers (7.4% ± 4.8%) comparable to controls (9.3% ± 5.3%). Additionally, 37% ± 30% demonstrated organized histology from 32 × 103cells under static conditions. Switching toα-MEM + 10% KSR during the differentiation phase increased formation efficiency to 85 ± 7%, along with elevated germ cell numbers, testosterone production (3.1 ± 0.9 ng ml-1) and generation ofγ-H2AX+spermatid-like cells (steps 8-11, 1.2% ± 2.2% of the total). Adding differentiation factors to theα-MEM increased spermatid-like cell numbers to 2.9% ± 5.9%, confirmed through positive staining for CREM, transition protein 1, and peanut agglutinin. Although, these remained diploid with irregular nuclear maturation. DEX supplementation had no additional effect, and immune cell depletion adversely impacted TO formation. The manipulability of TOs offers advantages in studying male infertility and exploring therapies, with scalability enabling high-throughput chemical screening and reducing animal usage in reproductive toxicity and drug discovery studies.

Butyrate Enhances γ-H2AX Induced by Benzo[a]pyrene.

Benzo[a]pyrene (BaP) is known to form DNA adduct following metabolic activation, which causes phosphorylation of histone H2AX (γ-H2AX). Recent studies have shown that histone deacetylase (HDAC) inhibitors enhanced BaP-induced CYP1A1 gene expression. In this study, we examined the relationship between the HDAC inhibitor-augmented metabolic activation and BaP-induced γ-H2AX. Sodium butyrate (SB), a typical HDAC inhibitor, enhanced BaP-induced γ-H2AX. The enhanced DNA damage was further confirmed by biased sinusoidal field gel electrophoresis, which detects DNA double-strand breaks. SB remarkably augmented BaP-induced CYP1A1 gene expression, and CYP1A1-overexpressing cells showed elevated generation of γ-H2AX. Furthermore, SB enhanced intracellular oxidation after treatment with BaP. These results suggested that SB-induced CYP1A1 upregulation facilitated BaP metabolism, which might result in excess DNA adducts or oxidative DNA damages, leading to augmentation of γ-H2AX.

Trichloroethylene exposure results in the phosphorylation of histone H2AX in a human hepatic cell line through cytochrome P450 2E1-mediated oxidative stress.

Trichloroethylene (TCE), a chlorinated hydrocarbon, was recently reclassified as a human carcinogen by the International Agency for Research on Cancer. Genotoxic events are known to be crucial steps in the initiation of cancer. The genotoxic properties of TCE have been examined in many studies using a standard battery of genotoxicity tests both in vitro and in vivo. However, consistent results have not been obtained, and studies investigating the mechanism behind the genotoxicity of this compound are lacking. In the present study, we examined the genotoxicity of TCE by assessing phosphorylated histone H2AX (γ-H2AX), a new sensitive and reliable marker of DNA damage, in WRL-68 cells, cultured human hepatocytes and mouse livers. Our results showed that TCE exposure results in the generation of γ-H2AX, both in vitro and in vivo. By investigating the in vitro mechanism, we found that TCE increases the levels of intracellular reactive oxygen species (ROS) and that this increase in ROS levels is attenuated in the presence of disulfiram, a specific cytochrome P450 2E1 (CYP2E1) inhibitor. Furthermore, γ-H2AX induced by TCE was also attenuated by CYP2E1 inhibitors and the antioxidant N-acetylcysteine. These results suggested that ROS, produced via cytochrome P450 2E1-mediated metabolic processing, is a major causal factor for γ-H2AX generation upon exposure to TCE.

Evaluation of inflammatory and genotoxic effects of smokeless tobacco using an organotypic in vitro human model of oral epithelium

Phosphorylation of histone H2AX (γ-H2AX) occurs following formation of DNA double strand breaks (DSBs). Other types of DNA damage also generate DSBs through DNA replication and repair, leading to the production of γ-H2AX. In the present study, we demonstrated that linear alkylbenzene sulfonates (LAS), the most widely used and non-genotoxic anionic surfactants, could generate γ-H2AX via a novel pathway. Breast adenocarcinoma MCF-7 cells were treated with five kinds of LAS with alkyl chains ranging from 10 to 14 carbon units (C10–C14LAS). The generation of DSBs and subsequent production of γ-H2AX increased in a manner that depended on the number of carbon units in LAS. γ-H2AX could also be generated with non-cytotoxic doses of LAS and was independent of the cell cycle, indicating the non-apoptotic and DNA replication-independent formation of DSBs. The generation of γ-H2AX could be attenuated by EGTA and ZnCl2, deoxyribonuclease-1 (DNase I) inhibitors, as well as by the knockdown of DNase I. LAS weakened the interaction between DNase I and actin, and the enhanced release of DNase I was dependent on the number of carbon units in LAS. DNase I released by the LAS treatment translocated to the nucleus, in which DNase I attacked DNA and generated γ-H2AX. These results suggested that the LAS-induced generation of γ-H2AX could be attributed to the translocation of DNase I to the nucleus through the disruption of actin, and not to LAS-induced DNA damage.

Abstract A33: γH2AX: A molecular marker of DNA damage response in smoking-induced pancreatic ductal adenocarcinoma

Abstract Introduction: Cigarette smoking is an important risk factor for the development pancreatic ductal adenocarcinoma (PDAC). Nicotine and nitrosamines cause pancreatic cell injury and contribute to a cascade of oncogenic events. Among multiple tobacco specific nitrosamines, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is considered to be the most carcinogenic and induces DNA double strand-breaks (DSBs). However, identification of potential prognostic biomarkers and experimental models of smoking-induced PDAC remain unknown. We have studied phosphorylated histone H2AX (γH2AX), a marker for double-stranded DNA breaks, as a new biomarker for detecting cigarette smoke-induced pathogenesis of PDAC. Experimental Procedure: We have characterized the expression of γH2AX protein in human pancreatic tissues, human pancreatic duct epithelial (HPDE)6, PDAC cell lines, and PanIn, primary PDAC (PDA) and liver metastasis (LMP) cell lines generated from a genetically engineered mice model of PDAC. Kaplan-Meier survival analysis was performed to determine the significance of γH2AX expression amongst smokers and non-smokers with PDAC. HPDE6 and PanIN cells were cultured for 4 days in the presence or absence of cigarette smoke (CS) (0-25μg/ml) or NNK (0-10μM). Cell cycle analysis and apoptosis were assessed through flow cytometry with the markers propidium iodide and annexin V respectively. HPDE6 and PanIN cells treated with CS or NNK for up to 50 days were analyzed for rapid activation of DNA damage response (DDR) pathway proteins γH2AX, RAD51, BRCA1, BRCA2, phospho-CHK2 and phospho-CHK1. Cells were treated with either CS or NNK along with a CHK2 inhibitor and analyzed for activation of γH2AX. To determine DNA integrity, accumulation of γH2AX was measured by ELISA in cells treated with CS or NNK. Immunofluorescence confocal microscopy was utilized to examine the role of γH2AX on nuclear foci formation with DNA repair complexes by quantifying the amount of co-localization. To replicate chronic smoke exposure, we treated HPDE6 and PanIn cells with 1 and 10 µg/ml CS or 10nM and 1µM NNK for 10 passages, and examined these cell lines for DSBs by comet assay, apoptosis markers by protein array and in vitro tumorigenicity by colony formation assay. Results: γH2AX expression was significantly higher (p<0.0001) in smokers when compared to non-smokers. Overall survival of patients with high γH2AX expression in their primary tumor was associated with significantly decreased median survival (11 months) when compared to low γH2AX expression (36 months, p=0.0007). HPDE6 cells exposed to CS or NNK, showed more than 80% survival. The surviving cells had altered morphology, acquired mesenchymal characteristics and displayed increased tumorigenicity suggesting that these cells acquire resistance to apoptosis after CS or NNK exposure. In addition, exposure of cells to CS or NNK resulted in generation of γH2AX and induction of phospho-CHK2 expression. RAD51 and phospho-CHK2 co-localize with γH2AX after DNA damage with CS or NNK exposure. Inhibition of CHK2 reduced expression of γH2AX. The product of the tumor suppressor gene BRCA2 co-localized with γH2AX indicating that γH2AX is necessary for the recruitment of DNA repair factors to the site of DNA damage. BRCA2 mutant cell line CAPAN1 exhibited a substantially reduced ability to form co-localization with RAD51 and γH2AX, consistent with a role of γH2AX in DNA damage repair. Conclusion: This work supports the role of γH2AX as a new molecular biomarker to assess the contribution of cigarette smoking-induced PDAC. γH2AX is necessary for DNA damage repair in PDAC and its activation is dependent on CHK2. Therapies that can block γH2AX expression by inhibiting upstream kinases may be a new potential therapeutic approach to treat and/or prevent PDAC. Citation Format: Kumaraswamy Honnenahally, Chanjuan Shi, Xi Chen, Jason Castellanos, Nipun Merchant, Timothy Blackwell, Nagaraj Nagathihalli. γH2AX: A molecular marker of DNA damage response in smoking-induced pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A33.

γ-H2AX induced by linear alkylbenzene sulfonates is due to deoxyribonuclease-1 translocation to the nucleus via actin disruption

Phosphorylation of histone H2AX (γ-H2AX) occurs following formation of DNA double strand breaks (DSBs). Other types of DNA damage also generate DSBs through DNA replication and repair, leading to the production of γ-H2AX. In the present study, we demonstrated that linear alkylbenzene sulfonates (LAS), the most widely used and non-genotoxic anionic surfactants, could generate γ-H2AX via a novel pathway. Breast adenocarcinoma MCF-7 cells were treated with five kinds of LAS with alkyl chains ranging from 10 to 14 carbon units (C10–C14LAS). The generation of DSBs and subsequent production of γ-H2AX increased in a manner that depended on the number of carbon units in LAS. γ-H2AX could also be generated with non-cytotoxic doses of LAS and was independent of the cell cycle, indicating the non-apoptotic and DNA replication-independent formation of DSBs. The generation of γ-H2AX could be attenuated by EGTA and ZnCl2, deoxyribonuclease-1 (DNase I) inhibitors, as well as by the knockdown of DNase I. LAS weakened the interaction between DNase I and actin, and the enhanced release of DNase I was dependent on the number of carbon units in LAS. DNase I released by the LAS treatment translocated to the nucleus, in which DNase I attacked DNA and generated γ-H2AX. These results suggested that the LAS-induced generation of γ-H2AX could be attributed to the translocation of DNase I to the nucleus through the disruption of actin, and not to LAS-induced DNA damage.

Evaluation of chemical phototoxicity, focusing on phosphorylated histone H2AX.

Histone H2AX is a minor component of nuclear histone H2A. The phosphorylation of histone H2AX at Ser 139, termed γ-H2AX, was originally identified as an early event after the direct formation of DNA double-strand breaks (DSBs) by ionizing radiation. Now, the generation of γ-H2AX is also considered to occur in association with secondarily formed DSBs by cellular processing such as DNA replication and repair at the site of the initial damage, including DNA adducts, crosslinks, and UV-induced photolesions. Therefore, γ-H2AX is currently attracting attention as a new biomarker for detecting various genotoxic insults. We have determined the toxic impact of various environmental stresses such as chemicals, light and/or their coexposure using γ-H2AX, and found that the γ-H2AX assay exhibited high sensitivity and a low false-positive rate as a detection system of genotoxic potential. In this review, we introduced our recent findings concerning the evaluation of chemical phototoxicity, focusing on γ-H2AX.

Silver ions enhance UVB-induced phosphorylation of histone H2AX.

Silver (Ag) is used in a wide range of industries including healthcare, food, cosmetics, and environmental industries due to its antibacterial properties. The rapidly expanding use of Ag has raised issues concerning its toxicity in humans. However, studies investigating the effects of Ag on humans are very limited, and the combined effects of Ag and other environmental factors have not yet been determined. Ultraviolet (UV) radiation in sunlight is the most prominent and ubiquitous physical stressor in our natural environment. In this study, we investigated the genotoxic potential of combined exposure to Ag(+) (AgNO3) and UVB in the human keratinocyte cell line, HaCaT, by measuring the generation of phosphorylated histone H2AX, which is currently attracting attention as a biomarker for the detection of genotoxic insults. We found that the generation of γ-H2AX was synergistically enhanced when cells were coexposed to Ag(+) and UVB. Furthermore, we showed that the enhanced generation of γ-H2AX could be attributed to the increased formation of UVB-induced cyclobutane pyrimidine dimers and (6-4) photoproducts. These lesions, if not repaired properly, are the major causal factor for skin carcinogenesis. Our results provide an important insight into influence of Ag on the genotoxic potency of sunlight.

Nonylphenol Polyethoxylates Degraded by Three Different Wavelengths of UV and Their Genotoxic Change—Detected by Generation of γ‐H2AX

UV rays in sunlight are an important factor in the degradation of chemicals. In this study, we investigated the degradation of nonionic surfactants, nonylphenol polyethoxylates (NPEOs) with 10 or 70 ethylene oxide (EO) units using UVA, B and C, and their genotoxic change based on phosphorylation of histone H2AX (γ-H2AX), a marker of DNA damage. NPEOs were degraded dependent on the energy of UV, that is, UVC having the highest energy was most effective, whereas UVA having the lowest energy caused little change. The EO side chain of NPEO(70) was broken near the benzene ring by UV, producing NPEOs with a shortened EO chain (around 10 units). The generation of γ-H2AX reflected the pattern of degradation; shortening of the EO chain changed NPEO(70) into an inducer for γ-H2AX, and degradation of NPEO(10) attenuated the genotoxicity. The γ-H2AX generated by NPEO(10) and UV-degraded NPEO(70) was independent of the cell cycle. The formation of DNA double strand breaks detected by gel electrophoresis was consistent with the results for γ-H2AX. These results suggested that UV rays can make NPEOs harmless or genotoxic according to the degradation of the EO side chain, the effects being dependent on wavelength.

UVB irradiation changes genotoxic potential of nonylphenolpolyethoxylates— remarkable generation of γ-H2AX with degradation of chemical structure

Nonylphenolpolyethoxylates (NPEOs) are non-ionic surfactants widely used for industrial and household purposes. In actual environments, NPEOs can be biodegraded, but the products are reported to be more persistent and toxic than the parent compounds. NPEOs are also exposed to sunlight and degraded. Studies on the photodegradation of NPEOs have focused mainly on chemical changes after exposure to light. Toxic changes of photodegraded products correlating to the chemical changes are not completely understood. In this study, we examined the genotoxicity of UVB-irradiated NPEOs having ethylene oxide units 15 and 70 in a human breast adenocarcinoma cell line, MCF-7, based on the phosphorylation of histone H2AX (γ-H2AX), a sensitive marker for DNA damage. We clarified that UVB irradiation drastically changed the genotoxic potential of NPEOs: NPEO(15)'s ability to generate γ-H2AX was significantly reduced, whereas non-genotoxic NPEO(70) became able to generate γ-H2AX. Flow cytometric analysis showed that the γ-H2AX generated by UVB-irradiated NPEO(70)was produced independent of cell cycle phases. In addition, its production involved the activation of ATM or DNA-PK, a general signalling pathway in response to DNA double strand breaks. High-performance liquid chromatography analysis indicated that the formation of NPEO intermediates with a short side-chain like NPEO(15) was the cause of the γ-H2AX generation. This study suggests the importance of taking the genotoxicity of photodegraded intermediates into consideration when conducting risk assessments of environmental pollutants.

AIF-mediated caspase-independent necroptosis requires ATM and DNA-PK-induced histone H2AX Ser139 phosphorylation

The alkylating DNA-damage agent N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) induces a form of caspase-independent necroptosis implicating the mitochondrial flavoprotein apoptosis-inducing factor (AIF). Following the activation of PARP-1 (poly(ADP-ribose) polymerase-1), calpains, BID (BH3 interacting domain death agonist), and BAX (Bcl-2-associated X protein), the apoptogenic form of AIF (tAIF) is translocated to the nucleus where, associated with Ser139-phosphorylated histone H2AX (γH2AX), it creates a DNA-degrading complex that provokes chromatinolysis and cell death by necroptosis. The generation of γH2AX is crucial for this form of cell death, as mutation of H2AX Ser139 to Ala or genetic ablation of H2AX abolish both chromatinolysis and necroptosis. On the contrary, reintroduction of H2AX-wt or the phosphomimetic H2AX mutant (H2AX-S139E) into H2AX−/− cells resensitizes to MNNG-triggered necroptosis. Employing a pharmacological approach and gene knockout cells, we also demonstrate in this paper that the phosphatidylinositol-3-OH kinase-related kinases (PIKKs) ATM (ataxia telangiectasia mutated) and DNA-dependent protein kinase (DNA-PK) mediate γH2AX generation and, consequently, MNNG-induced necroptosis. By contrast, H2AX phosphorylation is not regulated by ATR or other H2AX-related kinases, such as JNK. Interestingly, ATM and DNA-PK phosphorylate H2AX at Ser139 in a synergistic manner with different kinetics of activation. Early after MNNG treatment, ATM generates γH2AX. Further, DNA-PK contributes to H2AX Ser139 phosphorylation. In revealing the pivotal role of PIKKs in MNNG-induced cell death, our data uncover a milestone in the mechanisms regulating AIF-mediated caspase-independent necroptosis.

Titanium dioxide particles phosphorylate histone H2AX independent of ROS production

With the rising use of nano-sized particles in nanotechnology, harmful effects of TiO 2 particles which have been recognized as a safe material, are of increasing concern. In this study, we examined the genotoxicity of two different sized TiO 2 particles in the lung adenocarcinoma epithelial cell line A549 based on the phosphorylation of histone H2AX (γ-H2AX), recently regarded as a sensitive marker for DNA damage. TiO 2 particles generated γ-H2AX, which was more remarkable with the smaller particles. Flow cytometric analysis showed that the generation was independent of cell cycle phases and cells which incorporated larger amounts of TiO 2 particles had more significant γ-H2AX. Although there are some reports that the incorporation of nanomaterials into cells generates reactive oxygen species (ROS), the level of ROS was low even if large amounts of TiO 2 particles were taken-up. In addition, the generation of γ-H2AX by TiO 2 particles was more significant than that after treatment with hydrogen peroxide. On the other hand, the generation of γ-H2AX was attenuated by coating the surface of TiO 2 particles with bovine serum albumin. These results suggested that smaller TiO 2 particles were easy to incorporate into cells and generated cell cycle phase-independent γ-H2AX, which was dependent on the condition of the TiO 2 surface but not on the formation of ROS.

Nonylphenol polyethoxylates induce phosphorylation of histone H2AX

Nonylphenol polyethoxylates (NPEOs) are non-ionic surfactants widely used for industrial and household purposes. Since biodegraded short chain NPEOs were reported to elicit estrogenic activity in organisms, numerous studies have been carried out to assess the endocrine-disrupting potential of NPEOs; however, the genotoxicity of the compounds is not fully known, let alone the relationship between the genotoxic potential and number of ethylene oxide (EO) units of NPEOs. In this study, we examined the genotoxicity of NPEO( n) having various EO units ( n = 0, 5, 10, 15, 20, 30, 40 and 70) in a human breast adenocarcinoma cell line, MCF-7, based on the phosphorylation of histone H2AX (γ-H2AX), recently regarded as a sensitive marker for DNA damage. We clarified that NPEOs have the ability to form γ-H2AX via activation of ATM or DNA-PK, a general signaling pathway in response to DSBs, and this ability was strongly dependent on the number of EO units, that is, NPEO(0–15) having smaller numbers of EO units more readily generated γ-H2AX. Flow cytometric analysis revealed that the generation of γ-H2AX was independent of cell cycle phases. Although the mechanism by which the NPEOs generated γ-H2AX was not able to be elucidated in the present study, it was clear that the involvement of reactive oxygen species and apoptotic DNA fragmentation were not causal factors. The generation of γ-H2AX means the formation of DSBs, the worst type of DNA damage. The results indicated that attention should be paid to degradated short chain NPEOs and their genotoxicity.

UVB in solar-simulated light causes formation of BaP-photoproducts capable of generating phosphorylated histone H2AX

Polycyclic aromatic hydrocarbons (PAHs), wide-spread mutagenic and carcinogenic environmental pollutants, are consistently exposed to sunlight in the environment. Our previous paper showed that benzo[ a]pyrene (BaP) exposed to solar-simulated light (SSL) induced phosphorylation of histone H2AX (γ-H2AX) [T. Toyooka, G. Ohnuki, Y. Ibuki, Solar-simulated light-exposed benzo[a]pyrene induces phosphorylation of histone H2AX, Mutat. Res. 650 (2008) 132–139], a marker of DNA double strand breaks. In this study, we found the ultraviolet B (UVB) region of SSL to produce photomodified BaP with high cytotoxicity and the ability to generate γ-H2AX. Degradation of BaP by SSL, resulting in an increase in cytotoxicity and the generation of γ-H2AX, was decreased by UVB-masking using a glass plate. Exposure to UVB itself increased the cytotoxicity of BaP and amount of γ-H2AX generated. Other PAHs, 1,2-benzoanthracene and 1,2:5,6-dibenzoanthracene, which absorb UVB, also showed enhanced cytotoxicity and the promoted the generation of γ-H2AX after exposure to SSL, whereas naphthalene and chrysene, which have low absorption in the UVB region, did not. These findings suggested that UVB is important for the degradation of PAHs having absorbance in this region, but that the production of genotoxic intermediates during the degradation process needs to be considered. UVB is a two-edged blade in environments, effectively degrading toxic chemicals but also producing genotoxic compounds as reactive intermediates.

Cigarette sidestream smoke induces phosphorylated histone H2AX

Cigarette sidestream smoke (CSS) is a widespread environmental pollutant having highly genotoxic potency. In spite of the overwhelming evidence that CSS induces a wide range of DNA damage such as oxidative base damage and DNA adducts, evidence that CSS can result in DNA double strand breaks (DSBs) is little. In this study, we showed that CSS generated phosphorylated histone H2AX (γ-H2AX), recently considered as a sensitive marker of the generation of DSBs, in a human pulmonary epithelial cell model, A549. Treatment with CSS drastically induced discrete foci of γ-H2AX within the nucleus in a dose-dependent manner. CSS increased intracellular oxidation, and N-acetylcysteine (NAC), an antioxidant, significantly attenuated the formation of γ-H2AX, suggesting that reactive oxygen species produced from CSS partially contributed to the phosphorylation. The generation of γ-H2AX is considered to be accompanied the induction of DSBs. CSS in fact induced DSBs, which was also inhibited by NAC. DSBs are the worst type of DNA damage, related to genomic instability and carcinogenesis. Our results would increase the evidence of the strong genotoxicity of passive smoking.

Solar-simulated light-exposed benzo[ a]pyrene induces phosphorylation of histone H2AX

Polycyclic aromatic hydrocarbons (PAHs), wide-spread mutagenic and carcinogenic environmental pollutants, are consistently exposed to sunlight in the environment. The exposure causes structural change, resulting in the generation of a variety of photomodified products having different bioactivities compared with the parent compounds. In this study, we found that benzo[ a]pyrene (BaP) exposed to solar-simulated light (SSL)-induced phosphorylation of histone H2AX (γ-H2AX), which was recently identified as an early event after the induction of DNA double strand breaks (DSBs). Although BaP itself did not produce γ-H2AX, SSL-exposed BaP significantly generated γ-H2AX depending on the period of exposure. Furthermore, we revealed that reactive oxygen species produced by the SSL-exposed BaP mainly contributed to the generation of γ-H2AX. The appearance of γ-H2AX means the induction of the most serious form of DNA damage, DSBs, suggesting the potential risk of carcinogenesis.