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. Environ. Mol. Mutagen. 55:556–565, 2014. © 2014 Wiley Periodicals, Inc.     

Water soluble fraction of solar-simulated light-exposed crude oil generates phosphorylation of histone H2AX in human skin cells under UVA exposure

Crude oil contains compounds, which have toxic and cancer-causing properties to humans. The oil spilled in environments is usually exposed to sunlight; however, the toxicity of sunlight-exposed oil is poorly understood. In this study, we found that the water soluble fraction (WSF) of crude oil irradiated with solar-simulated light (SSL) generated phosphorylation of histone H2AX (gamma-H2AX) in human skin cells under UVA irradiation, which was due to the formation of DNA double strand breaks (DSBs). Crude oil was exposed to SSL for approximately 7 days. The WSF obtained from unexposed crude oil showed no toxicity, whereas the WSF obtained from crude oil pre-exposed to SSL induced acute cell death on exposure to UVA irradiation (induction of phototoxicity), which was more remarkable in human skin fibroblasts than human skin keratinocytes. gamma-H2AX was detected in both cell lines immediately after treatment with the WSF plus UVA. Interestingly, gamma-H2AX was detectable even at low SSL- and UVA-doses, which induced no cytotoxicity. The WSF of crude oil irradiated with SSL, generated DSBs under UVA irradiation, which were detected by biased sinusoidal field gel electrophoresis. This was confirmed using xrs-5 cells isolated from CHO-K1 cells, which are deficient in a repair enzyme for DSBs; the WSF plus UVA induced a more dramatic decrease in survival in xrs-5 cells than CHO-K1 cells. These findings demonstrate that exposure of crude oil to sunlight makes the WSF phototoxic, generating DSBs accompanying the appearance of gamma-H2AX in human skin cells.     

γH2AX formation kinetics in PBMCs of rabbits exposed to acute and fractionated radiation and attenuation of focus frequency through preadministration of a combination of podophyllotoxin and rutin hydrate

DNA damage can be assessed by the quantitation of γH2AX foci that form at DSB sites. This study examines the generation and persistence of γH2AX foci, variability in foci size after acute and fractionated radiation exposure, and the effect of pretreatment with a safe radioprotective formulation termed G‐003M on foci generation and persistence. G‐003M contains a combination of podophyllotoxin and rutin hydrate, and was administered intramuscularly to rabbits 1 hr prior to Co⁶⁰ gamma irradiation. Rabbits were assigned to one of the following treatment groups: untreated, G‐003M alone, irradiated (single dose 8 Gy, fractionated 2 Gy/day for 4 days or single dose 2 Gy) or G‐003M preadministration followed by radiation exposure. Foci continuously persisted for a week in peripheral blood mononuclear cells of rabbits exposed to a single 8 Gy dose. However, the number of foci gradually decreased after reaching a maximum at 1 h. In rabbits exposed to fractionated radiation, foci detected 1 hr after the final exposure were significantly larger (P < 0.001) than in rabbits exposed to a single 8 Gy dose, but disappeared completely after 24 h. In both groups, foci reappeared on days 11‐15 in terminally ill animals. G‐003M pretreatment significantly (P < 0.05) attenuated the formation of γH2AX foci in all irradiated rabbits. This study reveals that γH2AX focus assessment could be used to confirm radiation exposure, that focus size reflects the type of radiation exposure (acute or fractionated), that the re‐appearance of foci is a strong indicator of imminent death in animals, and that G‐003M provides protection against radiation. Environ. Mol. Mutagen. 57:455–468, 2016. © 2016 Wiley Periodicals, Inc.     

Comparison of PAX-2, RCC antigen, and antiphosphorylated H2AX antibody (gamma-H2AX) in diagnosing metastatic renal cell carcinoma by fine-needle aspiration

Diagnosing metastatic renal cell carcinoma (RCC) by fine-needle aspiration (FNA) can be challenging. Existing antibodies supporting a diagnosis of RCC, including CD10 and RCC-Ma, have problems with specificity and interpretation. In this report, we evaluate the use of two newer immunostains, PAX-2 and gamma-H2AX, which to our knowledge have not been studied in FNA material, in the diagnosis of metastatic RCC and in comparison with RCC-Ma. 29 cases of metastatic RCC were identified as well as a TMA of an additional 30 RCC cases. In the case cohort, RCC-Ma in a membranous pattern of staining identified 15/27 (56%) metastatic RCC, although interpretation was made difficult in many cases due to focality of staining and non-specific cytoplasmic staining. PAX-2 stained 23/29 (79%) of tumors in a nuclear stain, most strongly. Gamma-H2AX stained 19/26 (73%) of metastatic RCC strongly in a nuclear stain. In the TMA, strong, diffuse nuclear staining with gamma-H2AX was present in 22/30 RCC (73%). If weak staining was also included as positive, 26/30 (87%) were positive. PAX-2 stained RCC TMA with a lower percentage at 56%, including weaker staining intensity. Both PAX-2 and gamma-H2AX demonstrated patchy staining of normal renal tubules, PAX-2 to a greater extent. Both PAX-2 and gamma-H2AX are sensitive markers for the diagnosis of metastatic RCC, with improved ease of interpretation when compared with RCC-Ma. A combination of all 3 markers identified 87% of cases, and failure to stain for both PAX-2 and gamma-H2AX suggests against, but does not disprove, a diagnosis of RCC.     

Enhanced sensitivity of the C3H/10T1/2 cell transformation system to alkylating and chemotherapeutic agents by treatment with 12-O-tetradecanoylphorbol-13-acetate

The failure of the C3H/10T1/2 cell transformation system to respond to numerous known carcinogens has limited its applications for the detection and study of cancer-causing substances. Recent studies have found, however, that some carcinogens function as initiating agents for the process of transformation in these cells. Treatment with such agents is generally not sufficient to transform low-density asynchronous cultures of C3H/101/2 cells, but morphologic transformation will occur if such cultures are subsequently exposed to the potent tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). In the present study, the ability of TPA to enhance transformation was examined in cultures treated with a variety of chemical agents. The addition of TPA after chemical treatment enhanced the transformation of these cells by methylmethanesulfonate, ethylmethanesulfonate, N-methyl-N′-nitro-N-nitrosoguanidine, N-nitrosomethylurea, N-nitrosoethylurea, mitomycin C, 5-fluorodeoxyuridine, and 5-azacytidine. Treatment with amethopterin or benzo(e)pyrene did not produce significant numbers of foci in the presence or absence of TPA. TPA inhibited transformation by high concentrations of 3-methylcholanthrene and benzo(a)pyrene. Thus, numerous carcinogens function as initiating agents for these cells and the presence of TPA can dramatically increase the sensitivity of this cell transformation system.     

Direct quantification of gamma H2AX by cell‐based high throughput screening for evaluation of genotoxicity of pesticides in a human thyroid cell lines

Genotoxicity is thought to be the cause of many cancers. Genotoxicity due to environmental toxins may be partly responsible for the dramatic increase in the incidence of papillary thyroid cancer over the past two decades. Here, we present a fully automatable assay platform that directly quantifies the phosphorylation of nuclear histone gamma H2AX (γH2AX), a specific cellular marker for DNA double strand breaks (DSBs) via immunohistochemistry and laser scanning cytometry. It multiplexes γH2AX with total cell number measured as propidium iodide and calculates the percentage of cells with DSBs. Validation of this assay using NTHY‐ori‐3‐1 human thyroid cells and etoposide showed that it was an excellent choice for high throughput applications. We used the assay to test the genotoxic effects of the EPA Toxcast Phase 1 pesticide library of 309 compounds. Compounds were evaluated in dose response and the DSB was quantified. We found that 19 pesticides induce DSB in vitro, highlighting a need to further assess these pesticides for their long‐term oncogenic effects on the thyroid gland. Environ. Mol. Mutagen. 58:522–528, 2017. © 2017 Wiley Periodicals, Inc.     

Genetic and environmental influence on DNA strand break repair: A twin study

Accumulation of DNA damage deriving from exogenous and endogenous sources has significant consequences for cellular survival, and is implicated in aging, cancer, and neurological diseases. Different DNA repair pathways have evolved in order to maintain genomic stability. Genetic and environmental factors are likely to influence DNA repair capacity. In order to gain more insight into the genetic and environmental contribution to the molecular basis of DNA repair, we have performed a human twin study, where we focused on the consequences of some of the most abundant types of DNA damage (single‐strand breaks), and some of the most hazardous lesions (DNA double‐strand breaks). DNA damage signaling response (Gamma‐H2AX signaling), relative amount of endogenous damage, and DNA‐strand break repair capacities were studied in peripheral blood mononuclear cells from 198 twins (94 monozygotic and 104 dizygotic). We did not detect genetic effects on the DNA‐strand break variables in our study. Environ. Mol. Mutagen. 54:414–420, 2013. © 2013 Wiley Periodicals, Inc.     

Hydroxyurea induces chromosomal damage in G2 and enhances the clastogenic effect of mitomycin C in Fanconi anemia cells

Fanconi's anemia (FA) is a recessive disease; 16 genes are currently recognized in FA. FA proteins participate in the FA/BRCA pathway that plays a crucial role in the repair of DNA damage induced by crosslinking compounds. Hydroxyurea (HU) is an agent that induces replicative stress by inhibiting ribonucleotide reductase (RNR), which synthesizes deoxyribonucleotide triphosphates (dNTPs) necessary for DNA replication and repair. HU is known to activate the FA pathway; however, its clastogenic effects are not well characterized. We have investigated the effects of HU treatment alone or in sequential combination with mitomycin‐C (MMC) on FA patient‐derived lymphoblastoid cell lines from groups FA‐A, B, C, D1/BRCA2, and E and on lymphocytes from two unclassified FA patients. All FA cells showed a significant increase (P < 0.05) in chromosomal aberrations following treatment with HU during the last 3 h before mitosis. Furthermore, when FA cells previously exposed to MMC were treated with HU, we observed an increase of MMC‐induced DNA damage that was characterized by high occurrence of DNA breaks and a reduction in rejoined chromosomal aberrations. These findings show that exposure to HU during G2 induces chromosomal aberrations by a mechanism that is independent of its well‐known role in replication fork stalling during S‐phase and that HU interfered mainly with the rejoining process of DNA damage. We suggest that impaired oxidative stress response, lack of an adequate amount of dNTPs for DNA repair due to RNR inhibition, and interference with cell cycle control checkpoints underlie the clastogenic activity of HU in FA cells. Environ. Mol. Mutagen. 56:457–467, 2015. © 2015 Wiley Periodicals, Inc.     

Geminin ablation in vivo enhances tumorigenesis through increased genomic instability

Geminin, a DNA replication licensing inhibitor, ensures faithful DNA replication in vertebrates. Several studies have shown that geminin depletion in vitro results in rereplication and DNA damage, whereas increased expression of geminin has been observed in human cancers. However, conditional inactivation of geminin during embryogenesis has not revealed any detectable DNA replication defects. In order to examine its role in vivo, we conditionally inactivated geminin in the murine colon and lung, and assessed chemically induced carcinogenesis. We show here that mice lacking geminin develop a significantly higher number of tumors and bear a larger tumor burden than sham‐treated controls in urethane‐induced lung and azoxymethane/dextran sodium sulfate‐induced colon carcinogenesis. Survival is also significantly reduced in mice lacking geminin during lung carcinogenesis. A significant increase in the total number and grade of lesions (hyperplasias, adenomas, and carcinomas) was also confirmed by hematoxylin and eosin staining. Moreover, increased genomic aberrations, identified by increased ATR and γH2AX expression, was detected with immunohistochemistry analysis. In addition, we analyzed geminin expression in human colon cancer, and found increased expression, as well as a positive correlation with ATM/ATR levels and a non‐monotonic association with γH2AX. Taken together, our data demonstrate that geminin acts as a tumor suppressor by safeguarding genome stability, whereas its overexpression is also associated with genomic instability. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

TiO2 nanoparticles induce DNA double strand breaks and cell cycle arrest in human alveolar cells

TiO₂ nanoparticles (NPs) have the second highest global annual production (～3000 tons) among the metal‐containing NPs. These NPs are used as photocatalysts for bacterial disinfection, and in various other consumer products including sunscreen, food packaging, therapeutics, biosensors, surface cleaning agents, and others. Humans are exposed to these NPs during synthesis (laboratory), manufacture (industry), and use (consumer products, devices, medicines, etc.), as well as through environmental exposures (disposal). Hence, there is great concern regarding the health effects caused by exposure to NPs and, in particular, to TiO₂ NPs. In the present study, the genotoxic potential of TiO₂ NPs in A549 cells was examined, focusing on their potential to induce ROS, different types of DNA damage, and cell cycle arrest. We show that TiO₂ NPs can induce DNA damage and a corresponding increase in micronucleus frequency, as evident from the comet and cytokinesis‐block micronucleus assays. We demonstrate that DNA damage may be attributed to increased oxidative stress and ROS generation. Furthermore, genomic and proteomic analyses showed increased expression of ATM, P53, and CdC‐2 and decreased expression of ATR, H2AX, and Cyclin B1 in A549 cells, suggesting induction of DNA double strand breaks. The occurrence of double strand breaks was correlated with cell cycle arrest in G2/M phase. Overall, the results indicate the potential for genotoxicity following exposure to these TiO₂ NPs, suggesting that use should be carefully monitored. Environ. Mol. Mutagen. 56:204–217, 2015. © 2014 Wiley Periodicals, Inc.     

Follow-up evaluation of radiation-induced DNA damage in CSF disseminated high-grade glioma using phospho-histone H2AX antibody

Cytological examination of cerebrospinal fluid (CSF) is used not only for the diagnosis of spinal disease, but also to assess the postoperative effect of treatment. We experienced a case of high-grade glioma in disseminated CSF, and retrospectively examined the clinical, pathological and cytological features. We further investigated radiation-induced DNA damage in glioma cells using phospho-Histone H2AX antibody. A five-year-old boy received a clinical diagnosis of optic nerve glioma, and was followed-up for three months after chemotherapy. Magnetic resonance imaging was repeated, revealing abnormalities in other brain areas. The pathological diagnosis was anaplastic astrocytoma. CSF dissemination was detected, and increases in the number and mitosis of tumor cells were observed in CSF cytology. After radiotherapy the tumor cells in CSF decreased markedly. On cytomorphologic and immunocytochemical evaluation post-irradiation, tumor cells showed vacuolation of both the nucleus and cytoplasm, degeneration of nuclear chromatin, and alteration of the phospho-Histone H2AX expression, compared with tumor cells before the irradiation. CSF cytology is an effective means of evaluating DNA damage in tumor cells after irradiation, and may be useful in assessing the therapeutic response.     

Increased level of DNA damage in some organs of obese Zucker rats by γ‐H2AX analysis

In a recent study, we showed that lymphocytes of obese Italian children/adolescents displayed levels of double strand breaks (DSB), assayed as serine 139‐phosphorylated histone H2AX (γ‐H2AX), about eightfold higher than normal weight controls, and that 30% of this damage‐generated micronuclei. These findings suggested that obese children could be at increased risk of obesity‐mediated cancer later in life. We therefore aimed to assess the level of γ‐H2AX in a genetic animal model of obesity (Zucker rat) to identify a genotoxic/carcinogenic risk in some organs. The DSB marker was studied in 3‐ to 4‐week‐old rats and in 9‐ to 13‐week‐old rats. Paraffin‐embedded sections of heart, thyroid, liver, pancreas, lung, kidney, esophagus, and gut from the fa?/fa? (obese) and the fa+/fa? (lean) control animals were processed for immunohistochemistry detection of γ‐H2AX. Pancreas (0.0624 ± 0.0195), lung (0.1197 ± 0.0217), esophagus (0.1230 ± 0.0351), kidney (0.1546 ± 0.0149), and gut (0.1724 ± 0.0352) of 9‐ to 13‐week‐old obese rats showed a higher proportion of γ‐H2AX‐positive nuclei, than their lean counterparts (0.0092 ± 0.0033, 0.0416 ± 0.0185, 0.0368 ± 0.0088, 0.0686 ± 0.0318, and 0.0703 ± 0.0239, respectively). No difference was seen in the 3‐ to 4‐week‐old age group with regard to obesity, indicating that the DNA damage increased with older age of the rats. We hypothesize that the organs of the obese animals showing high levels of DSB could represent target tissues for the development of obesity‐related cancers. Environ. Mol. Mutagen. 58:477–484, 2017. © 2017 Wiley Periodicals, Inc.     

DNA damage foci: Meaning and significance

The discovery of DNA damage response proteins such as γH2AX, ATM, 53BP1, RAD51, and the MRE11/RAD50/NBS1 complex, that accumulate and/or are modified in the vicinity of a chromosomal DNA double‐strand break to form microscopically visible, subnuclear foci, has revolutionized the detection of these lesions and has enabled studies of the cellular machinery that contributes to their repair. Double‐strand breaks are induced directly by a number of physical and chemical agents, including ionizing radiation and radiomimetic drugs, but can also arise as secondary lesions during replication and DNA repair following exposure to a wide range of genotoxins. Here we aim to review the biological meaning and significance of DNA damage foci, looking specifically at a range of different settings in which such markers of DNA damage and repair are being studied and interpreted. Environ. Mol. Mutagen. 56:491–504, 2015. © Wiley Periodicals, Inc.     

Prooxidant activity of flavonoids: copper-dependent strand breaks and the formation of 8-hydroxy-2'-deoxyguanosine in DNA

The naturally occurring flavonoids caused strand scission of DNA in the presence of copper ion. Flavonoids such as myricetin, baicalein, and quercetin as well as ascorbic acid cleaved plasmid pBR322 DNA and calf thymus DNA potently. Addition of catalase protected DNA from the strand breaks caused by flavonoids. Treatment of calf thymus DNA with these flavonoids or ascorbate plus copper produced 8-hydroxy-2'-deoxyguanosine. Cuprous ion reduced by flavonoids and ascorbic acid may play a key role in the oxidative cleavage of DNA and the formation of base adduct. Mutagenic and carcinogenic action of flavonoids may be explained by the prooxidant effects of the compounds.     

Coexposure to benzo[a]pyrene and UVA induces DNA damage: first proof of double-strand breaks in a cell-free system

DNA damage induced by solar ultraviolet (UV) radiation plays an important role in the induction of skin cancer. Although UVA constitutes the majority of solar UV radiation, it is less damaging to DNA than UVB. The DNA damage produced by UVA radiation, however, can be augmented in the presence of a photosensitizer. We previously used benzo[a]pyrene (BaP), an environmental carcinogenic polycyclic aromatic hydrocarbon, as an exogenous photosensitizer, and demonstrated that combined exposure to BaP and UVA resulted in DNA double-strand breaks (DSBs) in cultured Chinese hamster ovary (CHO-K1) cells. In this study, we investigated whether coexposure to BaP and UVA induces DSBs in a cell-free system and whether reactive oxygen species (ROS) were involved in the generation of the DSBs. DSBs were induced by the coexposure both in the cell-free system (in vitro) and in CHO-K1 cells (in vivo), but not by treatment with BaP or UVA alone. DSB induction in vitro required higher doses of UVA and BaP than were required in vivo, suggesting that the mechanism of DSB induction differed. A similar difference in efficiency also was observed in the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) by coexposure to BaP and UVA in vitro and in vivo. A singlet oxygen ((1)O2) scavenger (NaN3) effectively inhibited the production of DSBs and 8-oxodG, suggesting that (1)O2 is a principal ROS generated by BaP and UVA both in vitro and in vivo. Furthermore, repair-deficient xrs-5 cells were more sensitive to coexposure with BaP and UVA than were CHO-K1 cells, but the two cell lines were equally sensitive to the combined treatment in the presence of NaN3. This result suggested that the cell death produced by coexposure to BaP and UVA was at least partly due to the DSBs generated by (1)O2. Our findings indicate that coexposure to BaP and UVA effectively induced DNA damage, especially DSBs, which results in phototoxicity and possibly photocarcinogenesis.     

γH2AX and p53 responses in TK6 cells discriminate promutagens and nongenotoxicants in the presence of rat liver S9

Previous work with a diverse set of reference chemicals suggests that an in vitro multiplexed flow cytometry‐based assay (MultiFlow? DNA Damage Kit—p53, γH2AX, Phospho‐Histone H3) can distinguish direct‐acting clastogens and aneugens from nongenotoxicants (Bryce SM et al. [ 2016 ]: Environ Mol Mutagen 57:171‐189). This work extends this line of investigation to include compounds that require metabolic activation to form reactive electrophiles. For these experiments, TK6 cells were exposed to 11 promutagens and 37 presumed nongenotoxicants in 96 well plates. Unless precipitation or foreknowledge about cytotoxicity suggested otherwise, the highest concentration was 1 mM. Exposure occurred for 4 hr after which time cells were washed to remove S9 and test article. Immediately following the wash and again at 24 hr, cell aliquots were added to wells of a microtiter plate containing the working detergent/stain/antibody cocktail. After a brief incubation, robotic sampling was employed for walk‐away flow cytometric data acquisition. Univariate logistic regression analyses indicated that γH2AX induction and p53 activation provide the greatest degree of discrimination between clastogens and nongenotoxicants. Multivariate prediction algorithms that incorporated both of these endpoints, in each combination of time points, were evaluated. The best performing models correctly predicted 9 clastogens out of 11 and 36 nongenotoxicants out of 37. These results are encouraging as they suggest that an efficient and highly scalable multiplexed assay can effectively identify clastogenic chemicals that require bioactivation. More work is planned with a broader range of chemicals, additional cell lines, and other laboratories to further evaluate the merits and limitations of this approach. Environ. Mol. Mutagen. 57:546–558, 2016. © 2016 Wiley Periodicals, Inc.     

Genotoxicity of 7H-dibenzo[c,g]carbazole and its tissue-specific derivatives in human hepatoma HepG2 cells is related to CYP1A1/1A2 expression

The goal of this study was to investigate the genotoxicity of 7H-dibenzo[c,g]carbazole (DBC), a ubiquitous environmental pollutant, and its methyl derivatives, 5,9-dimethylDBC (DiMeDBC), a strict hepatocarcinogen, and N-methylDBC (N-MeDBC), a specific sarcomagen in human hepatoma HepG2 cells, and to infer potential mechanisms underlying the biological activity of particular carcinogen. All dibenzocarbazoles, regardless the tissue specificity, induced significant DNA strand break levels and micronuclei in HepG2 cells; though a mitotic spindle dysfunction rather than a chromosome breakage was implicated in N-MeDBC-mediated micronucleus formation. While DBC and N-MeDBC produced stable DNA adducts followed with p53 protein phosphorylation at Ser-15, DiMeDBC failed. A significant increase in DNA strand breaks following incubation of exposed cells with a repair-specific endonuclease (Fpg protein) suggested that either oxidative DNA damage or unstable DNA-adducts might underlie DiMeDBC genotoxicity in human hepatoma cells. DiMeDBC and N-MeDBC increased substantially also the amount of CYP1A1/2 expression in HepG2 cells. Pretreatment of cells with substances affecting AhR-mediated CYP1A family of enzymes expression; however, diminished DiMeDBC and N-MeDBC genotoxicity. Our data clearly demonstrated differences in the mechanisms involved in the biological activity of DiMeDBC and N-MeDBC in human hepatoma cells; the genotoxicity of these DBC derivatives is closely related to CYP1A1/2 expression.