Real opinions on general medicine residency programs in Japan: Perspectives from medical students, residents, and young academic generalists.

Medical students and junior residents have five concerns about general medicine training, and senior residents and young academic generalists respond to these concerns. We hope that this paper will help to dispel some common concerns for those who wish to become specialists in general medicine.

[Investigating erythrocyte hemolysis assay use for proinflammatory potential prediction of silica particles].

OBJECTIVES: Crystalline silica, which is a causative agent of silicosis (an occupational disease), is manufactured in a variety of products (particles) with different particle characteristics, such as size and surface properties. In Japan, the products are currently uniformly controlled as crystalline silica, which is a substance subject to labeling and notification requirements. However, since the toxicity of silica particles reportedly varies depending on its characteristics, businesses are encouraged to conduct appropriate risk assessments for each product to prevent silicosis. Recently, silica particles have been reported to induce lysosomal membrane damage, leading to the activation of proinflammatory factors. An indirect method to evaluate lysosomal membrane damage known as the erythrocyte hemolysis assay, in which the erythrocyte membrane is assumed to be the lysosomal membrane, was performed. This study aimed to examine the possibility of constructing a screening system for proinflammatory potential prediction of silica particles based on their erythrocyte hemolytic activity. METHODS: Hemolysis assays were performed on the silica particles with different sizes, crystallinity, and surface functional groups using the erythrocytes from a healthy volunteer. Additionally, the hemolytic activity of other element particles was compared with that of the silica particles, and 27 types of commercially available crystalline silica particle products underwent screening trials. RESULTS: The hemolytic activity of silica particles was higher in crystalline than that in amorphous and increased with the decreasing size. The hemolytic reaction was particular to silica particles and rarely occurred in particles of other elements. Moreover, the hemolytic activity was significantly suppressed if the silica particles surface was modified with metal ions (Fe3+, Al3+). The hemolytic activities of the crystalline silica products used industrially significantly differed. CONCLUSIONS: This study revealed that particle properties, such as size, crystallinity, and surface functional groups, affect the hemolytic activity of silica particles. Particularly, the surface functional groups (silanol groups) that are unique to silica particles were considered to be strongly involved in hemolytic activities. Since grading the commercially available crystalline silica particle products based on the hemolytic rate was possible, hemolytic activity was suggested to be an evaluation index for predicting the proinflammatory potential of silica particles.

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.

2-mercaptobenzothiazole generates γ-H2AX via CYP2E1-dependent production of reactive oxygen species in urothelial cells.

Ortho (o)-toluidine is a widely known carcinogenic substance associated with cancers of the human bladder. A study on British chemical factory workers exposed to 2-mercaptobenzothiazole, phenyl-ß-naphthylamine, aniline, and o-toluidine demonstrated the crucial roles of o-toluidine, 2-mercaptobenzothiazole, and phenyl-ß-naphthylamine in the development of bladder cancer. As genotoxic events are crucial steps in the initiation of cancer, in the present study, we aimed to examine the genotoxic potential of the four chemicals using phosphorylated histone H2AX (γ-H2AX), which is a sensitive and reliable marker of DNA damage, in cultured human urothelial cells. Of the four chemicals, 2-mercaptobenzothiazole was a particularly potent DNA-damaging agent. Moreover, mechanistic studies revealed that γ-H2AX generation by 2-mercaptobenzothiazole was mainly associated with the generation of reactive oxygen species via cytochrome P450 2E1-mediated metabolism. The findings of this study may provide information that is important for the assessment of risks associated with chemicals as well as the interpretation of epidemiological studies investigating occupational bladder cancer.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone-Induced Histone Acetylation via α7nAChR-Mediated PI3K/Akt Activation and Its Impact on γ-H2AX Generation.

A typical tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is known as a strong carcinogen. We previously reported that metabolized NNK induced histone H2AX phosphorylation (γ-H2AX), a DNA damage-induced histone modification. In this study, we found that NNK globally acetylated histone H3, which affected γ-H2AX generation. Human lung adenocarcinoma A549 was treated with several doses of NNK. NNK induced dose-dependent global histone H3 acetylation (Ac-H3), at 2 to 12 h after the treatment, independent of the cell cycle. The Ac-H3 pattern was not affected by CYP2A13 overexpression unlike γ-H2AX, indicating no requirement of NNK metabolism to induce Ac-H3. Immunofluorescence staining of Ac-H3 was uniform throughout the nucleus, whereas γ-H2AX was formed as foci and did not coincide with Ac-H3. Nicotinic receptor antagonist methyllycaconitine inhibited Ac-H3 and also γ-H2AX. Phosphoinositide-3-kinase (PI3K)/Akt inhibitors, LY294002, wortmannin, and GSK690693, also suppressed both Ac-H3 and γ-H2AX, whereas KU-55933, an inhibitor of ataxia telangiectasia mutated (ATM) upstream of γ-H2AX, inhibited γ-H2AX but not Ac-H3. These results suggested that binding of NNK to the nicotinic acetylcholine receptor (α7nAChR) activated the PI3K/Akt pathway, resulting in Ac-H3. The activated pathway leading to Ac-H3 enhanced γ-H2AX, suggesting that NNK-induced DNA damage is impacted by the α7nAChR-mediated signal transduction pathway.

Long-wavelength UVA enhances UVB-induced cell death in cultured keratinocytes: DSB formation and suppressed survival pathway.

Solar UV radiation consists of both UVA and UVB. The wavelength-specific molecular responses to UV radiation have been studied, but the interaction between UVA and UVB has not been well understood. In this study, we found that long-wavelength UVA, UVA1, augmented UVB-induced cell death, and examined the underlying mechanisms. Human keratinocytes HaCaT were exposed to UVA1, followed by UVB irradiation. Irradiation by UVA1 alone showed no effect on cell survival, whereas the UVA1 pre-irradiation remarkably enhanced UVB-induced cell death. UVA1 delayed the repair of pyrimidine dimers formed by UVB and the accumulation of nucleotide excision repair (NER) proteins to damaged sites. Gap synthesis during NER was also decreased, suggesting that UVA1 delayed NER, and unrepaired pyrimidine dimers and single-strand breaks generated in the process of NER were left behind. Accumulation of this unrepaired DNA damage might have led to the formation of DNA double-strand breaks (DSBs), as was detected using gel electrophoresis analysis and phosphorylated histone H2AX assay. Combined exposure enhanced the ATM-Chk2 signaling pathway, but not the ATR-Chk1 pathway, confirming the enhanced formation of DSBs. Moreover, UVA1 suppressed the UVB-induced phosphorylation of Akt, a survival signal pathway. These results indicated that UVA1 influenced the repair of UVB-induced DNA damage, which resulted in the formation of DSBs and enhanced cell death, suggesting the risk of simultaneous exposure to high doses of UVA1 and UVB.

[Management of occupational health for adverse health effects of beryllium and its compounds in workplaces - Recent trends and issues in Japan].

OBJECTIVES: Beryllium is primarily used in its metallic form, in alloys, or in beryllium oxide ceramics. Its physical and mechanical properties make it useful for many applications across a range of industries. Because beryllium is recognized as a sensitizing and carcinogenic agent, the management of occupational health for workers who may be occupationally exposed to beryllium has long been an important issue in the world. Under these circumstances, the U.S. Occupational Safety and Health Administration (OSHA) had published a rule in January 2017, to prevent the development of chronic beryllium disease and lung cancer. This rule strengthens the regulations governing the use of beryllium and its compounds. With the announcement of the OSHA rule in January 2017, the purpose of this study is to gain insight into the health problems and industrial hygiene associated with the use of beryllium and share the issues related to the management of occupational health for persons working with beryllium in Japan. METHODS: We collected information regarding the beryllium industry, beryllium exposure, beryllium-induced health disorders, OSHA rule of January 2017, and regulations for beryllium use in Japan. After reviewing them, we discussed the issues concerning occupational health management of workers exposed to beryllium in Japan. RESULTS: It has been reconfirmed that in recent years, the most serious health problem due to beryllium exposure is chronic beryllium disease caused by beryllium sensitization. Management of occupational health that emphasizes reduction of beryllium sensitization and early detection of beryllium-sensitized workers is important. CONCLUSIONS: It was suggested that the following should be considered as the issues of management of occupational health of workers exposed to beryllium in Japan: (1) Collect epidemiologic data on health hazards from beryllium exposure in Japan. (2) Review the diagnostic items of special medical check-ups. (3) Review the definition of beryllium and its compounds in the Ordinance on Prevention of Hazards due to Specified Chemical Substances.

Comparative γ-H2AX analysis for assessment of the genotoxicity of six aromatic amines implicated in bladder cancer in human urothelial cell line.

Recently, it was reported that ten cases of bladder cancer occurred among employees, who handled several kinds of aromatic amines, at a Japanese chemical plant. The common aromatic amines were identified as ortho-toluidine, para-toluidine, aniline, ortho-chloroaniline, ortho-anisidine, and 2,4-dimethylaniline. All of these aromatic amines, except ortho-chloroaniline, have been found to be carcinogenic in animals and/or humans. Genotoxic events are known to be crucial steps in the initiation of cancer; information on the genotoxicity of these aromatic amines is insufficient and consistent results have not been obtained. In this study, we examined the genotoxicity of the six different aromatic amines associated with bladder cancer by assessing phosphorylated histone H2AX (γ-H2AX) in a cultured human urothelial cell line, 1T1. We showed that all six aromatic amines generated γ-H2AX. In addition, the γ-H2AX-inducing potential of these six aromatic amines was distinctly different; ortho-chloroaniline and 2,4-dimethylaniline showed particularly high potential, followed by ortho-toluidine, ortho-anisidine, para-toluidine ≒ aniline. The findings of this study may provide important information for the risk assessment of chemicals and for interpreting epidemiological studies on occupational bladder cancer.

Role of metallothionein-like cadmium-binding protein (MTLCdBP) in the protective mechanism against cadmium toxicity in the testis.

The role of metallothionein (MT)-like cadmium (Cd) binding protein (MTLCdBP) in protecting the testes against Cd toxicity was examined. In the acute Cd exposure treatment, cadmium chloride was intraperitoneally injected at 2 mg Cd/kg to Wistar male rats. In the chronic Cd toxicity treatments, 20 mg Cd/kg/d was orally administered for 5 d a week for 5, 10, and 15 wk. MT (-I,-II) and MTLCdBP were measured using ELISA and Cd-Hem methods, respectively. Testicular tissues were immunostained with antibodies of MT-I,-II, MT-III, and MTLCdBP. Expression of HO1, OGG, iNOS, COX2, and p53 was measured by RT-PCR. Cd concentration in the testis increased dose-dependently in response to Cd exposure. MTLCdBP concentration increased markedly with increasing Cd accumulation. Significant increases in expression of iNOS, HO1, COX2, and OGG1 were observed in the acute exposure treatment. In the chronic oral administration group, expression of MT-I, MT-II, MT-III, iNOS, HO1, and COX2 did not change. Positive immunostaining of MTLCdBP was observed in testicular interstitial tissue. In the testis protected from Cd toxicity, MTLCdBP induction increased significantly with increasing Cd accumulation. Our results suggest that MTLCdBP plays an important role in protecting the testis against Cd toxicity.

Reliability of Cancer Treatment Information on the Internet: Observational Study.

BACKGROUND: Finding the correct medical information in a flood of information from the internet is a significant issue for patients with cancer. OBJECTIVE: We investigated the reliability of the information on cancer treatment methods available on the internet based on an evaluation by medical oncologists, medical students, and cancer survivors. METHODS: Using Google and Yahoo as the search engines, we carried out the information search using 2 keywords, "cancer treatment" and "cancer cure," and the top 20 information sites were identified. A similar search was conducted on 5 types of cancer. The reliability of the information presented was rated on a 3-level scale (A, B, or C). Level A referred to reliable sites (providing information complying with the clinical practice guidelines for various types of cancer), Level B included sites not falling under either Level A or Level C, and Level C comprised dangerous or harmful sites (providing information on treatment not approved by the regulatory authority in Japan and bombastic advertisements without any relevant clinical evidence). The evaluation was conducted by medical oncologists, medical students, and cancer survivors. The consistency of the information reliability level rating between the medical students or cancer survivors with that of the medical oncologists was assessed by using the kappa value. RESULTS: A total of 247 sites were evaluated for reliability. The ratings provided by the medical students' group were as follows: Level A, 12.1% (30/247); Level B, 56.3% (139/247); and Level C, 31.6% (78/247). The ratings provided by the cancer survivors' group were as follows: Level A, 16.8% (41/244); Level B, 44.7% (109/244); and Level C, 38.5% (94/244). The ratings provided by the oncologists' group were as follows: Level A, 10.1% (25/247); Level B, 51.4% (127/247); and Level C, 38.5% (95/247). The intergroup rating consistency between the medical students' group and oncologists' group was 87.4% (216/247, kappa=0.77) and that between the cancer survivors' group and oncologists' group was 76.2% (186/244, kappa=0.61). CONCLUSIONS: Of the investigated sites providing information on cancer treatment on the internet, the percentage of sites that seemed to provide harmful information was much higher than that of sites providing reliable information. The reliability level rating was highly consistent between the medical students' group and the medical oncologists' group and also between the cancer survivors' group and the medical oncologists' group.

2,4-Dimethylaniline generates phosphorylated histone H2AX in human urothelial and hepatic cells through reactive oxygen species produced by cytochrome P450 2E1.

The Japanese Ministry of Health, Labour, and Welfare recently reported an outbreak of bladder cancer among workers who handled aromatic amines in Japan. 2,4-dimethylaniline (2,4-DMA) is one of the chemicals that workers are considered to have the most opportunities to be exposed. Genotoxic events are known to be crucial steps in the initiation of cancer. However, studies on the genotoxicity of 2,4-DMA are limited, particularly studies investigating the mechanism behind the genotoxicity by 2,4-DMA are completely lacking. We examined genotoxic properties of 2,4-DMA using phosphorylated histone H2AX (γ-H2AX), a sensitive and reliable marker of DNA damage, in cultured human urothelial and hepatic cells. Our results clearly showed that 2,4-DMA at a concentration range of 1-10 mM generates γ-H2AX in both cell lines, indicating that 2,4-DMA is genotoxic. During mechanistic investigation, we found that 2,4-DMA boosts intracellular reactive oxygen species, an effect clearly attenuated by disulfiram, a strong inhibitor of cytochrome P450 2E1 (CYP2E1). In addition, CYP2E1 inhibitors and the antioxidant, N-acetylcysteine, also attenuated γ-H2AX generation following exposure to 2,4-DMA. Collectively, these results suggest that γ-H2AX is formed following exposure to 2,4-DMA via reactive oxygen species produced by CYP2E1-mediated metabolism. Continuous exposure to genotoxic aromatic amines such as 2,4-DMA over a long period of time may have contributed to the development of bladder cancer. Our results provide important insights into the carcinogenicity risk of 2,4-DMA in occupational bladder cancer outbreaks at chemical plants in Japan.

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.

Effect of physicochemical character differences on the genotoxic potency of kaolin.

BACKGROUND: Kaolin is white clay mineral with the chemical composition Al2Si2O5(OH)4, and many varieties of kaolins having different crystal structures are utilized in industrial, cosmetic and medical fields. To evaluate the effect of physicochemical character differences on the genotoxicity of kaolin, two types of kaolin, kaolin-S with smooth, sphere-shaped crystals, and kaolin-P with clusters of thin pseudohexagonal plates, were used in the study. RESULTS: ICR mice were intratracheally instilled with the kaolins (0.05 and 0.2 mg/mouse), and comet assay was performed on their lungs. Both kaolins showed DNA damage in the lungs of the mice, however the DNA damaging potency was much higher with kaolin-P than that with kaolin-S. In order to clarify the mechanisms for the different genotoxic potency, we examined the incorporation rate and ROS generation of these two types of kaolin in alveolar epithelial A549 and macrophage-like RAW264 cells, using flow cytometric (FCM) analysis. Kaolin-P showed a higher incorporation rate into the mammalian cells and ROS generation than that of kaolin-S. Especially, RAW264 cells aggressively incorporated kaolins, and generated ROS, whereas almost no ROS generation was observed in A549 cells. In addition, inflammatory cytokines were quantified, using the ELISA method, to understand further genotoxic potency differences of kaolins. Concentrations of interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) in the media were increased by exposure to both kaolins, but in the case of kaolin-P, these inflammatory cytokines were significantly elevated. Based on these findings, differences of genotoxic potency may contribute to incorporation rates into immune cells. Furthermore, it is likely that immune cells and epithelial cells might closely interact with each other for the appearance of genotoxocity in vivo. In order to clarify the interaction between epithelial and immune cells, A549 and RAW264 were co-cultured and RAW264 cells only were exposed to kaolins, then subsequently A549 was applied to FCM analysis and comet assay. DNA damage observed in the A549 cells markedly increased in the presence of kaolin-exposed RAW264 cells compared to the single culture. CONCLUSION: From these observations, it is suggested that mechanisms of kaolin genotoxicity against epithelial cells are through the activation of macrophage cells. Therefore, it is thought that interactions between epithelial and immune cells would be very important for evaluation of the genotoxicity of fine particulate matter. We also showed here that co-culture models of epithelial and immune cells could be used as suitable models for evaluation of lung genotoxicity of fine particulate matter, including nanomaterials, as in vivo mimicking systems.

Silver nanoparticle-induced phosphorylation of histone H3 at serine 10 is due to dynamic changes in actin filaments and the activation of Aurora kinases.

The phosphorylation of histone H3 at serine 10 (p-H3S10) has been closely correlated with mitotic chromosome condensation. We previously reported that silver nanoparticles (AgNPs) significantly induced p-H3S10 independent of mitosis. In the present study, we examined the mechanisms underlying the induction of p-H3S10 by AgNPs. A treatment with AgNPs markedly induced p-H3S10 in a dose-dependent manner in three types of cell lines, and this was dependent on the cellular incorporation of AgNPs. The immunofluorescent staining of AgNP-induced p-H3S10 was thin and solid throughout the nucleus, and differed from that normally associated with mitosis. AgNPs induced the formation of globular actin in a dose-dependent manner. Latrunculin B (LatB) and phalloidin, inhibitors of actin polymerization and depolymerization, respectively, inhibited p-H3S10, suggesting that dynamic changes in actin filaments are related to AgNP-induced p-H3S10. Furthermore, p-H3S10 was mediated by Aurora kinase (AURK) pathways, which were suppressed by LatB and siRNA for cofilin 1, an actin-depolymerizing protein. AgNO3 (Ag ions) exerted similar effects to those of AgNPs. These results suggest that Ag ions released from AgNPs incorporated into inner cells changed the dynamics of actin filaments, and this was followed by the activation of AURKs, leading to the induction of p-H3S10.

1,2-Dichloropropane generates phosphorylated histone H2AX via cytochrome P450 2E1-mediated metabolism.

1,2-Dichloropropane (1,2-DCP), a synthetic chlorinated solvent, was recently classified as carcinogenic. Genotoxic events are known as a crucial step in the initiation of cancer. However, studies on the genotoxicity of 1,2-DCP are very limited, particularly studies investigating the mechanism behind DNA damage by 1,2-DCP. In this study, we examined the genotoxicity of 1,2-DCP using phosphorylated histone H2AX (γ-H2AX), a sensitive DNA damage marker. 1,2-DCP showed dose- (1-10mM: 4h) and time-dependent (1-24h: 5mM) γ-H2AX generation in cultured human hepatocytes (WRL-68) and cholangiocytes (MMNK-1). Additionally, γ-H2AX generation was observed in the livers of mice inhalationally exposed to 1,2-DCP at concentrations of 100, 200, and 400 ppm. During an in vitro mechanistic investigation, we found that γ-H2AX generation by 1,2-DCP was clearly attenuated in the presence of disulfiram and 4-methylpyrazole, a specific cytochrome P450 2E1 (CYP2E1) inhibitor. Furthermore, we showed that 1,2-DCP increased the levels of intracellular reactive oxygen species (ROS), with the increase significantly inhibited by CYP2E1 inhibitors. These results suggested that ROS produced via the cytochrome P450 2E1 metabolic process of 1,2-DCP was a major causal factor for γ-H2AX generation by treatment with 1,2-DCP.

New mechanism of γ-H2AX generation: Surfactant-induced actin disruption causes deoxyribonuclease I translocation to the nucleus and forms DNA double-strand breaks.

We previously showed that nonionic surfactants, nonylphenol polyethoxylates (NPEOs), induced phosphorylation of histone H2AX, forming γ-H2AX. In this study, we analyzed the mechanism of γ-H2AX generation by an NPEO with 15 ethylene oxide units (NPEO(15)). In MCF-7 breast carcinoma cells, NPEO(15) treatment induced γ-H2AX in a dose-dependent manner. EDTA and ZnCl2, two inhibitors of deoxyribonuclease I (DNase I), inhibited both the γ-H2AX and DNA double-strand breaks induced by NPEO(15). NPEO(15) disrupted filamentous actin and released free DNase I as detected by cell fractionation analysis. Based on immunofluorescence staining of DNase I and monitoring DNase I-GFP localization, DNase I was translocated from the cytosol to the nucleus of cells after treatment with NPEO(15). This translocation did not occur with the common DNA damage inducers ultraviolet B irradiation and hydrogen peroxide. Other surfactants, Tween 20, Triton X-100 and Nonidet P-40, also generated γ-H2AX. These results show that γ-H2AX induction by surfactants including NPEOs, occurs via a new mechanism involving release of free DNase I with actin disruption. This mechanism is distinct from the process of γ-H2AX generation caused by direct chemically induced DNA damage.

γ-H2AX is a sensitive marker of DNA damage induced by metabolically activated 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a nicotine-derived nitrosamine, is a potent pulmonary carcinogen present in tobacco smoke. DNA adducts induced by metabolically activated NNK cause carcinogenesis; however, the DNA adducts are difficult to detect in cultured cells because of low intrinsic metabolic enzyme activity. In this study, we indirectly detected NNK-induced DNA adducts via the phosphorylation of histone H2AX (γ-H2AX) in A549 human lung adenocarcinoma epithelial cells. NNK treatment dose-dependently induced γ-H2AX. This γ-H2AX induction was suppressed by ataxia telangiectasia mutated inhibitors, suggesting that DNA double-strand breaks (DSBs) are formed during replication and repair of DNA adducts; however, DSBs could not be directly detected by biased sinusoidal field gel electrophoresis (BSFGE). CYP2A13-overexpressing cells showed prolonged γ-H2AX induction compared with control cells, and DSBs could be detected by BSFGE in CYP2A13-overexpressing cells as a clear migration of double-stranded DNA. These findings suggest that γ-H2AX is a sensitive marker of DNA adducts and provides a possible system for genotoxicity screening of chemicals such as NNK, which need metabolic activation to induce DNA damage.

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.

Bacterial genotoxins promote inside-out integrin ß1 activation, formation of focal adhesion complexes and cell spreading.

Integrins are membrane bound receptors that regulate several cellular processes, such as cell adhesion, migration, survival and proliferation, and may contribute to tumor initiation/progression in cells exposed to genotoxic stress. The extent of integrin activation and its role in cell survival upon intoxication with bacterial genotoxins are still poorly characterized. These toxins induce DNA strand breaks in the target cells and activate the DNA damage response (DDR), coordinated by the Ataxia Telangectasia Mutated (ATM) kinase. In the present study, we demonstrate that induction of DNA damage by two bacterial genotoxins promotes activation of integrin ß1, leading to enhanced assembly of focal adhesions and cell spreading on fibronectin, but not on vitronectin. This phenotype is mediated by an ATM-dependent inside-out integrin signaling, and requires the actin cytoskeleton remodeler NET1. The toxin-mediated cell spreading and anchorage-independent survival further relies on ALIX and TSG101, two components of the endosomal sorting complex required for transport (ESCRT), known to regulate integrin intracellular trafficking. These data reveal a novel aspect of the cellular response to bacterial genotoxins, and provide new tools to understand the carcinogenic potential of these effectors in the context of chronic intoxication and infection.

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.