Tatsushi Toyooka

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.

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.

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.

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.

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

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.