Genes and Environment最新文献

Background: Overcoming species differences in metabolism between humans and animals remains a critical challenge in toxicological studies. Rat liver S9 fraction has long been the gold standard for exogenous metabolic activation in in vitro genotoxicity tests. Experiences with human S9 or human primary hepatocytes have suggested that the human materials are unsuitable for standardized testing due to high variability. Nevertheless, there is growing interest in genotoxicity evaluation using metabolic systems that more closely mimic human physiology.

Results: We developed an in-cell ELISA system to measure γH2AX as a DNA damage marker in stable human hepatocytes (γH2AX-SHE). HepaSH cells are consistently available human hepatocytes that stably express a range of metabolic enzymes and drug transporters in vitro. Due to their highly differentiated and non-proliferative nature, conventional genotoxicity endpoints such as micronuclei formation, chromosomal aberrations, or mutant colony assays are not applicable. We used γH2AX, a sensitive DNA damage marker, in this assay system. Indirect mutagens including benzo(a)pyrene, aristolochic acid, and 2-Amino-1-methyl-6-phenylimidazo(4,5-b)pyridine induced dose-dependent increases in γH2AX across all three HepaSH strains. Time-course analysis following benzo(a)pyrene exposure indicated that a treatment duration of 16 hours or longer was necessary to detect genotoxic responses. Prolonged exposure for 48 hours resulted in extensive cell death, which may interfere with γH2AX quantification.

Conclusions: We demonstrated that γH2AX-SHE can serve as a valuable tool for detecting DNA damage under conditions that mimic human metabolic activity. Based on the findings in this study, we recommend the following assay conditions for γH2AX-SHE: a 24-hour treatment period, a DMSO concentration not exceeding 1%, and careful interpretation of positive responses observed at highly cytotoxic doses - defined as approximately less than 60% cell survival - as these may lack biological relevance.

Background: Nanomaterials such as mesoporous silica and graphene oxide are increasingly used in industrial, medical, and cosmetic applications due to their unique physical and chemical properties. However, their potential genotoxicity remains poorly understood. To evaluate the associated health risks of mesoporous silica and graphene oxide, we assessed their cytotoxicity and genotoxicity in GDL1 cells using trypan blue exclusion and gpt mutation assays, followed by mutation frequency and spectrum analysis through gpt gene sequencing.

Results: A 24-hour exposure of mesoporous silica to GDL1 cells induced dose-dependent reductions in cell viability, as well as dose-dependent increases in gpt mutation frequencies at 0.06 and 0.09 mg/mL. Graphene oxide induced cytotoxicity at higher concentrations (0.2 and 0.4 mg/mL) and significantly increased gpt mutation frequency in the highest concentration exposure group compared to controls. Mutation spectrum analysis revealed a significant increase in G: C to A: T transitions in both the exposed groups. In addition, exposure to mesoporous silica significantly increased G: C to T: A transversions, while graphene oxide exposure significantly increased G: C to C: G transversions. Mutation hotspots at positions 64, 164, and 416 in the gpt gene were identified exclusively in the mesoporous silica-treated group, indicating material-specific mutagenesis. Mutations at position 401 were detected exclusively in the graphene oxide group, indicating this site as a potential mutation hotspot.

Conclusion: These results demonstrate that both mesoporous silica and graphene oxide exhibit cytotoxic and genotoxic potential in vitro. The mutation patterns suggest that oxidative DNA damage, as well as inflammation associated with oxidative stress, may contribute to the observed mutagenicity. The findings reported here provide valuable insights into the molecular mechanisms underlying the mutagenicity induced by these nanomaterials and contribute to the assessment of potential human health risks.

Background: Various immortalized cells and human fresh blood lymphocytes have been used in in vitro genotoxicity studies (e.g., micronucleus (MN) test). Although immortalized cells can be supplied stably, their properties are different from normal cells such as abnormal karyotype. Human fresh blood lymphocytes are representative human normal cells, but homogenous lymphocytes are difficult to supply stably and in a timely manner due to individual differences between donors. Here, we aimed to develop a novel in vitro MN test using human induced pluripotent stem cell (hiPSC)-derived T lymphocytes to overcome the above problems.

Results: hiPSCs were differentiated to T lymphocytes, which were confirmed to possess the ability to grow well in culture, a normal karyotype, and a spontaneous frequency of micronuclei. The genotoxicity of several reference positive / negative control substances was evaluated. The responses for all test substances, including clastogen, aneugen and negative substances, were consistent with published reports.

Conclusions: Our results demonstrated promising proof-of-principle data as an in vitro MN test and suggest that hiPSC-derived T lymphocytes have a potential to make a significant contribution to the improvement of in vitro genotoxicity studies.

The open symposium of the Japanese Environmental Mutagen and Genome Society (JEMS) entitled "The Science Behind Safety in Our Daily Lives," was held as a hybrid in-person and online meeting on June 14, 2025. The rapid advancement of science and technology continues to profoundly alter our lifestyles. We face potential risks from chemical, biological, and physical agents, including chemical substances, bacteria/viruses, and radioactive substances, particularly in pharmaceuticals, food, and indoor environments. Furthermore, natural disasters such as earthquakes and heavy rains not only cause physical damage, but can also lead to health hazards from chemical substances and radiation. This underscores the urgent need for robust systems that can effectively respond to health crises. This symposium aimed to improve public understanding of safety science in daily life, including in pharmaceuticals, food, and living environments. In this symposium, we invited five scientists who are expanding the frontiers of health sciences. We organized this public event to be open to everyone, not just members of the JEMS. Herein, the organizers present a summary of the symposium.

Background: DNA polymerase κ (Polk), a member of Y-family DNA polymerases, plays an important role in translesion DNA synthesis (TLS), allowing DNA replication forks to bypass DNA damage or DNA adducts to continue daughter strand synthesis. Polk is also believed to contribute to the replication-independent repair of DNA lesions such as cross-links. TLS circumvents stalls of DNA replication and promotes gap filling in DNA repair which would otherwise result in DNA double-strand breaks (DSBs) and cell death. Mitomycin C (MMC) is a widely used chemotherapeutic drug which generates DNA cross-links and induces DSBs. To clarify how Polk contributes to the prevention of MMC-induced DSB in various organs or tissues, immunohistochemical staining of γH2AX was conducted in catalytically inactivated Polk knock-in (Polk KI) mice and Polk wild-type (Polk⁺) mice treated with MMC or saline.

Results: The γH2AX induction by MMC was enhanced by inactivation of Polk across many organs or tissues to varying degrees. Obvious enhancement was observed in liver, bladder, adrenal cortex, thyroid, and spermatids, whereas less enhancement was shown in brain and retina. The results suggest that Polk plays a role in preventing DSBs caused by MMC in most organs or tissues. Elevated DSB frequencies were observed in both proliferative cells, such as bladder epithelium cells, and less or slowly proliferative cells, such as hepatocytes. Increased DSB levels in inactivated Polk KI mice relative to Polk⁺ mice were also observed in saline-treated mice in the adrenal cortex and other tissues.

Conclusion: Polk plays a systemic role in mitigating MMC-induced DSBs, likely through both DNA replication-dependent and -independent mechanisms. Furthermore, Polk appears to protect against DSBs caused by endogenous mutagens in some organs such as the adrenal cortex, prostate, and retina.

Background: Echinops spinosus (ES), known as spiny globe thistle, has been widely used in traditional medicine to treat various ailments, such as splenic and renal disorders. However, the genoprotective effect of ES has not been examined previously. This report assessed the in vitro and in vivo genoprotective effects of crude extract of Echinops spinosus (CEES) and its aqueous fraction (AFES) against ethyl methanesulfonate (EMS) in mice. This study applied a battery of genotoxic endpoints, including chromosomal aberrations (CAs), the comet assay, and the micronucleus (MN) assay. Further, GC-MS and HPLC analyses were employed to identify the primary and secondary metabolites in the plant samples, respectively. Total polyphenol and flavonoid contents (TPC and TFC) were also colorimetrically measured. In vitro experiments were conducted using cultured primary mouse bone marrow and spleen. These cells were treated with two concentrations of CEES or AFES (250 and 500 µg/mL; for 24 h), followed by EMS treatment (300 µg/mL; for two hours) before the harvest. For the in vivo experiments, mice were orally administered CEES and AFES (250, 500 mg/kg; for 7 days), with or without intraperitoneal injection with EMS (300 mg/kg; for 24 h).

Results: GC-MS analysis demonstrated 25 primary metabolites in AFES, and the nitrogenous compound bis(trimethylsilyl) ethylamine was the main constituent. HPLC analysis reported 17 and 14 secondary compounds in CEES and AFES, respectively, in which chlorogenic acid was the main constituent in both samples. Colorimetric analysis showed that CEES exhibited higher TPC and TFC compared to AFES. Genotoxic results showed that EMS increased the levels of CAs and comet tail formation in vitro bone marrow and splenic cultures. Further, EMS caused chromosomal damage, as indicated by a significant increase in the frequency of CAs and MN in vivo mouse bone marrow cells. Supplementation with CEES and AFES alleviated chromosomal and DNA damage induced by EMS, and this reduction was more pronounced in vivo than in vitro experiments.

Conclusion: High-polar constituents primarily mediated the antimutagenic activity of CEES and AFES. Meanwhile, other phytoconstituents in CEES, such as moderately polar and nonpolar constituents, synergistically potentiated the genoprotective activity, resulting in greater efficacy of CEES than AFES.

Background: Salvianolic acid B (Sal B), a natural polyphenol with potential therapeutic applications, has been reported to induce reactive oxygen species (ROS) generation. However, its underlying mechanism has not yet been fully elucidated. In this study, we investigated copper-mediated oxidative DNA damage induced by Sal B.

Results: Sal B significantly increased the level of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) in HL-60 cells, but not in H₂O₂-resistant HP100 cells. The formation of 8-oxodG was inhibited by a Cu(I)-specific chelator. These results suggested that Cu(I) and H₂O₂ play critical roles in this process. In calf thymus DNA, Sal B induced 8-oxodG formation in the presence of Cu(II), which was markedly enhanced in the presence of NADH. Using ³²P-5'-end-labeled DNA fragments, we showed that treatment with Sal B in combination with Cu(II) and NADH caused DNA strand breaks and site-specific base modification, especially at thymine and cytosine residues. These results suggest the involvement of ROS other than •OH and this was further supported by radical scavenger experiments. Furthermore, theoretical calculation data suggest that one of the catechol groups in Sal B is electron-donating. Collectively, these results indicate that Cu(II)-mediated autoxidation of the catechol group in Sal B generates Cu(I) and H₂O₂, likely leading to a Cu(I)-hydroperoxide complex formation and resultant oxidative DNA damage. NADH enhances ROS production and oxidative DNA damage by reducing oxidized Sal B and promoting its recycling.

Conclusions: The potential pro-oxidant risk of Sal B should be carefully evaluated when used as a therapeutic agent.