Idiopathic pulmonary fibrosis (IPF) represents another progressive and devastating interstitial lung disease found across populations, with a prominent male predominance accounting for approximately 70
Abstract Background Previous research using the Ames assay showed that licoricidin (LCD) and isoliquiritigenin (ILTG) can block mutations caused by N-methyl-N-nitrosourea (MNU) in Salmonella typhimurium (S. typhimurium) TA1535. With the aim to clarify the antimutagenic mechanism of these flavonoids, we evaluated the noncovalent interactions of test compounds (LCD, ILTG, ethidium bromide (EtBr), or Hoechst 33258) with calf thymus DNA (ctDNA) using a UV-Vis spectrophotometer and quantified the DNA adducts formed when treated with a mixture of MNU and the test compounds. Additionally, we measured the half-life of MNU and the amount of each test compound remaining in the reaction mixture. Results The spectral and thermodynamic parameters indicated considerable binding between flavonoids (LCD and ILTG) and DNA. Intercalative EtBr and the minor-groove binder Hoechst 33258 inhibited MNU-induced mutagenicity in S. typhimurium TA1535. O 6-Methylguanine (O 6-MeG) and N 3-methyladenine (N 3-MeA) were quantified in a mixture of ctDNA and the test compounds (LCD, ILTG, EtBr, and Hoechst 33258) using LC‒MS/MS. The amount of the DNA adducts decreased with increasing concentrations of the compounds. Moreover, the half-lives of MNU were similar in the presence and absence of flavonoids (LCD and ILTG). No new products were detected, and no significant changes in the flavonoids remaining in the reaction mixture were observed, indicating that LCD and ILTG did not react directly with MNU. Conclusions LCD and ILTG directly bind to DNA and suppress DNA adduct formation. However, interaction with DNA alone does not fully account for their antimutagenic activity, implying the involvement of other mechanisms.
Abstract Background Substitution of dietary saturated fat with seed oils highly enriched in n-6 polyunsaturated fatty acids (PUFAs) has been advocated as healthy strategy to offset elevated cholesterol levels. However, both n-6 as well as n-3 PUFAs, considered essential because vertebrates lack the enzymatic apparatus for their de novo synthesis, are the main source of endogenous DNA damage during the aging process due to their high oxidizability. The membrane pacemaker theory of aging is an extension to the oxidative theory of aging and postulates that higher PUFA content in membrane lipids determines the lifespan of different species. Objective We have examined whether a saturated fat-rich diet lacking the essential fatty acids versus a PUFA-rich diet differentially affects lipid profiles and membrane fatty-acid composition, as well as markers of oxidative protein and DNA damage and mitochondrial DNA (mtDNA) integrity in vivo. Methods Three-week-old male C57BL/6J mice were fed isocaloric, high-fat diets containing either coconut oil (SFA-rich) or soybean oil (PUFA-rich) for 12 weeks. Plasma and liver lipids were measured, and the fatty acid composition was analyzed in liver and erythrocyte membranes. Endogenous DNA damage was assessed using 1,N⁶-etheno-2’-deoxyadenosine (εdA) detection in blood and liver. mtDNA damage and lipid peroxidation derived protein adducts from liver were also examined. Results Mice were maintained on a SFA-rich diet for 12 weeks without exhibiting any symptoms of essential fatty acid deficiency (EFAD) as described in historical literature despite the massive synthesis of compensatory n-9 PUFAs. Furthermore, EFAD mice showed reduced levels of endogenous εdA and mtDNA damage as well as protein adducts originating from the primary n-6 PUFA lipid peroxidation product, 4-hydroxy-2-nonenal. However, some other lipid peroxidation-derived protein adducts, such as malondialdehyde and, surprisingly, 4-hydroxy-2-hexenal, were elevated on a SFA-rich diet. Conclusions A PUFA-rich diet, relative to the SFA-rich diet, is associated with increased lipid-peroxidation linked adducts and a greater degree of mtDNA damage in vivo, in parallel with membrane enrichment in n-6 PUFA. These findings provide clear evidence of the biological effects of a PUFA-rich diet on endogenous genotoxic stress.
The 48th Annual Meeting and International Conference of the Environmental Mutagen Society of India (EMSI) on 'Environmental Mutagenesis & Epigenomics in Relation to Human Health' was held at Jamshedpur Co-Operative College, in association with Kolhan University, Jharkhand, India, from January 29-31, 2026. There were 141 deliberations in total, with participation from researchers, academicians, Vice-Chancellors, and state government officials from India and eight other countries. The scientific topics, including environmental impact on humans and aquaculture, transgenerational plant protection, molecular insights into cancer research, plants with antimutagenic potential, and sustainable agriculture through the use of bio-pesticides and bio-fertilizers, broadly justified the conference theme. The molecular mechanisms of pathogenesis were discussed through lectures on signalling pathways, gene expression, and DNA damage and repair, highlighting targeted drug development. Additionally, in silico docking of synthetic drugs and nanoparticles was discussed in detail. Notably, nanotoxicology, microplastics, airborne particulate matter, and prenatal arsenic exposure were shown to have a significant impact on human health. As Jharkhand and neighbouring states depend largely on agricultural yield, discussions on the use of plant-based medicines, harnessing infection and immunity, and agricultural eco-toxicology suggested ways to protect farmers' health and the food chain from the overuse of chemicals. Altogether, the deliberations supported several Sustainable Development Goals and highlighted cost-effective agricultural modalities. These messages were disseminated to the public through local media via daily briefings. Notably, this EMSI conference provided a platform for scientific exchange that attracted administrators and pollution control regulators aimed at protecting human health by mitigating environmental exposure.
Symposium 3 of the 53rd Annual Meeting of the Japanese Environmental Mutagen and Genome Society (JEMS), entitled "Potential for Computational Genotoxicity," was held at Shujitsu University, Okayama, Japan, on December 8, 2024. The symposium discussed the application of advanced informatics technologies, such as (quantitative) structure-activity relationship ((Q)SAR) and error-corrected next-generation sequencing (ecNGS), to the field of genotoxicity within the framework of computational genotoxicity. In this symposium, we invited three scientists who are global leaders in the field of computational genotoxicity. This report summarizes the key discussions and presentations from the symposium. The organizers hope this summary will increase awareness of computational genotoxicity.
BACKGROUND: Although the associations between air pollution exposure and thyroid function have been reported, the interactive effects of the genes involved remain unknown. Therefore, we aimed to identify candidate genetic loci involved in thyroid function by interacting with annual average exposure to particulate matter with a diameter of 10 microns or smaller (PM10) in Korean adults. A total of 1,863 and 1,458 adults were included in the discovery and replication steps, respectively. The average annual concentration of PM10 exposure was considered, and the participants were classified into two groups (low-to-moderate exposure and high-exposure groups) for binary analyses. A genome-wide single-nucleotide polymorphism analysis using a PM10 exposure interaction study was performed to determine thyroid-stimulating hormone levels in Korean adults. RESULTS: Although no SNPs surpassed the stringent genome-wide significance threshold of Pint < 5E-08, one SNP (rs11781213) near MSRA reached a suggestive level of significance of Pint < 5E-07. Two genetic susceptibility loci (FAM84B/PCAT1 and STARD13) were replicated at a nominal significance level of Pint < 1E-05 for the discovery cohort, and Pint < 0.05 for the replication cohort. A genetic variant (rs7169081 G > A) between CGNL1 and GCOM1 was of functional interest. CONCLUSIONS: This is the first study reporting genome-wide-air pollution interaction results for thyroid function. The association between long-term PM10 exposure and thyroid hormone levels may be partly explained by identifying several suggestive loci, including MSRA, PCAT1, and GCOM1.
Abstract Background The growing interest and demand for alternative protein sources has propelled research and production of protein-rich microbial biomass products for human consumption. The potential genotoxicity of a protein-rich biomass derived from Xanthobacter sp. SoF1 (SoF1), an autotrophic hydrogen-oxidizing bacteria, was evaluated in the bone marrow of male NMRI mice in the current work. This study was conducted as the final step of a comprehensive toxicological safety assessment that has been previously published. While a genotoxic evaluation including a bacterial reverse mutation test, an in vitro chromosomal aberration assay in human lymphocytes, and an in vitro micronucleus test in human lymphocytes was previously conducted without incidence, an in vivo mammalian micronucleus test was lacking and thus, this study was performed to provide additional relevant in vivo insights into the genotoxicity of SoF1. Results No treatment-related adverse effects were observed and no increases in the frequencies of micronucleated immature erythrocytes (MPCEs) in male mice (5/group) at any dose level (0, 500, 1000, and 2000 mg/kg bw) of the test item by oral gavage were observed when compared with the concurrent negative and historical negative control groups. Conclusion The test item did not exhibit any clastogenic or aneugenic activity in the bone marrow of mice under the applied experimental conditions.
Abstract Background Familial adenomatous polyposis (FAP) is an autosomal dominantly inherited disease that results in the development of more than 100 polyps, premalignant lesions, in the colorectum. Therefore, patients with FAP are a high-risk group for colorectal cancer (CRC). The only standard method of preventing CRC is total colectomy. Thus, alternative methods to prevent the development of CRC are desired by patients. Epidemiological and animal studies suggested that green tea and its extracts, such as (−)-epigallocatechin gallate, may have the potential to prevent cancer development. Results In the present study, we evaluated the suppressive effects of green tea extract (GTE) on suppress colorectal polyps in FAP and conducted a double-blind clinical trial. Eighty patients were randomly assigned to the GTE group (1.5 g/day for 2 years) and an equal number were assigned to the placebo group. The primary endpoint of colon polyp enlargement tended to be reduced in the GTE group compared with the placebo group, with a risk ratio of 0.43 (95% confidence interval 0.12–1.49; p = 0.16). Conclusions Given that the risk ratio was less than 0.5 and few adverse events were observed, we believe that further research using GTE after calculating the necessary sample size is calculated on the based of this study, should be considered in a future large-scale clinical trial.
BACKGROUND: The 5-year survival of pancreatic ductal adenocarcinoma (PDAC) remains about 10% despite therapeutic advances, underscoring the need for effective preventive and early intervention strategies. Ellagic acid (EA), a naturally occurring polyphenol, has been associated with demonstrated antitumor activity in several malignancies. However, its potential role in preventing PDAC development remains unclear. In this study, we examined the chemopreventive potential and underlying mechanisms of EA in a hamster model of PDAC induced by a high-fat diet and N-nitrosobis(2-oxopropyl)amine. RESULTS: Dietary EA (0.1% w/w) was associated with significantly reduction in both the incidence and multiplicity of PDACs compared to controls. The proportion of histologically normal pancreatic ducts increased and the Ki-67 labeling index decreased in pancreatic intraepithelial neoplasia (PanIN) following EA exposure. In vitro, cell proliferation decreased in a dose-dependent manner, G1 arrest was induced, and invasion was diminished after EA treatment. Multiplex Western blotting revealed lower inhibition of the IL-6/STAT3 pathway. The proportion of pSTAT3-positive cells in hamster PanINs and PDACs was significantly lower in the EA-treated groups than in controls. Because a high-fat diet is known to elevate adipocytokines, and resistin (Res) has been implicated in STAT3 regulation, the Res/STAT3 axis was also examined. Res-associated promotion of invasion was observed but there was no proliferation in vitro, and pSTAT3 expression did not increase. Similarly, serum Res levels did not differ significantly across groups, suggesting a limited contribution of the Res/STAT3 pathway in this hamster model. CONCLUSIONS: EA treatment was associated with reduced pancreatic carcinogenesis in vivo. The IL-6/STAT3 pathway appears to be a primary molecular target under our experimental conditions, whereas the contribution of Res is likely minimal. These findings support the potential of EA as a preventive agent against pancreatic cancer.
Genetic effects due to long term exposure to low doses of ionizing radiation (LDIR) in humans are not well understood. Human population living in high level natural radiation areas (HLNRAs) of Kerala coast in India are continuously exposed to chronic LDIR emanating from monazite containing beach sand for many generations. The background radiation level in this area varies from < 1.0 to 45mGy/year. The people residing in HLNRAs sometimes receives background radiation dose which is approximately 10–40 times higher than the people living in adjacent normal level natural radiation areas (NLNRAs). This population provides a unique opportunity to identify, if present, a mutational signature due to chronic low-dose radiation exposure in humans. We have employed whole exome sequencing approach to determine germline mutational changes in the lymphocytes of healthy individuals from HLNRAs (mean background dose: 31.8 ± 5.4 mGy/year, mean age: 43.0 ± 5.9 years) and compared them with healthy individuals from NLNRAs (mean background dose: 0.9 ± 0.2 mGy/year, mean age: 43.0 ± 11.3 years). Our results revealed that the overall number of single nucleotide variants (SNVs) and insertions/deletions (indels) were not significantly different in HLNRA (7744 SNVs, 880 indels) and NLNRA (7951 SNVs, 856 indels) groups. A similar number of protein affecting mutations (PAMs) were observed in HLNRA (1925) and NLNRA (2082) individuals. Interestingly, several unique SNVs were identified in both the groups. In HLNRA, unique SNVs were overrepresented in genes involved in important biological pathways such as DNA repair (EXO1, PARP2, DDB1, POLQ, LIG1), epigenetic modification (KDM5D, SETDB2, KMT2B, BRD8, SIRT1), cell cycle progression (CDK14, CCND1) etc. Furthermore, significant predominance of C > T transitions which were unique to HLNRA group was observed preferentially at CpG dinucleotide regions. Analysis with REVEL and AloFT tools did not show any increase in potentially pathogenic mutations including those involved in carcinogenesis in HLNRA individuals exposed to chronic radiation. This study did not show any significant changes in genetic variants due to long term exposure to LDIR in human population living in HLNRAs of Kerala coast. However, presence of unique SNVs and C > T transitions in CpG islands of HLNRA individuals indicate the possible role of epigenetic mechanisms i.e. DNA methylation in response to chronic LDIR in this population. This study significantly enhances the current understanding of radiation induced genetic changes and associated cancer risk in human population.
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. 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. 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
Prediction of cytochrome P450 (CYP)-mediated metabolism is crucial for assessing the safety of chemicals. An in silico system to reproduce CYP1B1-mediated reactions has been developed by the reverse construction of ligand-accessible spaces from ligand assemblies as a fused grid Template* system. There are close similarities between Templates of CYP1B1 and previously established CYP1A1 (Genes Environ 2023) in the distribution area, Site of oxidation, and the available Width, except for the use of Position 52’ (Pier-sitting) and the lack of use of the bottom in the middle region (Rings F and Ea) on CYP1B1-Template. Experiments using various substrates of both enzymes further suggested the distinct localization of Bay-2 residues on Templates of CYP1A1 and CYP1B1. More than 260 reactions of CYP1A1/1B1 ligands were examined on the present CYP1A1- and CYP1B1-Template systems. From their placements on the Templates and rules for interaction modes, verifications of good and poor substrates, regio/stereo-selectivity, and inhibition became faithfully available for these ligands. To understand the structural basis of the inhibitory interaction, various inhibitors were applied to the Templates and verified modes of the steric interactions. Both the hangings at Position 32 of CYP1A1 ligands and the adherence at Positions 24–52 of CYP1B1 ligands are suggested to retard the dissociation of Bay-2 residues from ligand molecules. Dissociation interference of ligands with Bay-2 residue is thus possible to be a mechanism of ligand-mediated inhibition on CYP1A1 and CYP1B1.
Pharmaceuticals may contain trace impurities unrelated to therapeutic activity, whICH may occur as synthetic intermediates or degradation products. Among these impurities, mutagenic impurities are of particular concern, as even very low levels of exposure can increase the risk of cancer in humans. To address this issue, the International Council on Harmonization (ICH) developed the M7 guideline in 2014, whICH aim to provide a scientific and practical framework for the assessment and control of mutagenic impurities in pharmaceuticals. The M7 guideline has been updated twice, in 2017 (R1) and 2023 (R2), with the addition of addendum defining compound-specific acceptable intake levels and a comprehensive Q A document to promote harmonized implementation. Recent cases involving N-nitrosamine impurities have highlighted the need for improved risk assessment methods due to their high carcinogenic potential. The latest revision of the M7 guidelines (M7(R3)) will introduce a new approach to consider the quantitative carcinogenic potential of N-nitrosamines based on their structural characteristics.
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. 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. 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.
We previously determined the antimutagenic activity of the juice of Actinidia arguta (hereafter referred to as sarunashi-juice) using the Ames test. The anticarcinogenic effect of sarunashi-juice was observed in lung and skin tumorigenesis in mice. We hypothesized that tea prepared from the leaves and twigs of A. arguta (hereafter referred to as sarunashi-tea) might also have antimutagenic and anticarcinogenic properties. We investigated the antimutagenic activity of sarunashi-tea using the Ames test, and its preventive effects on the formation of aberrant crypt foci (ACF) induced by 1,2-dimethylhydrazine (DMH) in mice. Antimutagenic results against aflatoxin B1, benzo(a)pyrene (B(a)P), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) and 1-methyl-6-phenyl-1H-imidazo[4,5-b]pyridin-2-amine (PhIP) were observed with the administration of sarunashi-tea. The amounts of sarunashi-tea needed for 50
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.
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. 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. 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.
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). 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. 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.
