In March 2024, a food poisoning incident in Japan was traced to red yeast rice (beni-koji) supplements contaminated with puberulic acid (PA), a mycotoxin produced by Penicillium adametzioides. Although PA was implicated in renal dysfunction in both humans and rats, its mutagenic potential had not been clarified. Here, we comprehensively assessed the mutagenicity of PA using a tiered approach that combined in silico, in vitro, and in vivo methods. In silico quantitative structure-activity relationship analyses predicted PA to be mutagenic, and in vitro Ames tests confirmed the positive finding. In contrast, in vivo assays, including the transgenic rodent gene mutation assay in mice and the Pig-a assay in rats, demonstrated no induction of mutations in kidney, glandular stomach, and blood cells, even at high exposure levels. Collectively, these findings indicate that PA is mutagenic in vitro bacterial tests, but not in mammalian in vivo systems, suggesting that the overall concern for mutagenicity in humans is low.

Carminic acid, which is an anthracene derivative, is a dye used as a food additive. Although anthracene is considered not to be epigenetic modifier, some anthracene derivatives affect yeast flocculation that is under the epigenetic regulation. FLO assay can detect the expression of Saccharomyces cerevisiae FLO1 gene that mediates the flocculation phenotype influenced by epigenetic regulation. FLO reporter assays showed that 5-20 μM carminic acid induced the FLO1 promoter in wild type yeast as well as in the yeast transformed with human DNA methyltransferase genes, in the absence of rat liver homogenates. Furthermore, in mouse embryonic stem cell line EB3, carminic acid decreased the expression of Oct3/4, which is also under epigenetic regulation, and induced slight morphological changes. These results suggest that carminic acid, but not its metabolites, has the potential to alter the epigenetic state of the genome.

In March 2024, a health hazard associated with the consumption of food products containing red yeast rice (beni-koji), which could lead to renal dysfunction, was reported in Japan. Puberulic acid (PA) was identified as an unintentional contaminant in these products; however, information on PA toxicity remains limited. The toxicological profile of PA was evaluated in a 28-day subacute toxicity study in rats. Synthesized PA was administrated by gavage to 6-week-old Crl:CD(SD) rats at 0, 1, 3, or 10 mg/kg/day (male) or 0, 0.3, 1, or 3 mg/kg/day (female) over 28 days, and satellite groups were used to evaluate the reversibility over a 14-day period. Male rats in the 10 mg/kg group exhibited increased urinary glucose and serum creatinine levels compared to controls. Histopathological examination revealed vacuolation, necrosis, and regeneration of proximal tubules in kidneys of all rats in the male 10 mg/kg and female 3 mg/kg groups. After the 14-day recovery period, focal interstitial fibrosis was observed in one male rats from the high-dose group, whereas no renal lesions were detected in the remaining rats of either sex. These results suggest that PA-induced nephrotoxicity is largely reversible under the conditions studied, although residual chronic lesions may occur in severe cases. Apoptosis/necrosis and diffuse hyperplasia of the glandular stomach mucosa were observed in male 3 and 10 mg/kg and female 3 mg/kg groups but were absent after the recovery period. These results indicate that, under the study conditions, the no-observed-adverse-effect level for PA was 1 mg/kg/day for both sexes.

Numerous chemicals are associated with carcinogenesis through epigenetic alterations in cells. To detect global epigenetic changes induced by carcinogens, the housekeeping gene can serve as a reporter locus, offering a baseline for identifying shifts in epigenetic marks. To investigate this potential, we developed a simple, cost-effective, and quantitative reporter system to assess chemically induced epigenetic effects, utilizing the thymidine kinase (TK) gene mutation assay as a foundation. Using a standard genotoxicity test cell line, human lymphoblast TK6, we edited the CpG promoter loci of the endogenous TK gene using the CRISPR/dCas9-SunTag-DNMT3A system. This epi-genotoxicity assay, employing modified mTK6 cells, provides a simple method for quantifying chemically induced epigenetic effects. The assay successfully detects both increased TK reversion rates induced by DNMT inhibitors, such as 5-Aza-2'-deoxycytidine and GSK-3484862, and, for the first time, a significant reduction in TK revertant frequency caused by the non-genotoxic carcinogen 12-O-tetradecanoylphorbol-13-acetate (TPA). Chromatin immunoprecipitation and western blotting analyses revealed that TPA treatment led to a global decrease in H3K27Ac levels, likely driven by TPA-mediated inflammation. These results demonstrate the utility of the epi-genotoxicity assay as a valuable tool for evaluating dual-directional epigenetic changes triggered by chemical exposure.

The Ames mutagenicity test is an effective means of screening compounds for their carcinogenic potential. Here, we conducted Ames tests on 15 aryl, benzyl, and aliphatic ring N-nitrosamines. Then, by using two indicators of mutagenicity strength calculated from the Ames test results, namely, maximum specific activity (MSA; number of revertant colonies) and maximum fold increase (MFI; relative ratio of increased colonies), we examined the relationship between Ames mutagenicity strength and Carcinogenic Potency Categorization Approach (CPCA) potency category, which is a structure-activity-relationship-based prediction of the carcinogenic potency of nitrosamines. Eleven of the test compounds were Ames positive and four were negative. Of the 11 positive compounds, three were categorized as strong positive (MSA >= 1000), five as medium positive (100 <= MSA <1000), and three as weak positive (MSA <100). The compounds with an aliphatic ring showed a negative relationship between mutagenicity strength (i.e., MSA or MFI) and carcinogenic potential (i.e., CPCA category), whereas, the alpha-methyl aryl N-nitrosamines did not. Overall, MSA and MFI were found to be detailed indicators of the carcinogenic potency of the N-nitrosamines and can potentially be used to support CPCA categorization.

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.

Ensuring the in vitro Ames mutagenicity test data quality is important when data are applied to quantitative structure–activity relationship (QSAR) model development for regulatory uses. Here, more than 10,000 Ames test data used in the Ames/QSAR International Challenge Projects were curated with collecting detailed test information such as purity of the chemical substances and solvent used for the tests as well as five main strain information with and without metabolic activation. The summarized data will indicate the importance of the information, including metabolic activation and main strain data such as Salmonella typhimurium TA100, TA98, and TA1535. These curated data have already been provided to the participants of the second Ames/QSAR International Challenge Project and have helped to develop their QSAR models.

Abstract Background Primary aromatic amines (PAAs) present significant challenges in the prediction of mutagenicity using current standard quantitative structure activity relationship (QSAR) systems, which are knowledge-based and statistics-based, because of their low positive prediction values (PPVs). Previous studies have suggested that PAAs are metabolized into genotoxic nitrenium ions. Moreover, ddE, a relative-energy based index derived from quantum chemistry calculations that measures the stability nitrenium ions, has been correlated with mutagenicity. This study aims to further examine the ability of the ddE-based approach in improving QSAR mutagenicity predictions for PAAs and to develop a refined method to decrease false positive predictions. Results Information on 1,177 PAAs was collected, of which 420 were from public databases and 757 were from in-house databases across 16 laboratories. The total dataset included 465 Ames test-positive and 712 test-negative chemicals. For internal PAAs, detailed Ames test data were scrutinized and final decisions were made using common evaluation criteria. In this study, ddE calculations were performed using a convenient and consistent protocol. An optimal ddE cutoff value of -5 kcal/mol, combined with a molecular weight ≤ 500 and ortho substitution groups yielded well-balanced prediction scores: sensitivity of 72.0%, specificity of 75.9%, PPV of 65.6%, negative predictive value of 80.9% and a balanced accuracy of 74.0%. The PPV of the ddE-based approach was greatly reduced by the presence of two ortho substituent groups of ethyl or larger, as because almost all of them were negative in the Ames test regardless of their ddE values, probably due to steric hindrance affecting interactions between the PAA and metabolic enzymes. The great majority of the PAAs whose molecular weights were greater than 500 were also negative in Ames test, despite ddE predictions indicating positive mutagenicity. Conclusions This study proposes a refined approach to enhance the accuracy of QSAR mutagenicity predictions for PAAs by minimizing false positives. This integrative approach incorporating molecular weight, ortho substitution patterns, and ddE values, substantially can provide a more reliable basis for evaluating the genotoxic potential of PAAs.

Histone modifications are catalyzed and recognized by specific proteins to regulate dynamic DNA metabolism processes. NSD2 is a histone H3 lysine 36 (H3K36)-specific methyltransferase that is associated with both various transcription regulators and DNA repair factors. Specifically, it has been implicated in the repair of DNA double-strand breaks (DSBs); however, the role of NSD2 during DSB repair remains enigmatic. Here, we show that NSD2 does not accumulate at DSB sites and that it is not further mobilized by DSB formation. Using three different DSB repair reporter systems, which contained the endonuclease site in the active thymidine kinase gene (TK) locus, we demonstrated separate dose-dependent effects of NSD2 on homologous recombination (HR), canonical-non-homologous end joining (c-NHEJ), and non-canonical-NHEJ (non-c-NHEJ). Endogenous NSD2 has a role in repressing non-c-NHEJ, without affecting DSB repair efficiency by HR or total NHEJ. Furthermore, overexpression of NSD2 promotes c-NHEJ repair and suppresses HR repair. Therefore, we propose that NSD2 has functions in chromatin integrity at the active regions during DSB repair.

Background Carcinogenic risk assessment studies have been repeatedly improved and are still being debated to find a goal. Evaluation might be changed if new approaches would be applied to some chemicals which means that new approaches may change the final assessment. In this paper, the risk assessment of a chemical, in particular the proper carcinogenicity, is examined using the long-banned food additive, 2-(2-furyl)-3-(5-nitro-2-furyl)-acrylamide, AF-2, as a case study. Results First, Ames tests were carried out using strains TA1535, TA100, TA1538, and TA98 and their nitroreductase-deficient strains YG7127, YG7128, YG7129, and YG7130. The results showed that mutagenic activity was reduced by about 50% in the nitroreductase-deficient strains, indicating that part of the mutagenic activity shown in Ames test was due to bacterial metabolism. Second, in vivo genotoxicity tests were conducted, including the one that had not been developed in 1970’s. Both a micronucleus test and a gene mutation assay using transgenic mice were negative. Third, assuming it is a genotoxic carcinogen, the virtual safety dose of 550 μg/day was calculated from the TD 50 in rats with a probability of 10 −5 . Conclusion AF-2 has been shown to be carcinogenic to rodents and has previously been indicated to be genotoxic in vitro. However, the present in vivo genotoxicity study, it was negative in the forestomach, a target organ for cancer, particularly in the gene mutation assay in transgenic mice. Considering the daily intake of AF-2 in the 1970s and its virtually safety dose, the carcinogenic risk of AF-2 could be considered acceptable.

The fat mass and obesity-associated protein FTO is an “eraser” of N6-methyladenosine, the most abundant mRNA modification. FTO plays important roles in tumorigenesis. However, its activities have not been fully elucidated and its possible involvement in DNA damage - the early driving event in tumorigenesis - remains poorly characterized. Here, we have investigated the role of FTO in the DNA damage response (DDR) and its underlying mechanisms. We demonstrate that FTO responds to various DNA damage stimuli. FTO is overexpressed in mice following exposure to the promutagens aristolochic acid I and benzo[a]pyrene. Knockout of the FTO gene in TK6 cells, via CRISPR/Cas9, increased genotoxicity induced by DNA damage stimuli (micronucleus and TK mutation assays). Cisplatin- and diepoxybutane-induced micronucleus frequencies and methyl methanesulfonate- and azathioprine-induced TK mutant frequencies were also higher in FTO KO cells. We investigated the potential roles of FTO in DDR. RNA sequencing and enrichment analysis revealed that FTO deletion disrupted the p38 MAPK pathway and inhibited the activation of nucleotide excision repair and cell-cycle-related pathways following cisplatin (DNA intrastrand cross-links) treatment. These effects were confirmed by western blotting and qRT-PCR. FTO deletion impaired cell-cycle arrest at the G2/M phase following cisplatin and diepoxybutane treatment (flow cytometry analysis). Our findings demonstrated that FTO is involved in several aspects of DDR, acting, at least in part, by impairing cell cycle progression.

Mutagenicity, the ability of chemical agents to cause mutations and potentially lead to cancer, is a critical aspect of substance safety assessment for protecting human health and the environment. Metabolic enzymes activate multiple mutagens in living organisms, thus in vivo animal models provide highly important information for evaluating mutagenicity in human. Rats are considered suitable models as they share a similar metabolic pathway with humans for processing toxic chemical and exhibit higher responsiveness to chemical carcinogens than mice. To assess mutagenicity in rats, transgenic rodents (TGRs) are widely used for in vivo gene mutation assays. However, such assays are labor-intensive and could only detect transgene mutations inserted into the genome. Therefore, introducing a technology to directly detect in vivo mutagenicity in rats would be necessary. The next-generation sequencing (NGS) based error-corrected sequencing technique is a promising approach for such purposes. We investigated the applicability of paired-end and complementary consensus sequencing (PECC-Seq), an error-corrected sequencing technique, for detecting in vivo mutagenicity in the rat liver samples. PECC-Seq allows for the direct detection of ultra-rare somatic mutations in the genomic DNA without being constrained by the genomic locus, tissue, or organism. We tested PECC-Seq feasibility in rats treated with diethylnitrosamine (DEN), a mutagenic compound. Interestingly, the mutation and mutant frequencies between PECC-Seq and the TGR assay displayed a promising correlation. Our results also demonstrated that PECC-Seq could successfully detect the A:T > T:A mutation in rat liver samples, consistent with the TGR assay. Furthermore, we calculated the trinucleotide mutation frequency and proved that PECC-Seq accurately identified the DEN treatment-induced mutational signatures. Our study provides the first evidence of using PECC-Seq for in vivo mutagenicity detection in rat liver samples. This approach could provide a valuable alternative to conventional TGR assays as it is labor- and time-efficient and eliminates the need for transgenic rodents. Error-corrected sequencing techniques, such as PECC-Seq, represent promising approaches for enhancing mutagenicity assessment and advancing regulatory science.

We assessed the genotoxicity of 30 food-flavoring chemicals used in Japan that have not been investigated before. These 30 food-flavoring chemicals have representative chemical structures belonging to 18 chemical classes. The Ames and chromosomal aberration (CA) tests (in vitro tests) were first conducted in accordance with the “Food Additive Risk Assessment Guidelines” of the Japan Food Safety Commission. If the in vitro test yielded a positive result, an in vivo micronucleus test or a transgenic mouse gene mutation assay was performed to verify the in vitro test results. Of the 30 food-flavoring chemicals, 3 yielded a positive result in both Ames and CA tests. Another 11 chemicals yielded positive results in the CA test. However, none of the chemicals yielding positive in vitro test results yielded positive results in the in vivo tests. These findings indicate no genotoxicity concerns of the food-flavoring chemicals belonging to the abovementioned 18 chemical classes used in Japan unless there are other structural modifications.

Bisphenol-A (BPA) is an important environmental contaminant with adverse health effects suspected to be mediated through epigenetic mechanisms. We had reported that the FLO1-dependent flocculation of transgenic yeast expressing human DNA methyltransferase (DNMT yeast) is a useful tool in epigenotoxicology studies. In this report, we have investigated the effects of BPA in the presence of metabolic activation (S-9 mix) on the transcription level of the FLO1 gene in the DNMT yeast. In the presence of metabolic activation, BPA inhibited the intensity of green fluorescence reporter protein (GFP) driven by the FLO1 promoter. A metabolite of BPA, 4-methyl-2,4-bis(p-hydroxyphenyl) pent-1-ene (MBP), also exhibited similar inhibitory effect. Furthermore, BPA in the presence of S-9 mix had only a weak while MBP had no inhibitory effects on the expression of modified GFP reporter gene under the control of FLO1 promoter with reduced CpG motifs. Aforementioned behavior was confirmed by the inhibition of flocculation as well as FLO1 gene mRNA expression. In addition, the global DNA methylation level in the human HEK293 cells was also reduced by MBP. These results indicate that BPA metabolites have inhibitory effect on DNA methylation. Our approach offers a novel in vitro method for screening for chemicals that can alter the epigenome by a mechanism dependent on their metabolic activation.

Sodium azide is a strong mutagen that has been successfully employed in mutation breeding of crop plants. In biological systems, it is metabolically converted to the proximate mutagen azidoalanine, which requires further bioactivation to a putative ultimate mutagen that remains elusive. The nature of the DNA modifications induced by azides leading to mutations is also unknown. Other mutagenic organic azido compounds seem to share the same bioactivation pathway to the ultimate mutagenic species as they induce point mutations dependent on the same DNA repair pathways. We investigated mutations induced by the representative mutagen 3-azido-1,2-propanediol (azidoglycerol, AZG) in the human TK6 cell line. Until now, azides have been considered to be non-mutagens and non-carcinogens in mammals, including humans, as judged only by the conventional clastogenicity chromosomal aberration types of bioassays. Here, we show the potent mutagenicity of AZG in cultured human cells, comparable to alkylating agents such as methyl methanesulfonate at concentrations with similar lethality. The potent ability of an organic azide to induce base substitutions in a mammalian system raises an alert with respect to human exposure to organic and inorganic azido compounds.

Background Multiwalled carbon nanotubes (MWCNTs) are suspected lung carcinogens because their shape and size are similar to asbestos. Various MWCNT types are manufactured; however, only MWNT-7 is classified into Group 2B by The International Agency for Research on Cancer. MWNT-7’s carcinogenicity is strongly related to inflammatory reactions. On the other hand, inconsistent results on MWNT-7 genotoxicity have been reported. We previously observed no significant differences in both Pig-a (blood) and gpt (lung) mutant frequencies between MWNT-7-intratracheally treated and negative control rats. In this study, to investigate in vivo MWNT-7 genotoxicity on various endpoints, we attempted to develop a lung micronucleus assay through ex vivo culture targeting the cellular fraction of Clara cells and alveolar Type II (AT-II) cells, known as the initiating cells of lung cancer. Using this system, we analyzed the in vivo MWNT-7 genotoxicity induced by both whole-body inhalation exposure and intratracheal instillation. We also conducted an erythrocyte micronucleus assay using the samples obtained from animals under intratracheal instillation to investigate the tissue specificity of MWNT-7 induced genotoxicities. Results We detected a significant increase in the incidence of micronucleated cells derived from the cellular fraction of Clara cells and AT-II cells in both MWNT-7-treated and positive control groups compared to the negative control group under both whole-body inhalation exposures and intratracheal instillation. Additionally, the erythrocyte micronucleus assay detected a significant increase in the incidence of micronucleated reticulocytes only in the positive control group. Conclusions Our findings indicated that MWNT-7 was genotoxic in the lungs directly exposed by both the body inhalation and intratracheal instillation but not in the hematopoietic tissue.