Carcinogenic risk of food additive AF-2 banned in Japan: a case study on reassessment of genotoxicity.

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 TD50 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.

Detection of in vivo mutagenicity in rat liver samples using error-corrected sequencing techniques.

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.

FTO regulates the DNA damage response via effects on cell-cycle progression.

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.

Genotoxicity assessment of food-flavoring chemicals used in Japan.

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.

In vivo genotoxicity assessment of a multiwalled carbon nanotube in a mouse ex vivo culture.

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.

[Investigation of Foreign Particles in Moderna COVID-19 Vaccine].

Particular batches of Moderna mRNA Coronavirus Disease 2019 (COVID-19) vaccine were recalled after foreign particles were found in some vaccine vials at the vaccination site in Japan in August 2021. We investigated the foreign particles at the request of the Ministry of Health, Labour and Welfare. Energy dispersive X-ray spectroscopy analysis suggested that the foreign particles found in the vials recalled from the vaccination sites were from stainless steel SUS 316L, which was in line with the findings of the root cause investigation by the manufacturer. The sizes of the observed particles ranged from <50 µm to 548 µm in the major axis. Similar foreign particles were also detected in 2 of the 5 vaccine vials of the same lot stored by the manufacturer, indicating that the foreign particles have already been administered to some people via vaccine. Observation of the vials of the same lot by digital microscope found smaller particles those were not detected by visual inspection, suggesting that more vials were affected. Contrarily, visual inspection and subvisible particulate matter test indicated no foreign particles in the vials of normal lots. Possible root cause and strategies to prevent such a deviation were discussed from technical and regulatory aspects.

Bisphenol-A reduces DNA methylation after metabolic activation.

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.

Survival of SARS-CoV-2 and bovine coronavirus on common surfaces of living environments.

Aerosols or saliva containing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can contaminate living environments, and viruses can be indirectly transmitted. To understand the survival potential of the virus, the viral titers of bovine coronavirus (BCoV), as a model virus, and SARS-CoV-2 were measured on porous and non-porous surfaces. The amount of infectious BCoV recovered remained relatively high on non-porous substrates. However, it quickly decreased on several non-porous surfaces such as nitrile rubber. The time taken to reach the limit of detection on non-woven masks, as a porous substrate, was longer than that of non-porous substrates. On porous substrates other than non-woven masks, the amount of virus recovered quickly decreased, and then remained at a low level. Representative substrates were tested with SARS-CoV-2. The decrease in the amount of infectious virus recovered was similar to that of BCoV, although that of SARS-CoV-2 was more rapid. RNA derived from SARS-CoV-2 was also detected using real-time PCR, and it remained on surfaces much longer than infectious virus, on all substrates. Therefore, it is important to measure the viral titer to avoid the overestimation of infectious virus contamination in the environments. Our results suggest that the surface structure was not directly related to viral survivability.

Potent mutagenicity of an azide, 3-azido-1,2-propanediol, in human TK6 cells.

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.

Evaluation of the analytical performance of anti-SARS-CoV-2 antibody test kits distributed or developed in Japan.

Background: With the spread of COVID-19, anti-SARS-CoV-2 antibody tests have been utilized. Herein we evaluated the analytical performance of anti-SARS-CoV-2 antibody test kits using a new reference standard prepared from COVID-19 patient sera. Methods: Fifty-seven kits in total (16 immunochromatography types, 11 ELISA types and 30 types for automated analyzers) were examined. By measuring serially diluted reference standards, the maximum dilution factor showing a positive result and its precision were investigated. Results: The measured cut-off titers varied largely depending on the antibody kit; however, the variability was small, with the titers obtained by each kit being within twofold in most cases. Conclusion: The current results suggest that a suitable kit should be selected depending on the intended purpose.

Emerging technologies and their impact on regulatory science.

There is an evolution and increasing need for the utilization of emerging cellular, molecular and in silico technologies and novel approaches for safety assessment of food, drugs, and personal care products. Convergence of these emerging technologies is also enabling rapid advances and approaches that may impact regulatory decisions and approvals. Although the development of emerging technologies may allow rapid advances in regulatory decision making, there is concern that these new technologies have not been thoroughly evaluated to determine if they are ready for regulatory application, singularly or in combinations. The magnitude of these combined technical advances may outpace the ability to assess fit for purpose and to allow routine application of these new methods for regulatory purposes. There is a need to develop strategies to evaluate the new technologies to determine which ones are ready for regulatory use. The opportunity to apply these potentially faster, more accurate, and cost-effective approaches remains an important goal to facilitate their incorporation into regulatory use. However, without a clear strategy to evaluate emerging technologies rapidly and appropriately, the value of these efforts may go unrecognized or may take longer. It is important for the regulatory science field to keep up with the research in these technically advanced areas and to understand the science behind these new approaches. The regulatory field must understand the critical quality attributes of these novel approaches and learn from each other's experience so that workforces can be trained to prepare for emerging global regulatory challenges. Moreover, it is essential that the regulatory community must work with the technology developers to harness collective capabilities towards developing a strategy for evaluation of these new and novel assessment tools.

Comparison of the frequencies of ENU-induced point mutations in male germ cells and inherited germline mutations in their offspring.

BACKGROUND: Gene mutations induced in germ cells may be transmitted to the next generation and cause adverse effects such as genetic diseases. Certain mutations may result in infertility or death in early development. Thus, the mutations may not be inheritable. However, the extent to which point mutations in male germ cells are transmitted to the next generation or eliminated during transmission is largely unknown. This study compared mutation frequencies (MFs) in sperm of N-ethyl-N-nitrosourea (ENU)-treated gpt delta mice and de novo MFs in the whole exome/genome of their offspring. RESULTS: Male gpt delta mice were treated with 10, 30, and 85 mg/kg of ENU (i.p., weekly × 2) and mated with untreated females to generate offspring. We previously reported a dose-dependent increase in de novo MFs in the offspring estimated by whole exome sequencing (WES) (Mutat. Res., 810, 30-39, 2016). In this study, gpt MFs in the sperm of ENU-treated mice were estimated, and the MFs per reporter gene were converted to MFs per base pair. The inherited de novo MFs in the offspring (9, 26 and 133 × 10- 8/bp for 10, 30, and 85 mg/kg ENU-treated groups, respectively) were comparable to those of the converted gpt MFs in the sperm of ENU-treated fathers (6, 16, and 69 × 10- 8/bp). It indicated that the gpt MFs in the ENU-treated father's sperm were comparable to the inherited de novo MFs in the offspring as estimated by WES. In addition, de novo MFs in the offspring of 10 mg/kg ENU-treated and control fathers were estimated by whole genome sequencing (WGS), because WES was not sufficiently sensitive to detect low background MF. The de novo MF in the offspring of the ENU-treated fathers was 6 × 10- 8/bp and significantly higher than that of the control (2 × 10- 8/bp). There were no significant differences in de novo MFs between gene-coding and non-coding regions. WGS analysis was able to detect ENU-induced characteristic de novo base substitutions at a low dose group. CONCLUSIONS: Despite a difference between exome/genome and exogenous reporter genes, the results indicated that ENU-induced point mutations in male germ cells could be transmitted to the next generation without severe selection.

In vivo and in vitro mutagenicity of perillaldehyde and cinnamaldehyde.

BACKGROUND: Perillaldehyde and cinnamaldehyde are natural substances found in plants that are used as flavoring ingredients. Due to the α,ß-unsaturated aldehydes in their structures, these compounds are expected to be DNA reactive. Indeed, several reports have indicated that perillaldehyde and cinnamaldehyde show positive in in vitro and in vivo genotoxicity tests. However, their genotoxic potentials are currently disputed. To clarify the mutagenicity of perillaldehyde and cinnamaldehyde, we conducted in silico quantitative structure-activity relationship (QSAR) analysis, in vitro Ames tests, and in vivo transgenic rodent gene mutation (TGR) assays. RESULTS: In Ames tests, perillaldehyde was negative and cinnamaldehyde was positive; these respective results were supported by QSAR analysis. In TGR assays, we treated Muta™ Mice with perillaldehyde and gpt-delta mice with cinnamaldehyde up to the maximum tested doses (1000 mg/kg/day). There was no increase in gene mutations in the liver, glandular stomach, or small intestine following all treatments except the positive control (N-ethyl-N-nitrosourea at 100 mg/kg/day). CONCLUSIONS: These data clearly show no evidence of in vivo mutagenic potentials of perillaldehyde and cinnamaldehyde (administered up to 1000 mg/kg/day) in mice; however, cinnamaldehyde is mutagenic in vitro.

Follow-up genotoxicity assessment of Ames-positive/equivocal chemicals using the improved thymidine kinase gene mutation assay in DNA repair-deficient human TK6 cells.

Genotoxicity testing plays an important role in the safety assessment of pharmaceuticals, pesticides and chemical substances. Among the guidelines for various genotoxicity tests, the in vitro genotoxicity test battery comprises the bacterial Ames test and mammalian cell assays. Several chemicals exhibit conflicting results for the bacterial Ames test and mammalian cell genotoxicity studies, which may stem from the differences in DNA repair capacity or metabolism, between different cell types or species. For better understanding the mechanistic implications regarding conflict outcomes between different assay systems, it is necessary to develop in vitro genotoxicity testing approaches with higher specificity towards DNA-damaging reagents. We have recently established an improved thymidine kinase (TK) gene mutation assay (TK assay) i.e. deficient in DNA excision repair system using human lymphoblastoid TK6 cells lacking XRCC1 and XPA (XRCC1-/-/XPA-/-), the core factors of base excision repair (BER) and nucleotide excision repair (NER), respectively. This DNA repair-deficient TK6 cell line is expected to specifically evaluate the genotoxic potential of chemical substances based on the DNA damage. We focussed on four reagents, N-(1-naphthyl)ethylenediamine dihydrochloride (NEDA), p-phenylenediamine (PPD), auramine and malachite green (MG) as the Ames test-positive chemicals. In our assay, assessment using XRCC1-/-/XPA-/- cells revealed no statistically significant increase in the mutant frequencies after treatment with NEDA, PPD and MG, suggesting the chemicals to be non-genotoxic in humans. The observations were consistent with that of the follow-up in vivo studies. In contrast, the mutant frequency was markedly increased in XRCC1-/-/XPA-/- cells after treatment with auramine. The results suggest that auramine is the genotoxic reagent that preferentially induces DNA damages resolved by BER and/or NER in mammals. Taken together, BER/NER-deficient cell-based genotoxicity testing will contribute to elucidate the mechanism of genotoxicity and therefore play a pivotal role in the accurate safety assessment of chemical substances.

Epigenetic effect of the mycotoxin fumonisin B1 on DNA methylation.

Mycotoxin fumonisin B1 (FB1) is a secondary metabolite that is produced by certain Fusarium species. Although numerous studies demonstrate toxic and carcinogenic effects of FB1, the underlying mechanisms have not been fully elucidated. In this study, we evaluated the epigenetic effects of FB1 for the first time using FLO assays, which detect epigenetic changes that affect the flocculation gene (FLO1) promoter activity in budding yeast. FLO assays showed increased reporter activities of the FLO1 promoter in the presence of 10 and 20 µM FB1. FB1 (20 µM) treatments also promoted flocculation. In subsequent in vitro methylation assays of a bacterial DNA methyltransferase (DNMT), FB1 treatments increased DNMT activities. Moreover, global DNA methylation was significantly increased in HEK293 cells treated with 100 µM FB1. Taken together, these results suggest that FB1 exposure leads to unique epigenetic alterations due to increased DNMT activities and demonstrate that FB1 may be an important risk factor for epigenetic dysfunction-associated human diseases including cancer.

New homozygous gpt delta transgenic rat strain improves an efficiency of the in vivo mutagenicity assay.

BACKGROUND: Gene mutation assays in transgenic rodents are useful tools to investigate in vivo mutagenicity in a target tissue. Using a lambda EG10 transgene containing reporter genes, gpt delta transgenic mice and rats have been developed to detect point mutations and deletions. The transgene is integrated in the genome and can be rescued through an in vitro packaging reaction. However, the packaging efficiency is lower in gpt delta rats than in mice, because of the transgene in gpt delta rats being heterozygous and in low copy number. To improve the packaging efficiency, we herein describe a newly developed homozygous gpt delta rat strain. RESULTS: The new gpt delta rat has a Wistar Hannover background and has been successfully maintained as homozygous for the transgene. The packaging efficiency in the liver was 4 to 8 times higher than that of existing heterozygous F344 gpt delta rats. The frequency of gpt point mutations significantly increased in the liver and bone marrow of N-nitroso-N-ethylurea (ENU)- and benzo[a]pyrene (BaP)-treated rats. Spi- deletion frequencies significantly increased in the liver and bone marrow of BaP-treated rats but not in ENU-treated rats. Whole genome sequencing analysis identified ≥ 30 copies of lambda EG10 transgenes integrated in rat chromosome 1. CONCLUSIONS: The new homozygous gpt delta rat strain showed a higher packaging efficiency, and could be useful for in vivo gene mutation assays in rats.

Development of a new quantitative structure-activity relationship model for predicting Ames mutagenicity of food flavor chemicals using StarDrop™ auto-Modeller™.

BACKGROUND: Food flavors are relatively low molecular weight chemicals with unique odor-related functional groups that may also be associated with mutagenicity. These chemicals are often difficult to test for mutagenicity by the Ames test because of their low production and peculiar odor. Therefore, application of the quantitative structure-activity relationship (QSAR) approach is being considered. We used the StarDrop™ Auto-Modeller™ to develop a new QSAR model. RESULTS: In the first step, we developed a new robust Ames database of 406 food flavor chemicals consisting of existing Ames flavor chemical data and newly acquired Ames test data. Ames results for some existing flavor chemicals have been revised by expert reviews. We also collected 428 Ames test datasets for industrial chemicals from other databases that are structurally similar to flavor chemicals. A total of 834 chemicals' Ames test datasets were used to develop the new QSAR models. We repeated the development and verification of prototypes by selecting appropriate modeling methods and descriptors and developed a local QSAR model. A new QSAR model "StarDrop NIHS 834_67" showed excellent performance (sensitivity: 79.5%, specificity: 96.4%, accuracy: 94.6%) for predicting Ames mutagenicity of 406 food flavors and was better than other commercial QSAR tools. CONCLUSIONS: A local QSAR model, StarDrop NIHS 834_67, was customized to predict the Ames mutagenicity of food flavor chemicals and other low molecular weight chemicals. The model can be used to assess the mutagenicity of food flavors without actual testing.

Weight of evidence approach using a TK gene mutation assay with human TK6 cells for follow-up of positive results in Ames tests: a collaborative study by MMS/JEMS.

BACKGROUND: Conflicting results between bacterial mutagenicity tests (the Ames test) and mammalian carcinogenicity tests might be due to species differences in metabolism, genome structure, and DNA repair systems. Mutagenicity assays using human cells are thought to be an advantage as follow-up studies for positive results in Ames tests. In this collaborative study, a thymidine kinase gene mutation study (TK6 assay) using human lymphoblastoid TK6 cells, established in OECD TG490, was used to examine 10 chemicals that have conflicting results in mutagenicity studies (a positive Ames test and a negative result in rodent carcinogenicity studies). RESULTS: Two of 10 test substances were negative in the overall judgment (20% effective as a follow-up test). Three of these eight positive substances were negative after the short-term treatment and positive after the 24 h treatment, despite identical treatment conditions without S9. A toxicoproteomic analysis of TK6 cells treated with 4-nitroanthranilic acid was thus used to aid the interpretation of the test results. This analysis using differentially expressed proteins after the 24 h treatment indicated that in vitro specific oxidative stress is involved in false positive response in the TK6 assay. CONCLUSIONS: The usefulness of the TK6 assay, by current methods that have not been combined with new technologies such as proteomics, was found to be limited as a follow-up test, although it still may help to reduce some false positive results (20%) in Ames tests. Thus, the combination analysis with toxicoproteomics may be useful for interpreting false positive results raised by 24 h specific reactions in the assay, resulting in the more reduction (> 20%) of false positives in Ames test.

Effect of sampling time on somatic and germ cell mutations induced by acrylamide in gpt delta mice.

BACKGROUND: Acrylamide (AA) is a rodent carcinogen and classified by the IARC into Group 2A (probable human carcinogen). AA has been reported to induce mutations in transgenic rodent gene mutation assays (TGR assays), the extent of which is presumed to depend on exposure length and the duration of expression after exposure. In particular, it is not clear in germ cells. To investigate mutagenicity with AA in somatic and germ cells at different sampling times, we conducted TGR assays using gpt delta transgenic mice. RESULTS: The male gpt delta mice at 8 weeks of age were treated with AA at 7.5, 15 and 30 mg/kg/day by gavage for 28 days. Peripheral blood was sampled on the last day of the treatment for micronucleus tests and tissues were sampled for gene mutation assays at day 31 and day 77, those being 3 and 49 days after the final treatment (28 + 3d and 28 + 49d), respectively. Another group of mice was treated with N-Ethyl-N-nitrosourea (ENU) at 50 mg/kg/day by intraperitoneal administration for 5 consecutive days and tissues were sampled at the day 31 and day 77 (5 + 26d and 5 + 72d). Frequencies of micronucleated erythrocytes in the peripheral blood significantly increased at AA doses of 15 and 30 mg/kg/day. Two- to three-fold increases in gpt mutation frequencies (MFs) compared to vehicle control were observed in the testes and lung treated with 30 mg/kg/day of AA at both sampling time. In the sperm, the gpt MFs and G:C to T:A transversions were significantly increased at 28 + 3d, but not at 28 + 49d. ENU induced gpt mutations in these tissues were examined at both 5 + 26d and 5 + 72d. A higher mutant frequency in the ENU-treated sperm was observed at 5 + 72d than that at 5 + 26d. CONCLUSIONS: The gpt MFs in the testes, sperm and lung of the AA-treated mice were determined and compared between different sampling times (3 days or 49 days following 28 day-treatment). These results suggest that spermatogonial stem cells are less sensitive to AA mutagenicity under the experimental condition. Prolonged expression time after exposure to AA to detect mutagenicity may be effective in somatic cells but not in germ cells.

Visualizing the spatial localization of ciclesonide and its metabolites in rat lungs after inhalation of 1-µm aerosol of ciclesonide by desorption electrospray ionization-time of flight mass spectrometry imaging.

Inhaled ciclesonide (CIC), a corticosteroid used to treat asthma that is also being investigated for the treatment of corona virus disease 2019, hydrolyzes to desisobutyryl-ciclesonide (des-CIC) followed by reversible esterification when exposed to fatty acids in lungs. While previous studies have described the distribution and metabolism of the compounds after inhalation, spatial localization in the lungs remains unclear. We visualized two-dimensional spatial localization of CIC and its metabolites in rat lungs after administration of a single dose of a CIC aerosol (with the mass median aerodynamic diameter of 0.918-1.168 µm) using desorption electrospray ionization-time of flight mass spectrometry imaging (DESI-MSI). In the analysis, CIC, des-CIC, and des-CIC-oleate were imaged in frozen lung sections at high spatial and mass resolutions in negative-ion mode. MSI revealed the coexistence of CIC, des-CIC, and des-CIC-oleate on the airway epithelium, and the distribution of des-CIC and des-CIC-oleate in peripheral lung regions. In addition, a part of CIC independently localized on the airway epithelium. These results suggest that distribution of CIC and its metabolites in lungs is related to both the intended delivery of aerosols to pulmonary alveoli and peripheral regions, and the potential deposition of CIC particles on the airway epithelium.