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Nature Protocols Mar 2023The comet assay is a versatile method to detect nuclear DNA damage in individual eukaryotic cells, from yeast to human. The types of damage detected encompass DNA strand... (Review)
Review
The comet assay is a versatile method to detect nuclear DNA damage in individual eukaryotic cells, from yeast to human. The types of damage detected encompass DNA strand breaks and alkali-labile sites (e.g., apurinic/apyrimidinic sites), alkylated and oxidized nucleobases, DNA-DNA crosslinks, UV-induced cyclobutane pyrimidine dimers and some chemically induced DNA adducts. Depending on the specimen type, there are important modifications to the comet assay protocol to avoid the formation of additional DNA damage during the processing of samples and to ensure sufficient sensitivity to detect differences in damage levels between sample groups. Various applications of the comet assay have been validated by research groups in academia, industry and regulatory agencies, and its strengths are highlighted by the adoption of the comet assay as an in vivo test for genotoxicity in animal organs by the Organisation for Economic Co-operation and Development. The present document includes a series of consensus protocols that describe the application of the comet assay to a wide variety of cell types, species and types of DNA damage, thereby demonstrating its versatility.
Topics: Animals; Humans; Comet Assay; DNA Damage; Pyrimidine Dimers; Eukaryotic Cells; DNA
PubMed: 36707722
DOI: 10.1038/s41596-022-00754-y -
International Journal of Molecular... Feb 2020During almost 40 years of use, the micronucleus assay (MN) has become one of the most popular methods to assess genotoxicity of different chemical and physical factors,... (Review)
Review
During almost 40 years of use, the micronucleus assay (MN) has become one of the most popular methods to assess genotoxicity of different chemical and physical factors, including ionizing radiation-induced DNA damage. In this minireview, we focus on the position of MN among the other genotoxicity tests, its usefulness in different applications and visibility by international organizations, such as International Atomic Energy Agency, Organization for Economic Co-operation and Development and International Organization for Standardization. In addition, the mechanism of micronuclei formation is discussed. Finally, foreseen directions of the MN development are pointed, such as automation, buccal cells MN and chromothripsis phenomenon.
Topics: DNA Damage; Forecasting; Humans; Lymphocytes; Micronuclei, Chromosome-Defective; Micronucleus Tests; Mutagenicity Tests; Mutagens; Radiation, Ionizing
PubMed: 32102335
DOI: 10.3390/ijms21041534 -
Regulatory Toxicology and Pharmacology... Dec 2021Agrochemical safety assessment has traditionally relied on the use of animals for toxicity testing, based on scientific understanding and test guidelines developed in... (Review)
Review
Agrochemical safety assessment has traditionally relied on the use of animals for toxicity testing, based on scientific understanding and test guidelines developed in the 1980s. However, since then, there have been significant advances in the toxicological sciences that have improved our understanding of mechanisms underpinning adverse human health effects. The time is ripe to 'rethink' approaches used for human safety assessments of agrochemicals to ensure they reflect current scientific understanding and increasingly embrace new opportunities to improve human relevance and predictivity, and to reduce the reliance on animals. Although the ultimate aim is to enable a paradigm shift and an overhaul of global regulatory data requirements, there is much that can be done now to ensure new opportunities and approaches are adopted and implemented within the current regulatory frameworks. This commentary reviews current initiatives and emerging opportunities to embrace new approaches to improve agrochemical safety assessment for humans, and considers various endpoints and initiatives (including acute toxicity, repeat dose toxicity studies, carcinogenicity, developmental and reproductive toxicity, exposure-driven approaches, inhalation toxicity, and data modelling). Realistic aspirations to improve safety assessment, incorporate new technologies and reduce reliance on animal testing without compromising protection goals are discussed.
Topics: Acute Disease; Agrochemicals; Animal Testing Alternatives; Carcinogenicity Tests; Dose-Response Relationship, Drug; Guidelines as Topic; Mutagenicity Tests; Research Design; Risk Assessment; Species Specificity; Time Factors
PubMed: 34678328
DOI: 10.1016/j.yrtph.2021.105068 -
Regulatory Toxicology and Pharmacology... Aug 2023Consumer use of cannabidiol (CBD) for personal wellness purposes has garnered much public interest. However, safety-related data on CBD in the public domain are limited,...
Consumer use of cannabidiol (CBD) for personal wellness purposes has garnered much public interest. However, safety-related data on CBD in the public domain are limited, including a lack of quality studies evaluating its genotoxic potential. The quality of available studies is limited due to the test material used (e.g., low CBD purity) and/or study design, leading some global regulatory agencies to highlight genotoxicity as an important data gap for CBD. To address this gap, the genotoxic potential of a pure CBD isolate was investigated in a battery of three genotoxicity assays conducted according to OECD testing guidelines. In an in vitro microbial reverse mutation assay, CBD up to 5000 μg/plate was negative in Salmonella typhimurium strains TA98, TA100, TA1535, and TA1537, and Escherichia coli strain WP2 uvrA, with and without metabolic activation. Testing in an in vitro micronucleus assay was negative in human TK6 cells up to 10-11 μg/mL, with and without metabolic activation. Finally, an in vivo micronucleus assay conducted in male and female rats was negative for genotoxicity up to 1000 mg/kg-bw/d. Bioanalysis of CBD and its primary metabolite, 7-carboxy CBD, confirmed a dose-related increase in plasma exposure. Together, these assays indicate that CBD is unlikely to pose a genotoxic hazard.
Topics: Rats; Male; Humans; Female; Animals; Mutagenicity Tests; Cannabidiol; Micronucleus Tests; Salmonella typhimurium; DNA Damage; Escherichia coli
PubMed: 37271419
DOI: 10.1016/j.yrtph.2023.105425 -
Mutation Research. Genetic Toxicology... 2020The safety of D-10-camphorsulfonic acid (CSA) was evaluated by genotoxicity testing and in a subchronic 90-day study in rats. Ames test and in vitro micronucleus test...
The safety of D-10-camphorsulfonic acid (CSA) was evaluated by genotoxicity testing and in a subchronic 90-day study in rats. Ames test and in vitro micronucleus test results, either in the absence or the presence of metabolic activation, were negative. Administration of CSA to Wistar rats in the drinking water (0.05, 0.20, or 1.00 mg/mL), for 90 days caused neither test-item-related mortality nor adverse clinical signs. The only macroscopic change seen at necropsy was enlarged testes in the high-dose animals. The 0.20 mg/mL (25 mg/kg bw/day) dose level was considered to be the no observed adverse effect level (NOAEL). A total intake calculation for consumers was performed, based on the intended maximal amount of 0.5 ppm CSA in feed, published transfer factors, and conservative tissue consumption data, resulting in 0.29 μg/kg bw/day. Therefore, the NOAEL is approximately 80,000 × the maximum estimated human exposure, a margin that is more than adequate to ensure consumer safety.
Topics: Animals; Camphor; Chromosome Aberrations; Female; Humans; Male; Micronucleus Tests; Mutagenicity Tests; No-Observed-Adverse-Effect Level; Rats, Wistar
PubMed: 33198938
DOI: 10.1016/j.mrgentox.2020.503257 -
Journal of Pharmacopuncture Dec 2022Verbenalin is a compound found in herbs such as and . This study investigated whether verbenalin is safe by analyzing its mutagenicity.
OBJECTIVES
Verbenalin is a compound found in herbs such as and . This study investigated whether verbenalin is safe by analyzing its mutagenicity.
METHODS
To examine the mutagenic potential of verbenalin, a bacterial reverse mutation test (Ames test) was conducted with and strains. Experiments with and without metabolic activity were performed.
RESULTS
The mean colony number was less than double that of the control. Growth inhibition and precipitation of verbenalin were not apparent in all strains at different concentrations regardless of metabolic activity.
CONCLUSION
Verbenalin did not show any signs of mutagenicity in this study. Additional toxicity studies including repeated oral toxicity, reproductive toxicity, and carcinogenicity tests are needed.
PubMed: 36628351
DOI: 10.3831/KPI.2022.25.4.364 -
International Journal of Molecular... Dec 2021Natural dibenzo-α-pyrones (DAPs) can be viewed from two opposite angles. From one angle, the gastrointestinal metabolites urolithins are regarded as beneficial, while... (Review)
Review
Natural dibenzo-α-pyrones (DAPs) can be viewed from two opposite angles. From one angle, the gastrointestinal metabolites urolithins are regarded as beneficial, while from the other, the emerging mycotoxin alternariol and related fungal metabolites are evaluated critically with regards to potential hazardous effects. Thus, the important question is: can the structural characteristics of DAP subgroups be held responsible for distinct bioactivity patterns? If not, certain toxicological and/or pharmacological aspects of natural DAPs might yet await elucidation. Thus, this review focuses on comparing published data on the two groups of natural DAPs regarding both adverse and beneficial effects on human health. Literature on genotoxic, estrogenic, endocrine-disruptive effects, as well as on the induction of the cellular anti-oxidative defense system, anti-inflammatory properties, the inhibition of kinases, the activation of mitophagy and the induction of autophagy, is gathered and critically reviewed. Indeed, comparing published data suggests similar bioactivity profiles of alternariol and urolithin A. Thus, the current stratification into hazardous toxins and healthy urolithins seems debatable. An extrapolation of bioactivities to the other DAP sub-class could serve as a promising base for further research. Conclusively, urolithins should be further evaluated toward high-dose toxicity, while alternariol derivatives could be promising chemicals for the development of therapeutics.
Topics: Alternaria; Animals; Anti-Inflammatory Agents, Non-Steroidal; Coumarins; Estrogens; Gastrointestinal Microbiome; Humans; Lactones; Mitophagy; Mutagenicity Tests; Mycotoxins
PubMed: 34884865
DOI: 10.3390/ijms222313063 -
Mutation Research. Genetic Toxicology... Jun 2021Mining has a direct impact on the environment and on the health of miners and is considered one of the most hazardous occupations worldwide. Miners are exposed to... (Review)
Review
Mining has a direct impact on the environment and on the health of miners and is considered one of the most hazardous occupations worldwide. Miners are exposed to several occupational health risks, including genotoxic substances, which may cause adverse health effects, such as cancer. This review summarizes the relation between DNA damage and mining activities, focusing on coal and uranium miners. The search was performed using electronic databases, including original surveys reporting genetic damage in miners. Additionally, a temporal bibliometric analysis was performed using an electronic database to create a map of cooccurrence terms. The majority of studies were performed with regard to occupational exposure to coal, whereas genetic damage was assessed mainly through chromosomal aberrations (CAs), micronuclei (MNs) and comet assays. The bibliometric analysis demonstrated associations of coal exposure with silicosis and pneumoconiosis, uranium miners with lung cancer and tumors and some associated factors, such as age, smoking, working time and exposure to radiation. Significantly higher DNA damage in miners compared to nonexposed groups was observed in most of the studies. The timeline reveals that classic biomarkers (comet assay, micronucleus test and chromosomal aberrations) are still important tools to assess genotoxic/mutagenic damage in occupationally exposed miners; however, newer studies concerning genetic polymorphisms and epigenetic changes in miners are being conducted. A major challenge is to investigate further associations between miners and DNA damage and to encourage further studies with miners of other types of ores.
Topics: Animals; Chromosome Aberrations; Coal; Coal Mining; Comet Assay; DNA Damage; Humans; Micronuclei, Chromosome-Defective; Micronucleus Tests; Miners; Occupational Exposure; Uranium
PubMed: 33985692
DOI: 10.1016/j.mrgentox.2021.503348 -
International Journal of Environmental... Mar 2020Genotoxicity screening tests aim to evaluate if and to what extent a compound in contact with the human body (e.g., a drug molecule, a compound from the environment)... (Review)
Review
Genotoxicity screening tests aim to evaluate if and to what extent a compound in contact with the human body (e.g., a drug molecule, a compound from the environment) interacts with DNA. The comet assay is a sensitive method used to predict the risk of DNA damage in individual cells, as it quantifies the tape breaks, being the alkaline version (pH > 13) the most commonly used in the laboratory. Epithelial cells serve as biomatrices in genotoxicity assessments. As ca. 80% of solid cancers are of epithelial origin, the quantification of the DNA damage upon exposure of epithelial cells to a drug or drug formulation becomes relevant. Comet assays run in epithelial cells also have clinical applications in human biomonitoring, which assesses whether and to what extent is the human body exposed to environmental genotoxic compounds and how such exposure changes over time. Ocular mucosa is particularly exposed to environmental assaults. This review summarizes the published data on the genotoxicity assessment in estimating DNA damage in epithelial cells with a special focus on ocular cell lines. General comet assay procedures for ex vivo and in vivo epithelium samples are also described.
Topics: Cell Line; Comet Assay; DNA Damage; Eye; Humans; Mutagenicity Tests
PubMed: 32204489
DOI: 10.3390/ijerph17062046 -
Genes and Environment : the Official... Jul 2021Perillaldehyde and cinnamaldehyde are natural substances found in plants that are used as flavoring ingredients. Due to the α,β-unsaturated aldehydes in their...
BACKGROUND
Perillaldehyde and cinnamaldehyde are natural substances found in plants that are used as flavoring ingredients. Due to the α,β-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.
PubMed: 34271990
DOI: 10.1186/s41021-021-00204-3