A scoping review of recent advances in the application of comet assay to Allium cepa roots.

The comet assay is a sensitive method for the evaluation of DNA damages and DNA repair capacity at single-cell level. Allium cepa is a well-established plant model for toxicological studies. The aim of this scoping review was to investigate the recent application of the comet assay in Allium cepa root cells to assess the genotoxicity. To explore the literature a search was performed selecting articles published between January 2015 and February 2023 from Web of Science, PubMed, and Scopus databases using the combined search terms "Comet assay" and "Allium cepa". All the original articles that applied the comet assay to Allium cepa root cells were included. Of the 334 records initially found, 79 articles were identified as meeting the inclusion criteria. Some studies reported results for two or more toxicants. In these cases, the data for each toxicant were treated separately. Thus, the number of analyzed toxicants (such as chemicals, new materials, and environmental matrices) was higher than the number of selected papers and reached 90. The current use of the Allium-comet assay seems to be directed towards two types of approach: the direct study of the genotoxicity of compounds, mainly biocides (20% of analyzed compounds) and nano- and microparticles (17%), and assessing a treatment's ability to reduce or eliminate genotoxicity of known genotoxicants (19%). Although the genotoxicity identified by the Allium-comet assay is only one piece of a larger puzzle, this method could be considered a useful tool for screening the genotoxic potential of compounds released into the environment.

Reconstructed human intestinal comet assay, a possible alternative in vitro model for genotoxicity assessment.

The aim of the present study was to evaluate the compatibility of reconstructed 3D human small intestinal microtissues to perform the in vitro comet assay. The comet assay is a common follow-up genotoxicity test to confirm or supplement other genotoxicity data. Technically, it can be performed utilizing a range of in vitro and in vivo assay systems. Here, we have developed a new reconstructed human intestinal comet (RICom) assay protocol for the assessment of orally ingested materials. The human intestine is a major site of food digestion and adsorption, first-pass metabolism as well as an early site of toxicant first contact and thus is a key site for evaluation. Reconstructed intestinal tissues were dosed with eight test chemicals: ethyl methanesulfonate (EMS), ethyl nitrosourea (ENU), phenformin hydrochloride (Phen HCl), benzo[a]pyrene (BaP), 1,2-dimethylhydrazine hydrochloride (DMH), potassium bromate (KBr), glycidamide (GA), and etoposide (Etop) over a span of 48 h. The RICom assay correctly identified the genotoxicity of EMS, ENU, KBr, and GA. Phen HCl, a known non-genotoxin, did not induce DNA damage in the 3D reconstructed intestinal tissues whilst showing high cytotoxicity as assessed by the assay. The 3D reconstructed intestinal tissues possess sufficient metabolic competency for the successful detection of genotoxicity elicited by BaP, without the use of an exogenous metabolic system. In contrast, DMH, a chemical that requires liver metabolism to exert genotoxicity, did not induce detectable DNA damage in the 3D reconstructed intestinal tissue system. The genotoxicity of Etop, which is dependent on cellular proliferation, was also undetectable. These results suggest the RICom assay protocol is a promising tool for further investigation and safety assessment of novel ingested materials. We recommend that further work will broaden the scope of the 3D reconstructed intestinal tissue comet assay and facilitate broader analyses of genotoxic compounds having more varied modes of actions.

Synephrine and caffeine combination promotes cytotoxicity, DNA damage and transcriptional modulation of apoptosis-related genes in human HepG2 cells.

The abusive consumption of thermogenic supplements occurs worldwide and deserves special attention due to their use to stimulate weight loss and prevent obesity. Thermogenic formulations usually contain Synephrine (SN) and Caffeine (CAF), stimulating compounds extracted from natural sources, but no genetic toxicology studies have predicted this hazardous combination potential. This study examined the toxicogenomic responses induced by SN and CAF, either alone or in combination, in the human hepatic cell line HepG2 in vitro. SN (0.03-30 µM) and CAF (0.6-600 µM) alone did neither decrease cell viability nor induce DNA damage, as assessed using the MTT and comet assays, respectively. SN (3 µM) and CAF (30-600 µM) were combined at concentrations similar to those found in commercial dietary supplements. SN/CAF at 3:90 and 3:600 µM ratios significantly decreased cell viability and increased DNA damage levels in HepG2 cells. CAF (600 µM) and the SN/CAF association at 3:60, 3:90, and 3:600 µM ratios promoted cell death by apoptosis, as demonstrated by flow cytometry. Similar results were observed in gene expression (RT-qPCR): SN/CAF up-regulated the expression of apoptosis- (BCL-2 and CASP9) and DNA repair-related (XPC) genes. SN/CAF at 3:90 µM also downregulated the expression of cell cycle control (CDKN1A) genes. In conclusion, the SN/CAF combination reduces cell viability by inducing apoptosis, damages DNA, and modulates the transcriptional expression of apoptosis-, cell cycle-, and DNA repair-related genes in human hepatic (HepG2) cells in vitro. These effects can be worrisome to consumers of thermogenic supplements.

Genetic damage in coal and uranium miners.

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.

Melatonin supplementation over different time periods until ageing modulates genotoxic parameters in mice.

The ageing process is a multifactorial phenomenon, associated with decreased physiological and cellular functions and an increased propensity for various degenerative diseases. Studies on melatonin (N-acetyl-5-methoxytryptamine), a potent antioxidant, are gaining attention since melatonin production declines with advancing age. Hence, the aim of this study was to evaluate the effects of chronic melatonin consumption on genotoxic and mutagenic parameters of old Swiss mice. Herein, 3-month-old Swiss albino male mice (n = 240) were divided into eight groups and subdivided into two experiments: first (three groups): natural ageing experiment; second (five groups): animals that started water or melatonin supplementation at different ages (3, 6, 12 and 18 months) until 21 months. After 21 months, the animals from the second experiment were euthanized to perform the comet assay, micronucleus test and western blot analysis. The results demonstrated that melatonin prolonged the life span of the animals. Relative to genomic instability, melatonin was effective in reducing DNA damage caused by ageing, presenting antigenotoxic and antimutagenic activities, independently of initiation age. The group receiving melatonin for 18 months had high levels of APE1 and OGG1 repair enzymes. Conclusively, melatonin presents an efficient antioxidant mechanism aiding modulating genetic and physiological alterations due to ageing.

Cytotoxic and Genotoxic Assessment of Silicon Dioxide Nanoparticles by Allium and Comet Tests.

Silicon nanoparticles gained a great interest due to its use in biomedical research. It is considered as safe and has been used in nanomedicine. But literature still states its toxicity depending upon the size and dose of silicon nanoparticles. So, current study was aimed to evaluate the cytotoxicity and genotoxicity of silicon dioxide nanoparticles (SiO2NPs) by Allium anaphase-telophase and Comet tests. Characterization of SiO2NPs showed the particle size as 16.12 ± 3.07 nm. The mean diameter of SiO2NPs was having range of 404.66 ± 93.39 nm in solution. Highest total anomalies (18.80 ± 0.45) were observed at 100 µg/mL, whereas least (11.2 ± 0.84) were observed by the 12.5 µg/mL concentration. There was concentration-response association in increased CAs and DNA damage. The highest concentration (100 µg/mL) of SiO2NPs induced the significant DNA damage (149.67 ± 1.15), whereas the least was observed by the negative control (2.67 ± 0.58). The current study revealed the cytotoxic and genotoxic effects of SiO2NPs on the root meristem cells of A. cepa.

Protective effects of Cordyceps extract against UVB­induced damage and prediction of application prospects in the topical administration: An experimental validation and network pharmacology study.

ETHNOPHARMACOLOGICAL RELEVANCE: UVB is a high energy source that causes the major risk factor for sunburn and skin tumor. However, photochemical interactions lead to beneficial effects such as synthesis of vitamin D and corticosteroids. Therefore, a reasonable therapeutic regime is advocated to reduce UVB injuries but makes use of synthesizing sunlight metabolite. Many natural compounds improving plant cells resistant to oxidative stress by the harnessing of solar energy may be also used to protect human cells. Although many nature plants have shown photoprotective effects on skin, the mechanisms underlying of the effects are still ambiguous. AIM OF THE STUDY: This study evaluates the protective effects of cultivated Cordyceps against UVB-induced damage in human keratinocytes and identifies the photoprotective mechanisms using a transcriptomic network approach. MATERIALS AND METHODS: Cordyceps extract compositions were investigated by HPLC analysis. Cell survival, reactive oxygen species (ROS) generation, H2O2 content, aquaporin 3 (AQP3) level and DNA damage were determined upon UVB irradiation in the presence of Cordyceps extract. In addition, next-generation sequencing was used to profile transcriptomic alteration of 20 mJ/cm2 UVB and non-UV. Finally, a network pharmacology method was applied to study Cordyceps extract-related natural compounds and their UVB-induced differentially change targets using the Cytoscape 3.7.1 software. RESULTS: Adenosine and mannitol were the major contents in Cordyceps extract. Cordyceps caused a significant diminished in intracellular UVB-induced oxidative stress, including ROS production and intracellular H2O2 content. Besides, AQP3 which mediated intracellular signal transmission and transported H2O2 into cells was significantly increased in the presence of Cordyceps extract against UVB irradiation. In addition, DNA repair effect of Cordyceps extract after UV irradiation was proven to be effective by comet assay. Moreover, KEGG analysis showed steroid hormone biosynthesis, ovarian steroidogenesis, fat digestion and absorption were enriched in top 3 between 20 mJ/cm2 UVB and non-UV. Gene ontology (Go) analysis showed that steroid metabolic process, sterol metabolic process, and cholesterol metabolic process were enriched in top3 biology process. By using network analysis, 125 potential bioactive ingredients in Cordyceps and 201 targets were identified. Finally, signal pathway analyses suggested that the protective effects of Cordyceps compounds against low dose UVB­induced changes might target PPAR signaling pathway, cholesterol metabolism, and ovarian steroidogenesis. CONCLUSION: Cordyceps extract may be an ideal product for external use of skin which could not only avoid UVB-induced adverse effects, but also could application of metabolite products by UVB such us steroid hormone and vitamin D3.

Nephrotoxicity: Topical issue.

Drug-induced kidney injury is one of the most significant adverse events and dose limiting factor in chemotherapy as well a major cause of prospective drug attrition during pharmaceutical development. Moreover, kidney injury can also occur as a consequence of exposures to environmental xenobiotics such as heavy metals, fungal toxins and nanomaterials. The lack of adequate in vitro human kidney models that mimic more realistically the in vivo conditions and the absence of suitable and robust, cost-effective and predictive cell-based in vitro assays contribute to an underestimation of the kidney toxic potential of new drugs and xenobiotics. Therefore, a rapid screening system capable to detect potential nephrotoxicity at early stages of drug discovery is an urgent need. Here we provide an overview of human cell lines currently used as a surrogate in vitro kidney models in nephrotoxicity studies, including their advantages and limitations. In addition, the capacity of the single cell gel electrophoresis (SCGE)/comet assay as a potential tool in kidney toxicants screening is discussed. Despite a limited number of studies using the comet assay to evaluate the drug-induced kidney damage potential, a considerable variability in SCGE methodology (e.g. lysis, unwinding, and electrophoresis conditions) has been observed. Before the comet assay can be included in nephrotoxicity testing, a basic guideline has to be developed. To test its feasibility, additional in vitro experiments including inter-laboratory validation studies based on this guideline have to be performed.

Folate, genomic stability and colon cancer: The use of single cell gel electrophoresis in assessing the impact of folate in vitro, in vivo and in human biomonitoring.

Intake of folate (vitamin B9) is strongly inversely linked with human cancer risk, particularly colon cancer. In general, people with the highest dietary intake of folate or with high blood folate levels are at a reduced risk (approx. 25%) of developing colon cancer. Folate acts in normal cellular metabolism to maintain genomic stability through the provision of nucleotides for DNA replication and DNA repair and by regulating DNA methylation and gene expression. Folate deficiency can accelerate carcinogenesis by inducing misincorporation of uracil into DNA, by increasing DNA strand breakage, by inhibiting DNA base excision repair capacity and by inducing DNA hypomethylation and consequently aberrant gene and protein expression. Conversely, increasing folate intake may improve genomic stability. This review describes key applications of single cell gel electrophoresis (the comet assay) in assessing genomic instability (misincorporated uracil, DNA single strand breakage and DNA repair capacity) in response to folate status (deficient or supplemented) in human cells in vitro, in rodent models and in human case-control and intervention studies. It highlights an adaptation of the SCGE comet assay for measuring genome-wide and gene-specific DNA methylation in human cells and colon tissue.

Assessing relative sensitivity of marine and freshwater bivalves following exposure to copper: Application of classical and novel genotoxicological biomarkers.

Determination of relative sensitivity of biota following exposures to contaminants including metals is important for environmental protection. Copper (Cu), although biologically essential can be highly toxic to biota if present at higher concentrations in the natural environment. Given its ubiquitous presence within coastal and inland water bodies, we compared Cu-induced genotoxicity in two ecologically important mussel species, the freshwater Dreissena polymorpha (DP) and marine Mytilus galloprovincialis (MG), along with its tissue specific accumulation. Novel biomarker in terms of induction of gamma H2AX (γ-H2AX) foci, along with comet assay and induction of micronuclei (MN) were used to determine DNA damage response (DDR) in these two species following exposure to a range of Cu concentrations (18, 32, 56 µg L-1) for 10 days. Concentration-dependent increases in Cu concentration in gill tissue, as determined by Inductively Coupled Plasma Mass Spectrometry (ICP-MS), were paralleled by a greater degree of genotoxicity. An induction of γ-H2AX foci was present in all Cu exposure concentrations, proving this technique to be a sensitive and suitable biomarker of genotoxicity in bivalves. The multi-biomarker approach adopted here suggests firstly that in parallel with MG, which is widely used to assess the health of marine and coastal environment, DP is also suitable representative of inland water bodies, and that there is a similar mechanism of action for the induction of genotoxicity between the two species, following exposure to Cu. Secondly, for genotoxicity assessment a battery of responses could simultaneously be assessed in these two bivalve species. Finally, for adequate protection of the environment it is vital to adopt a multi-biomarker, multi-species approach to determine adverse biological effects to gain a holistic understanding of the real threat posed by contaminants to hydrosphere.

Safety of sucrose esters from Physalis peruviana L. in a 28-day repeated-dose study in mice.

Although extracts and consumed foods from Physalis species contain sucrose esters from their glandular trichomes, there is no experimental data available on their toxicological effects. As peruvioses A and B isolated from Physalis peruviana L. calyces have proved to be effective anti-inflammatory and immunomodulatory compounds, this work aimed to investigate their sub-acute toxicity study and genotoxicity. For this, CD-1(ICR) mice were treated intraperitoneally with peruvioses at doses of 2.5, 5, and 10mg/kg/day for 28 consecutive days, to simulate therapeutic and over-therapeutic dosage levels. At the end of the treatment, animals were sacrificed and their organs weighted, and blood and tissue samples were collected. Toxicological endpoints included clinical signs; food consumption; body and organ weights; hematological and biochemical parameters; as well as macroscopic and microscopic examination of tissues. The results showed no significant differences between treated animals and control group at macroscopic, histological, molecular, and biochemical levels. In addition, a combination of mammalian erythrocyte micronucleus test, comet assay in peripheral blood cells, and Ames test, did not reveal genotoxic effects induced by peruvioses. Taken together, our data suggests that peruvioses A and B can be safely employed to treat inflammatory diseases.

Validation of Comet assay in Oregon-R and Wild type strains of Drosophila melanogaster exposed to a natural radioactive environment in Brazilian semiarid region.

Natural radiation of geological origin is a common phenomenon in Brazil, a country where radioactive agents such as uranium may be often found. As an unstable atom, uranium undergoes radioactive decay with the generation of a series of decay by-products, including radon, which may be highly genotoxic and trigger several pathological processes, among which cancer. Because it is a gas, radon may move freely between cracks and gaps in the ground, seeping upwards into the buildings and in the environment. In this study, two Drosophila melanogaster Meigen (Diptera, Drosophilidae) strains called Oregon-R and Wild (collected in a non-radioactive environment) were exposed to atmospheric radiation in the Lajes Pintadas city, in the semiarid zone of northeastern Brazil. After six days of environmental exposure, the organisms presented genetic damage significantly higher than that of the negative control group. The genotoxic effects observed reinforce the findings of other studies carried out in the same region, which warn about the environmental risks related to natural radioactivity occurrence. The results also validate the use of the Comet assay in hemocytes of D. melanogaster as a sensitive test to detect genotoxicity caused by natural radiation, and the use of a recently collected D. melanogaster strain in the environmental of radon.

Earthworm Comet Assay for Assessing the Risk of Weathered Petroleum Hydrocarbon Contaminated Soils: Need to Look Further than Target Contaminants.

Earthworm toxicity assays contribute to ecological risk assessment and consequently standard toxicological endpoints, such as mortality and reproduction, are regularly estimated. These endpoints are not enough to better understand the mechanism of toxic pollutants. We employed an additional endpoint in the earthworm Eisenia andrei to estimate the pollutant-induced stress. In this study, comet assay was used as an additional endpoint to evaluate the genotoxicity of weathered hydrocarbon contaminated soils containing 520 to 1450 mg hydrocarbons kg-1 soil. Results showed that significantly higher DNA damage levels (two to sixfold higher) in earthworms exposed to hydrocarbon impacted soils. Interestingly, hydrocarbons levels in the tested soils were well below site-specific screening guideline values. In order to explore the reasons for observed toxicity, the contaminated soils were leached with rainwater and subjected to earthworm tests, including the comet assay, which showed no DNA damage. Soluble hydrocarbon fractions were not found originally in the soils and hence no hydrocarbons leached out during soil leaching. The soil leachate's Electrical Conductivity (EC) decreased from an average of 1665 ± 147 to 204 ± 20 µS cm-1. Decreased EC is due to the loss of sodium, magnesium, calcium, and sulphate. The leachate experiment demonstrated that elevated salinity might cause the toxicity and not the weathered hydrocarbons. Soil leaching removed the toxicity, which is substantiated by the comet assay and soil leachate analysis data. The implication is that earthworm comet assay can be included in future eco (geno) toxicology studies to assess accurately the risk of contaminated soils.

Jaundice, phototherapy and DNA damage in full-term neonates.

OBJECTIVE: Phototherapy is the standard therapeutic approach for neonatal hyperbilirubinemia. Oxidative effects of phototherapy may have potential harms, including DNA damage. Unconjugated bilirubin (UCB) might also possess antigenotoxic potential. Intensive phototherapy is more efficacious than conventional phototherapy in treating hyperbilirubinemia. This study aimed to assess the impact of hyperbilirubinemia and the two different types of phototherapy on DNA damage in peripheral blood mononuclear cells of neonates. STUDY DESIGN: The study was conducted on term neonates with non-hemolytic hyperbilirubinemia and control healthy neonates. Genotoxicity was assessed using single-cell gel electrophoresis (Comet assay) in peripheral mononuclear cells. Blood samples were obtained at enrollment in all infants and after intensive or conventional phototherapy in jaundiced infants. RESULT: DNA damage did not significantly differ between jaundiced and non-jaundiced neonates (11.4±8.7 and 10.9±8.3 arbitrary units (AU), respectively, P=0.58). It increased significantly after exposure to phototherapy compared with prephototherapy values (45.6±14.7 vs 11.4±8.7 AU, respectively, P<0.001). The duration of phototherapy correlated positively with markers of DNA damage (r=0.86, P<0.001); however, the intensity of used light did not significantly impact genotoxicity. CONCLUSION: Hyperbilirubinemia does not influence DNA damage, whereas both conventional and intensive phototherapy are associated with DNA damage in term infants with hyperbilirubinemia.

Assessment of DNA damage using comet assay in middle-aged overweight/obese subjects after following a hypocaloric diet supplemented with cocoa extract.

UNLABELLED: Nutrient excess and unbalanced diets can result in overproduction of reactive oxygen species (ROS), which are associated with oxidative stress. Cocoa extract contains antioxidants that inhibit the harmful effects of ROS. This trial analysed the effect of cocoa extract consumption integrated as a bioactive compound into ready-to-eat meals, on oxidative stress at the level of DNA in overweight/obese subjects. Fifty volunteers [57.26(5.24) years, 30.59(2.33)kg/m(2)] participated in a 4-week double-blind, randomised, placebo-controlled parallel nutritional intervention. Half of the volunteers received meals supplemented with 1.4 g/day cocoa extract, while the other half received control meals, both within a 15% energy restriction diet. Lymphocytes were isolated and endogenous strand breaks, oxidised bases and resistance to H2O2-induced damage were measured by the comet assay. The intake of ready-to-eat meals supplemented with cocoa extract did not show relevant changes in the oxidative status of DNA. However, in the cocoa group, oxidised bases negatively correlated with methyl epicatechin-O-sulphate (r = -0.76; P = -0.007) and epicatechin sulphate (r = -0.61; P = -0.046). When volunteers of both groups were analysed together, a marginal decrease (P = 0.072) in oxidised bases was observed, which attributed to weight loss. Subjects who started the intervention with higher levels of damage showed a greater reduction in oxidised bases after 4 weeks (P = 0.040) compared to those who had lower baseline levels. In conclusion, even if 1.4 g of cocoa supplementation for 4 weeks did not show notable changes in terms of antioxidant status of DNA, the energy restriction showed a slightly decrease in oxidised bases and this was seen to a greater extent in subjects who started the intervention with higher levels of damage. On the other hand, the inverse associations found between oxidised bases and some cocoa-derived metabolites suggest that a protective effect might be seen in a longer period of time or in subjects with higher baseline DNA damage. TRIAL REGISTRATION: www.clinicaltrials.gov (NCT01596309).

Sex-specific dose-response analysis of genotoxicity in cyproterone acetate-treated F344 rats.

Cyproterone acetate (CPA), a synthetic hormonal drug, induces rat liver tumors in a sex-specific manner, with five-fold higher doses needed to induce liver tumors in male rats compared to females. In order to evaluate the potential of the in vivo alkaline Comet assay to predict the sex-specific carcinogenicity of CPA, CPA-induced direct DNA damage (DNA strand breaks and alkali-labile sites) were evaluated in the livers of both male and female F344 rats. In addition, secondary oxidative DNA damage was measured concurrently utilizing the human 8-oxoguanine-DNA-N-glycosylase (hOGG1) and EndonucleaseIII (EndoIII)-modified in vivo alkaline Comet assays and the reticulocyte micronucleus (MN) frequency was analyzed in peripheral blood. Groups of 5 seven-week-old male and female F344 rats received olive oil or 10, 25, 50 or 100 mg/kg bw CPA in olive oil by gavage at 0, 24, and 45 h and were sacrificed at 48 h. CPA-induced direct DNA damage in rat liver showed the same sex-specific pattern as its hepatotumorigenicity: a five-fold-higher dose of CPA was needed to induce a statistically significant increase in direct DNA damage in livers of males compared to females. However, peripheral blood MN frequency was weak in both sexes and CPA-induced oxidative DNA damage was generally greater in male than female rat livers. Taken together, our results demonstrate concordance in the sex-specificity of CPA in the in vivo alkaline Comet assay and cancer bioassay, while the induction of oxidative DNA damage by CPA was not directly correlated with its tumorigenicity.

Dose-time dependency of hyperbaric hyperoxia-induced DNA strand breaks in human immune cells visualized with the comet assay.

PURPOSE: Hyperbaric oxygen exposure may induce dose-dependent DNA damage in peripheral blood mononuclear cells (PBMCs), and repetitive exposures of man may have protective cellular effects. METHOD: PBMCs, freshly isolated from non-divers and pure oxygen divers, were exposed to ambient air (21kPa) and hyperoxia at different levels: 100kPa, 240kPa, 400kPa and 600kPa) for up to 6.5 hours in an experimental pressure chamber. DNA double-strand breaks were studied in the comet assay by calculating the "tail moment" and an alternative "Yes or No" method for damaged nuclei. Previously, the experimental procedure had been optimized for human cell experiments: Pre-tests assured that DNA damage could be considered to be oxygen-induced; and cell viability remained over 95% during exposure time. RESULTS: Visible DNA damage increased with the partial pressure of oxygen (pO2) and exposure time dose-dependently. Linear regressions revealed r2 between 0.61 and 0.98 with the Yes/No method, and significant differences in slopes from control. Tail moment showed similar results, but with less accuracy. The PBMCs of oxygen divers exposed to 400kPa pO2 (up to six hours) showed a significant lower slope in the linear regression. CONCLUSION: Oxygen induces dose-dependent DNA double-strand breaks, and the Yes/No discrimination is superior to the tail moment in linearity and accuracy. Oxygen diver PBMCs seem to be more resistant to hyperbaric oxygen.

Protective effects of raw and cooked blackcurrant extract on DNA damage induced by hydrogen peroxide in human lymphoblastoid cells.

CONTEXT: Blackcurrant (Ribes nigrum L.) is a classical fruit that has long been used to make juice, liqueur and sometimes medicines in Europe. The beneficial effects of blackcurrant, which are inhibition of lipopolysaccharide-stimulated inflammatory, anticarcinogenesis and other health effects, have been reported. OBJECTIVE: Previously, we reported the antimutagenic activities of blackcurrant using a yeast gene mutation assay. In this study, we investigated whether this antimutagenicity of blackcurrant was confirmed in human cells. MATERIALS AND METHODS: We prepared four types of aqueous blackcurrant extracts (BCE) from mature and premature with or without heat treatment by microwave. Antioxidant activities of BCE were measured by the DPPH radical scavenger assay. In the DPPH radical scavenger assay, the maximum concentration of BCE was 1.6 mg/reaction. We investigated the antigenotoxic activities of BCE by the comet assay and micronucleus test using the human lymphoblastoid cell line TK6. In the comet assay, TK6 was treated with 300 µM H2O2 without or with BCE at concentrations of 0.5, 1.0, 2.0 and 3.0 mg/mL. In the micronucleus test, TK6 was treated with 1 mg/mL BCE without or with H2O2. RESULTS: All BCEs exhibited more than 90% of inhibition rates of DPPH radicals at the maximum concentration of BCE. DNA damage and micronuclei induced by H2O2 significantly decreased in the each BCE-treated condition. CONCLUSION: The results suggest that BCE treatment can reduce the genomic instability induced by H2O2 in human cells. We consider that these antigenotoxic effects are related to polyphenols, l-ascorbic acid and other antioxidant compounds.

Evaluation of the mutagenicity and antimutagenicity of soy phytoestrogens using micronucleus and comet assays of the peripheral blood of mice.

Studies show that soy imparts many favorable properties in the human body, including the prevention of chronic diseases such as osteoporosis, heart disease, cancer, and diabetes. Soy is rich in isoflavones, and it is a candidate for the chemoprevention of diseases owing to its low toxicity. In this study, a soy phytoestrogen (with high levels of the isoflavones genistin and daidzein) was tested in mice to investigate its mutagenicity and genotoxicity using micronucleus and comet assays of mouse peripheral blood. Phytoestrogen (0.083, 0.83 and 8.3 mg/kg body weight) was evaluated with and without the chemotherapeutic agent cyclophosphamide. For the micronucleus assay, blood was collected before treatment and after 24 and 48 h. For the comet assay, blood was collected only after 24 h. Phytoestrogen was not mutagenic and reduced cyclophosphamide-induced DNA damage. The results from the comet assay revealed a reduction of DNA damage; however, phytoestrogen did induce genotoxic damage during the 24-h treatment. This genotoxic damage could have been repaired and was therefore not identified in the micronucleus assay, which detects mutations. The results suggested that the reduction of DNA damage observed in associated treatments could also reduce the side effects of chemotherapy. Moreover, they suggested that phytoestrogen might be a candidate of interest for the chemoprevention of cancer because it protects against DNA damage.