In vivo assessment of DNA damage and protective effects of extracts from Miconia species using the comet assay and micronucleus test

The genus Miconia comprises approximately 1000 species belonging to the Melastomataceae family. Several crude plant extracts from Miconia and their isolated compounds have shown biological activities, such as analgesic and anti-neoplastic action; however, no studies concerning their effects on DNA are available. The present study aimed to evaluate, in vivo, the genotoxic and mutagenic effects of four species of plants from Miconia genus using the comet assay and micronucleus test. Their possible protective effects were also evaluated in experiments associating the plant extracts with cyclophosphamide (CPA). The methanolic extracts of Miconia albicans, Miconia cabucu, Miconia rubiginosa, Miconia stenostachya and the chloroformic extract of M. albicans were investigated. For genotoxic and mutagenic evaluations, three concentrations were tested, 200, 400 and 540 mg/kg body weight (bw), based on the solubility limit of the extract in distilled water. For the protective effects, only the highest dose was evaluated against 40 mg/kg bw of CPA. Blood was removed from mice tails pre- (T0) and post-treatment (T1-30 h) for the micronucleus test and 24 h post-treatment for the comet assay. The Student's t-test was used to compare data obtained at T0 and T1, the analysis of variance-Tukey test was used to compare between groups in the micronucleus test and the Kruskal-Wallis and Dunn's test were used to compare different groups in the comet assay. All the extracts induced alterations in DNA migration (comet assay); however, no mutagenic effect was observed in the micronucleus assay. All extracts showed a protective effect against CPA in both assays. Our study showed that the use of crude extracts could be more advantageous than the use of isolated compounds. The interaction between phytochemicals in the extracts showed efficacy in reducing mutagenicity and improving the protective effects.     

Genotoxicity of industrial solid waste leachates in Drosophila melanogaster

The potential toxicity of industrial solid wastes is a major environmental concern. The present study evaluated the genotoxicity of industrial waste leachates on the gut cells of Drosophila melanogaster (Oregon R+), using a modified alkaline comet assay. Leachates were prepared from control soil and solid wastes generated by a flashlight battery factory, a pigment plant, and a tannery, using different pHs (7.0, 4.93, and 2.88). Newly emerged first instar Drosophila larvae (22 +/- 2 hr) were transferred to standard Drosophila diet containing 0.05%, 0.1%, 0.5%, 1.0%, and 2.0% of the leachates, and allowed to grow. At 96 +/- 2 hr, the anterior midgut of control and treated larvae was dissected out; single cell suspensions were prepared; and the comet assay was performed on the cells. All the leachates produced significant (P < 0.05), dose-dependent increases in DNA damage, in the gut cells. Leachates prepared at pH 7.0 were significantly less genotoxic than leachates prepared at pH 4.93 or 2.88. A comparison of the comet parameters among the exposed groups indicated that leachates of the pigment plant solid waste produced the least DNA damage, while leachates prepared from the flashlight battery factory solid waste were the most genotoxic. The present study indicates that leachates of solid wastes from flashlight battery factories, pigment plants, and tanneries possess genotoxic activity and that D. melanogaster is a useful in vivo model for assessing the genotoxicity of these potential environmental contaminants.     

Synergistic in vitro antimalarial activity of plant extracts used as traditional herbal remedies in Mali

In Mali, where malaria is endemic, plants are extensively used for treating periodic fevers and malaria. According to the advice of traditional medicine, plants are often mixed during the preparation of febrifugal decoctions. In previous studies, we demonstrated the potent in vitro antimalarial activity of extracts isolated from four plants commonly used in traditional remedies: Mitragyna inermis (Willd.) O. Kuntze, Rubiaceae, Nauclea latifolia (Sm.), Rubiaceae, Guiera senegalensis (Gmel.), Combretaceae, and Feretia apodanthera (Del.), Rubiaceae. In the present work, we evaluate the potent in vitro synergistic antimalarial interaction between these extracts, using standard isobologram analysis. Then, we evaluate their cytotoxicity on human monocytes and their mutagenic activity on an in vitro system of two beta-carboline alkaloids isolated from Guiera senegalensis (harman and tetrahydroharman). Three combinations demonstrate a strong, synergistic, inhibitory effect on in vitro plasmodial development and are devoid of cytotoxicity towards human cells. These results justify their use in association in traditional medicine. Moreover, tetrahydroharman, isolated from G. senegalensis, presents interesting antimalarial activity, no cytotoxicity and is not genotoxic in the Salmonella Ames test with and without metabolic activation.     

Mutagens in human breast lipid and milk: the search for environmental agents that initiate breast cancer

Epidemiological studies indicate the involvement of environmental factors in the etiology of breast cancer, but have not provided clear indications of the nature of the agents responsible. Several environmental carcinogens are known to induce mammary tumors in rodents, and the abundance of adipose tissue in the human breast suggests that the epithelial cells, from which breast tumors commonly arise, could be exposed to lipid-soluble carcinogens sequestered by the adipose tissue. In this report we review our studies in which we have examined human mammary lipid, obtained from elective reduction mammoplasties from healthy donors, and human milk from healthy mothers, for the presence of components with genotoxic activity in several in vitro assays. A significant proportion of lipid extracts induced mutations in bacteria and micronuclei in mammalian cells. They also caused DNA damage, detected as single-strand breaks in the alkaline single-cell gel electrophoresis (comet) assay, in both the MCL-5 cell line and in primary cultures of human mammary epithelial cells. Genotoxic activity was also found in a significant proportion of extracts of human breast milk. Viable cells recovered from milk samples showed evidence of DNA damage and were susceptible to comet formation by genotoxic agents in vitro. Genotoxic activity was found to be less prevalent in milk samples from countries of lower breast cancer incidence (the Far East) compared with that in samples from the UK. The agents responsible for the activity in milk appear to be moderately polar lipophilic compounds and of low molecular weight. Identification of these agents and their sources may hold clues to the origins of breast cancer.     

The effect of smoking on DNA effects in the comet assay: a meta-analysis

The comet assay (alkaline single-cell gel electrophoresis, SCG or SCGE) is frequently used in biomonitoring to detect genotoxic effects in humans exposed at the workplace or in their environment. Because of its ready accessibility, blood is most frequently used in such studies. Many studies investigated cigarette smoking either as a genotoxic exposure itself or as a potential confounding factor in occupational studies. However, although smoking is considered to be a relevant exposure towards various genotoxins, conflicting results have been reported in the comet assay studies. The actual reasons for this discrepancy are not known. To further evaluate evidence for smoking-related DNA effects in the comet assay, we now used a meta-analysis approach based on a literature search. We identified 38 studies from 37 publications which were suited for a formal meta-analysis based on the standardized mean difference (SMD) between the study groups. The evaluation of these 38 studies indicated higher levels of DNA damage in smokers than in non-smokers [under a random effects model, SMD = 0.55, 95% confidence interval = (0.16-0.93)]. Subdividing these studies into studies investigating the effect of smoking as a genotoxic exposure (Type A studies, n = 12) and studies investigating smoking as a potential confounder in occupational studies (Type B, n = 26) indicated a significant difference only in Type A studies but not in Type B studies. Furthermore, studies using image analysis or image length measurements (n = 23) only indicated a tendency for a genotoxic effect of smoking, whereas studies using an arbitrary score (n = 15) found a significantly higher level of DNA damage in smokers.     

Cytotoxic and genotoxic effects of extracts from Annona montana M. fruit

Pulverized immature fruits of Annona montana M. are commonly used for biomedical applications, especially in the treatment of dysentery. The aim of this study was to investigate whether hexane, ethyl acetate or methanol extracts of A. montana M. can be cytotoxic to human tumor cells or exhibit genotoxic effects on normal human cells. The organic extracts were shown to inhibit survival of tumor cells originating from human astrocytoma, breast, colon, lung and prostate cancers. Furthermore, they were found to induce DNA damage in normal human lymphocytes, as shown by comet assay. The genotoxic effects observed suggest that traditional folk medicines prepared from extracts of the immature fruit of A. montana should be used with caution.     

An in vitro evaluation of extracts from some medicinal plants in Kenya against herpes simplex virus

The extracts from 21 medicinal plants commonly used in traditional remedies in Kenya were screened for antiviral activity against wild type 7401H strain herpes simplex virus type 1. The plant extracts exhibited antiviral activity against the virus in the plaque and yield reduction assays. The results reveal that twelve plants may contain constituents that could be exploited for the management of HSV infections. Although the extracts used in these experiments contain a complex matrix of a large number of compounds the results indicate that useful compounds can be isolated for further exploitation.     

Genetic polymorphisms and the effect of cigarette smoking in the comet assay

A potential genotoxic effect of cigarette smoking has repeatedly been investigated with the comet assay (alkaline single cell gel electrophoresis) and conflicting results have been reported. Besides differences in the methodology and the study design used, genetic differences between the subjects investigated might contribute to the variability of test results. Considering genetic polymorphisms of genes involved in metabolism or DNA repair has led to a better discrimination of smoking-related genotoxic effects in some cases but also led to discrepant results. We therefore evaluated our baseline comet assay effects obtained for nonsmokers and smokers in relation to selected genetic polymorphisms. Our study group comprised 52 nonsmokers and 51 smokers who were strictly selected to exclude potential confounding factors. We chose polymorphisms in the genes GSTM1 and CYP1A1 (Ile462Val) because they take part in the metabolism of genotoxins contained in tobacco smoke. In a subgroup of 32 nonsmokers and 31 smokers we also studied polymorphisms in XPD (Lys751Gln), XRCC1 (Arg399Gln) and XRCC3 (Thr241Val) because they are part of DNA repair pathways involved in the repair of tobacco-related DNA damage. Freshly collected peripheral whole blood samples were tested in the alkaline (pH > 13) comet assay. In all experiments a reference standard (untreated V79 cells) was included to correct for assay variability. An independent second evaluation was carried out for all experiments. None of these approaches revealed a significant difference between nonsmokers and smokers.     

Characterization of the genotoxic potential of formaldehyde in V79 cells

Formaldehyde (FA) is known to be genotoxic and mutagenic in proliferating mammalian cells in vitro. The present study was performed to further characterize its genotoxic potential in the V79 Chinese hamster cell line. The induction of DNA strand breaks and DNA-protein cross-links (DPXs) was measured by the comet assay in relationship to the induction of sister chromatid exchanges (SCEs) and micronuclei (MN). Induction of DNA strand breaks was found neither with the standard protocol of the alkaline comet assay nor with modifications using extended electrophoresis times or proteinase K. The concentration-effect relationship for the genotoxic effects was characterized by fitting different curves to the data. A two-phase regression model fitted best in comparison with a linear or a quadratic model and indicated practical thresholds for the induction of SCE and MN. For the induction of DPX as measured by the comet assay, neither a linear concentration-response relationship nor any of the tested models fitted well to the data. Three repeated treatments with genotoxic concentrations of FA with a 3-h interval led to enhanced levels of DPX and MN while the same treatments with a 24-h interval did not enhance FA genotoxicity but suggested adaptive protection against the DNA-damaging action of FA.     

Antioxidant, genotoxic and antigenotoxic activity of daphne gnidium leaf extracts

ABSTRACT: BACKGROUND: Plants play a significant role in maintaining human health and improving the quality of human life. They serve humans well as valuable components of food, as well as in cosmetics, dyes, and medicines. In fact, many plant extracts prepared from plants have been shown to exert biological activity in vitro and in vivo. The present study explored antioxidant and antigenotoxic effects of Daphne gnidium leaf extracts. METHODS: The genotoxic potential of petroleum ether, chloroform, ethyl acetate, methanol and total oligomer flavonoid (TOF) enriched extracts from leaves of Daphne gnidium, was assessed using Escherichia coli PQ37. Likewise, the antigenotoxicity of the same extracts was tested using the "SOS chromotest test". Antioxidant activities were studied using non enzymatic and enzymatic method: NBT/Riboflavine and xantine oxidase. RESULTS: None of the different extracts produced a genotoxic effect, except TOF extract at the lowest tested dose. Our results showed that D. gnidium leaf extracts possess an antigenotoxic effect against the nitrofurantoin a mutagen of reference. Ethyl acetate and TOF extracts were the most effective in inhibiting xanthine oxidase activity. While, methanol extract was the most potent superoxide scavenger when tested with the NBT/Riboflavine assay. CONCLUSIONS: The present study has demonstrated that D. gnidium leaf extract possess antioxidant and antigenotoxic effects. These activities could be ascribed to compounds like polyphenols and flavonoid. Further studies are required to isolate the active molecules.     

Antigenotoxic properties of Terminalia arjuna bark extracts.

Compounds possessing antimutagenic properties (polyphenols, tannins, vitamins, etc.) have been identified in fruits, vegetables, spices, and medicinal plants. Terminalia arjuna (Combretaceae), a tropical woody tree occurring throughout India and known locally as Kumbuk, is a medicinal plant rich in tannins and triterpenes that is used extensively in Ayurvedic medicine as a cardiac tonic. The aim of the present collaborative work was to test six solvent extracts from the bark of Terminalia arjuna for antigenotoxic activity using in vitro short-term tests. Terminalia arjuna extracts were obtained by sequential extraction using acetone, methanol, methanol + HCl, chloroform, ethyl acetate, and ethyl ether. The antigenotoxic properties of these extracts were investigated by assessing the inhibition of genotoxicity of the directacting mutagen 4-nitroquinoline-N-oxide (4NQO) using the "comet" assay and the micronucleus (MN) test. Human peripheral blood leukocytes were incubated with different concentrations of the six extracts (from 5 to 100 microg/ mL) and with 4NQO (1 and 2 microg/mL, for the "comet" assay and MN test, respectively). Each extract/4NQO combination was tested twice; in each experiment, positive control (4NQO alone) and negative control (1% DMSO) were set. "Comet" assay results showed that acetone and methanol extracts were highly effective in reducing the DNA damage caused by 4NQO, whereas the acidic methanol, chloroform, ethyl acetate, and ethyl ether extracts showed less marked or no antigenotoxic activity. In the MN test, a decrease in 4NQO genotoxicity was observed by testing this mutagen in the presence of acetone, methanol, chloroform, and ethyl acetate extracts, even though the extent of inhibition was not always statistically significant.     

Adaptation to alkylation damage in DNA measured by the comet assay

The alkylating mutagens N-methyl-N-nitrosourea (MNU) and methyl methanesulfonate (MMS) were studied for their potential to induce DNA strand breaks and abasic (AP) sites in meristematic nuclei of Vicia faba root tips by the comet assay. The alkaline unwinding/neutral electrophoresis (A/N) and alkaline unwinding/alkaline electrophoresis (A/A) protocols were used for detection of DNA damage. With the A/N comet assay, less DNA damage was seen after conditioning pretreatment with a low dose prior to a high challenging dose of alkylating mutagens as compared to application of the high dose only, whereas a nearly additive effect was seen when the A/A comet assay was used. Adaptation was even more obvious when AP sites were revealed by the AP-endonuclease activity of exonuclease III. The adaptation observed with the A/N comet assay was abolished by pretreatment with the protein synthesis inhibitor cycloheximide. These data suggest that the comet assay is able to detect on molecular level a phenomenon resembling clastogenic adaptation.     

Correlation of apoptosis with comet formation induced by tea polyphenols in human leukemia cells.

Induction of apoptosis is an important approach to cancer control. Apart from morphological changes in cells, apoptosis is characterized by fragmentation of nuclear DNA. The characteristic DNA ladder formation that is observed on gel electrophoresis does not reflect the DNA breakdown in individual cells; contributions from small subpopulations are usually overlooked. On the other hand, alkaline comet assay as measured by single cell gel electrophoresis accurately measures DNA fragmentation at a single cell level. The comet assay was originally developed as a cytogenetic test to measure the genotoxicity of various chemicals. However, the comet image generated by an apoptotic cell is different from that obtained with a cell treated for a short time with a genotoxic agent. In the present study using human leukemic cells, typical apoptotic features such as morphological characteristics, FACS analysis, caspase activation, and expression of apoptosis-related genes as induced by tea polyphenols have been found to correlate with the comet tail formation. It is apparent from the high degree of correlation observed between the comet tail moment and each parameter of apoptosis that the comet assay can accurately reflect the measure of DNA fragmentation and, hence, can be used to detect a cell undergoing apoptosis.     

Assessment of DNA damage in lymphocytes of workers exposed to X-radiation using the micronucleus test and the comet assay.

The mutagenic and carcinogenic effects of genotoxic agents on exposed people have constituted an increasing concern. Therefore, the objective of this work was to assess DNA damage in lymphocytes of workers exposed to X-radiation using the cytokinesis-blocked micronucleus test and the comet assay (single-cell gel electrophoresis), and to compare these two techniques in the monitoring of exposed populations. The cytokinesis-blocked micronucleus test and the comet assay were employed in the monitoring of 22 workers occupationally exposed to X-radiation in a hospital in southern Brazil. The frequency of dicentric bridges was also measured. The results of both assays and the frequency of dicentric bridges revealed a significant increase in genetic effects on the cells of exposed individuals. Age was significantly correlated with micronucleus frequency and damage index in the comet assay. The concomitant analysis of dicentric bridges when determining micronucleus frequency does not require much extra work, and may serve as a reference to the type of mutagenic effect (clastogenic or aneugenic). The combination of the alkaline comet assay with the cytokinesis-blocked micronucleus test appears to be very informative for the monitoring of populations chronically exposed to genotoxic agents.     

In vitro evaluation of the genotoxic and cytotoxic effects of artesunate, an antimalarial drug, in human lymphocytes

Artesunate is one of the main antimalarial drugs used in several countries. It is a semisynthetic compound derived from artemisinin, a substance extracted from the Chinese plant, Artemisia annua L. Despite the widespread use of artesunate as an antimalarial drug, there is a lack of data regarding its genotoxic effects in human lymphocytes. Therefore, in this study, we used the comet assay and micronucleus test to evaluate the possible genotoxic effects of artesunate in cultured human lymphocytes. In addition, cell death by necrosis and apoptosis was also assessed. Cells exposed to different concentrations of artesunate showed a significant concentration-dependent increase (P < 0.05) in DNA damage index and micronuclei frequency. A significant increase in the frequency of apoptotic and necrotic cells was also observed. Our results showed that artesunate is a genotoxic and cytotoxic compound in cultured human lymphocytes.     

Genotoxicity of pesticide mixtures present in the diet of the French population

Consumers may be simultaneously exposed to several pesticide residues in their diet. A previous study identified the seven most common pesticide mixtures to which the French population was exposed through food consumption in 2006. The aim of this study was to investigate if the seven mixtures are potentially cytotoxic and genotoxic and if so, whether compounds in a same mixture have a combined effect. The cytotoxicity and genotoxicity of the seven mixtures were investigated with a new assay (γ‐H2AX) using four human cell lines (ACHN, SH‐SY5Y, LS‐174T, and HepG2). Mixtures were tested at equimolar concentrations and also at concentrations reflecting their actual proportion in the diet. Irrespective of the cell line tested, parallel cytotoxicity of the seven mixtures was observed. Only one mixture was genotoxic for the HepG2 cells at concentrations = 3 μM in equimolar proportion and at 30 μM in actual proportion. Caspase 3/7 activity, the comet assay, and reactive oxygen species production were also investigated using the same mixture and HepG2 cells. Our results suggest that pesticide metabolites from the mixture generated by HepG2 cells were responsible for the observed damage to DNA. Among the five compounds in the genotoxic mixture, only fludioxonil and cyprodinil were genotoxic for HepG2 cells alone at concentrations = 4 and 20 μM, respectively. Our data suggest a combined genotoxic effect of the mixture at low concentrations with a significantly higher effect of the mixture of pesticides than would be expected from the response to the individual compounds. Environ. Mol. Mutagen. 2012. © 2012 Wiley Periodicals, Inc.     

Comparative biomonitoring study of workers at a waste disposal site using cytogenetic tests and the comet (single-cell gel) assay

Workers exposed to environmental pollutants at a waste disposal site were studied for genotoxic effects with cytogenetic tests and the comet (alkaline single-cell gel) assay. Analyses were performed on peripheral blood samples of 44 workers at a waste disposal site (DM) and 47 subjects of a control group (VE) matched for gender, age, and smoking habits. Chromosomal aberrations were evaluated in 1,000 lymphocytes per individual, sister chromatid exchanges in 50 cells, and DNA migration (tail moment) was determined in 100 leukocytes. Structural chromosome aberrations were more frequent in DM than in VE, but only the frequency of acentric fragments and the percentage of aberrant cells (excluding gaps) was significantly increased. No significant difference was found for the mean frequency of SCE. A statistically significant difference was also seen with the comet assay. The mean tail moment was higher in DM than in VE. However, no correlation was found between cytogenetic data and the effects in the comet assay. The results of our study indicate that DNA effects in the comet assay represent an independent endpoint which might be useful for the biomonitoring of genotoxic effects in addition to established tests. Environ. Mol. Mutagen. 32:17–24, 1998 © 1998 Wiley-Liss, Inc.     

Development and validation of a modified comet assay to phenotypically assess nucleotide excision repair

There is an increasing need for simple and reliable approaches to phenotypically assess DNA repair capacities. Therefore, a modification of the alkaline comet assay was developed to determine the ability of human lymphocyte extracts to perform the initial steps of the nucleotide excision repair (NER) process, i.e. damage recognition and incision. Gel-embedded nucleoids from A549 cells, pre-exposed to 1 microM benzo[a]pyrene-diol-epoxide, were incubated with cell extracts from frozen or freshly isolated lymphocytes. The rate at which incisions are introduced and the subsequent increase in tail moment is indicative for the repair capacity of the extracts. Freshly prepared extracts from lymphocytes of human volunteers (n = 8) showed significant inter-individual variations in their DNA repair capacity, which correlated with the removal of bulky DNA lesions over a period of 48 h determined by (32)P-post-labelling (R(2) = 0.76, P = 0.005). Repeated measurements revealed a low inter-assay variation (11%). Storage of cell extracts for more than 3 weeks significantly reduced (up to 80%) the capacity to incise the damaged DNA as compared to freshly isolated extracts. This reduction was completely restored by addition of ATP to the extracts before use, as it is required for the incision step of NER. In contrast, extracts freshly prepared from frozen lymphocyte pellets can be used without loss of repair activity. DNA repair deficient XPA-/- and XPC-/- fibroblasts were used to further validate the assay. Although some residual capacity to incise the DNA was observed in these cells, the repair activity was restored to normal wild-type levels when a complementary mixture of both extracts (thereby restoring XPA and XPC deficiency) was used. These results demonstrate that this repair assay can be applied in molecular epidemiological studies to assess inter-individual differences in NER.     

Antioxidant and antimicrobial activities of ethyl acetate extract, fractions and compounds from stem bark of Albizia adianthifolia (Mimosoideae)

Background

Plants play a significant role in maintaining human health and improving the quality of human life. They serve humans well as valuable components of food, as well as in cosmetics, dyes, and medicines. In fact, many plant extracts prepared from plants have been shown to exert biological activity in vitro and in vivo. The present study explored antioxidant and antigenotoxic effects of Daphne gnidium leaf extracts.

Methods

The genotoxic potential of petroleum ether, chloroform, ethyl acetate, methanol and total oligomer flavonoid (TOF) enriched extracts from leaves of Daphne gnidium, was assessed using Escherichia coli PQ37. Likewise, the antigenotoxicity of the same extracts was tested using the ??SOS chromotest test??. Antioxidant activities were studied using non enzymatic and enzymatic method: NBT/Riboflavine and xantine oxidase.

Results

None of the different extracts produced a genotoxic effect, except TOF extract at the lowest tested dose. Our results showed that D. gnidium leaf extracts possess an antigenotoxic effect against the nitrofurantoin a mutagen of reference. Ethyl acetate and TOF extracts were the most effective in inhibiting xanthine oxidase activity. While, methanol extract was the most potent superoxide scavenger when tested with the NBT/Riboflavine assay.

Conclusions

The present study has demonstrated that D. gnidium leaf extract possess antioxidant and antigenotoxic effects. These activities could be ascribed to compounds like polyphenols and flavonoid. Further studies are required to isolate the active molecules.     

Evaluation of genome damage and its relation to oxidative stress induced by glyphosate in human lymphocytes in vitro

In the present study we evaluated the genotoxic and oxidative potential of glyphosate on human lymphocytes at concentrations likely to be encountered in residential and occupational exposure. Testing was done with and without metabolic activation (S9). Ferric‐reducing ability of plasma (FRAP), thiobarbituric acid reactive substances (TBARS) and the hOGG1 modified comet assay were used to measure glyphosate's oxidative potential and its impact on DNA. Genotoxicity was evaluated by alkaline comet and analysis of micronuclei and other nuclear instabilities applying centromere probes. The alkaline comet assay showed significantly increased tail length (20.39 μm) and intensity (2.19%) for 580 μg/ml, and increased tail intensity (1.88%) at 92.8 μg/ml, compared to control values of 18.15 μm for tail length and 1.14% for tail intensity. With S9, tail length was significantly increased for all concentrations tested: 3.5, 92.8, and 580 μg/ml. Using the hOGG1 comet assay, a significant increase in tail intensity was observed at 2.91 μg/ml with S9 and 580 μg/ml without S9. Without S9, the frequency of micronuclei, nuclear buds and nucleoplasmic bridges slightly increased at concentrations 3.5 μg/ml and higher. The presence of S9 significantly elevated the frequency of nuclear instabilities only for 580 μg/ml. FRAP values slightly increased only at 580 μg/ml regardless of metabolic activation, while TBARS values increased significantly. Since for any of the assays applied, no clear dose‐dependent effect was observed, it indicates that glyphosate in concentrations relevant to human exposure do not pose significant health risk. Environ. Mol. Mutagen. 2009. © 2009 Wiley‐Liss, Inc.