Evaluation of genotoxicity and DNA protective effects of mangiferin,a glucosylxanthone isolated from Mangifera indica L. stem bark extract

Mangiferin is a glucosylxantone isolated from Mangifera indica L. stem bark. Several studies have shown its pharmacological properties which make it a promising candidate for putative therapeutic use. This study was focused to investigate the in vitro genotoxic effects of mangiferin in the Ames test, SOS Chromotest and Comet assay. The genotoxic effects in bone marrow erythrocytes from NMRI mice orally treated with mangiferin (2000 mg/kg) were also evaluated. Additionally, its potential antimutagenic activity against several mutagens in the Ames test and its effects on CYP1A1 activity were assessed. Mangiferin (50–5000 μg/plate) did not increased the frequency of reverse mutations in the Ames test, nor induced primary DNA damage (5–1000 μg/mL) to Escherichia coli PQ37 cells under the SOS Chromotest. It was observed neither single strand breaks nor alkali-labile sites in blood peripheral lymphocytes or hepatocytes after 1 h exposition to 10–500 μg/mL of mangiferin under the Comet assay. Furthermore, micronucleus studies showed mangiferin neither induced cytotoxic activity nor increased the frequency of micronucleated/binucleated cells in mice bone marrow. In short, mangiferin did not induce cytotoxic or genotoxic effects but it protect against DNA damage which would be associated with its antioxidant properties and its capacity to inhibit CYP enzymes.     

Carbamate insecticide methomyl confers cytotoxicity through DNA damage induction

Carbamate insecticide methomyl could induce genotoxic effects, including micronuclei, chromosome aberrations and sister-chromatid exchanges. However, methomyl induction of cytotoxicity through DNA damage is largely unknown. Here we identify cytotoxicity and potential genotoxicity of methomyl in vitro. We have employed alkaline comet assay, γH2AX foci formation and DNA ladder assay to detected DNA damage and apoptosis of Drosophila S2, HeLa and HEK293 cells. The alkaline comet assay was used to evaluate total DNA single strand breaks (SSBs) in the target cells exposed in vitro to sublethal concentrations of methomyl. As expected, methomyl induced significant concentration-dependent increases in DNA damage of target cells compared with the negative control, as measured by increases in tail length (μm), tail DNA (percentage of the comet tail) and tail moment (arbitrary units). In agreement with the comet assay data, the percentage of γH2AX positive reaction in HeLa cells also revealed methomyl caused DNA double strand breaks (DSBs) in a time-dependent manner. Moreover, methomyl induced a significant increase of apoptosis in Drosophila S2, HeLa and HEK293 cells in a concentration- and time-dependent manner, as determined by Urea PAGE DNA fragmentation analysis. In conclusion, methomyl is a strongly genotoxic agent that induces cell DNA damage and apoptosis in vitro at these sublethal concentrations.     

Ochratoxin A reduces aflatoxin B1 induced DNA damage detected by the comet assay in Hep G2 cells

Mycotoxins aflatoxin B1 (AFB1) and ochratoxin A (OTA) can be present together in food commodities. These food contaminants are considered to be genotoxins, acting by different mechanisms. The aim of this work was to characterize combined genotoxic in vitro effects of both mycotoxins in Hep G2 cells. For this purpose, cytotoxicity was first determined in isolated and combined treatments in order to determine the dose range of genotoxicity studies. Co-exposure of cells to OTA + AFB1 for 24 h resulted in additive effects. Genotoxicity was determined in Hep G2 cells by the modified comet assay with restriction enzymes (endo III and FPG). Significant reactive oxygen species formation was detected in both single and combined treatments. AFB1 was genotoxic after 3 h with external metabolic activation (S9 mix) and after 24 h without metabolic activation. Co-exposure to OTA significantly decreased DNA damage induced by AFB1, not only in breaks and apurinic sites but also in FPG-sensitive sites. The apparent contradiction between additive cytotoxic effects and antagonic genotoxic effects may be explained if AFB1 and OTA compete for the same CYPs, yielding more ROS but less AFB1 adducts.     

Nicotine,cotinine, and β-nicotyrine inhibit NNK-induced DNA-strand break in the hepatic cell line HepaRG

Recent in vitro work using purified enzymes demonstrated that nicotine and/or a nicotine metabolite could inhibit CYPs (CYP2A6, 2A13, 2E1) involved in the metabolism of the genotoxic tobacco nitrosamine NNK. This observation raises the possibility of nicotine interaction with the mechanism of NNK bioactivation. Therefore, we hypothesized that nicotine or a nicotine metabolite such as cotinine might contribute to the inhibition of NNK-induced DNA strand breaks by interfering with CYP enzymes. The effect of nicotine and cotinine on DNA strand breaks was evaluated using the COMET assay in CYP competent HepaRG cells incubated with bioactive CYP-dependent NNK and CYP-independent NNKOAc (4-(acetoxymethylnitrosoamino)-1-(3-pyridyl)-1-butanone). We report a dose-dependent reduction in DNA damage in hepatic-derived cell lines in the presence of nicotine and cotinine. Those results are discussed in the context of the in vitro model selected.     

Effects of borneol on the level of DNA damage induced in primary rat hepatocytes and testicular cells by hydrogen peroxide

The aim of this paper was to evaluate genotoxic effects of borneol and its ability to change DNA-damaging effects of H₂O₂ in rat hepatocytes and testicular cells. Both in vitro and ex vivo approaches were used in the case of hepatocytes. Testicular cells were tested only ex vivo, i.e. shortly after isolation from rats supplemented by borneol. Cytotoxicity of borneol increased in in vitro conditions in a concentration-dependent manner and it was associated with DNA-damaging effects at toxic concentrations. While non-toxic concentrations of borneol applied in vitro protected cells against H₂O₂-induced DNA damage and interfered only partly with rejoining of H₂O₂-induced DNA strand breaks, cytotoxic concentrations of borneol manifested synergy with H₂O₂, i.e. enhanced DNA-damaging effects of H₂O₂. On the other side, borneol given to rats in drinking water decreased the level of DNA damage induced by H₂O₂ in both hepatocytes and testicular cells. Our results show that though at higher concentrations (2-h treatment with >2 mM borneol >0.3084 mg/ml) borneol acts cytotoxically and genotoxically on primary hepatocytes cultured in vitro, if given to rats during 7 days in a daily concentration of 17.14 or 34.28 mg/kg it reduces genotoxicity of H₂O₂ in both hepatocytes and testicular cells.     

In vitro and in vivo genotoxic effects of straight versus tangled multi-walled carbon nanotubes

Some multi-walled carbon nanotubes (MWCNTs) induce mesothelioma in rodents, straight MWCNTs showing a more pronounced effect than tangled MWCNTs. As primary and secondary genotoxicity may play a role in MWCNT carcinogenesis, we used a battery of assays for DNA damage and micronuclei to compare the genotoxicity of straight (MWCNT-S) and tangled MWCNTs (MWCNT-T) in vitro (primary genotoxicity) and in vivo (primary or secondary genotoxicity). C57Bl/6 mice showed a dose-dependent increase in DNA strand breaks, as measured by the comet assay, in lung cells 24?h after a single pharyngeal aspiration of MWCNT-S (1–200?μg/mouse). An increase was also observed for DNA strand breaks in lung and bronchoalveolar lavage (BAL) cells and for micronucleated alveolar type II cells in mice exposed to aerosolized MWCNT-S (8.2–10.8?mg/m³) for 4 d, 4?h/d. No systemic genotoxic effects, assessed by the γ-H2AX assay in blood mononuclear leukocytes or by micronucleated polychromatic erythrocytes (MNPCEs) in bone marrow or blood, were observed for MWCNT-S by either exposure technique. MWCNT-T showed a dose-related decrease in DNA damage in BAL and lung cells of mice after a single pharyngeal aspiration (1–200?μg/mouse) and in MNPCEs after inhalation exposure (17.5?mg/m³). In vitro in human bronchial epithelial BEAS-2B cells, MWCNT-S induced DNA strand breaks at low doses (5 and 10?μg/cm²), while MWCNT-T increased strand breakage only at 200?μg/cm². Neither of the MWCNTs was able to induce micronuclei in vitro. Our findings suggest that both primary and secondary mechanisms may be involved in the genotoxicity of straight MWCNTs.     

Cadmium induces apoptosis and genotoxicity in rainbow trout hepatocytes through generation of reactive oxygene species

Cadmium poses a serious environmental threat in aquatic ecosystems but the mechanisms of its toxicity remain unclear. The purpose of this work was first to determine whether cadmium induced apoptosis in trout hepatocytes, second to determine whether or not reactive oxygen species (ROS) were involved in cadmium-induced apoptosis and genotoxicity. Hepatocytes exposed to increasing cadmium concentrations (in the range of 1-10 microM) showed a molecular hallmark of apoptosis which is the fragmentation of the nuclear DNA into oligonucleosomal-length fragments, resulting from an activation of endogenous endonucleases and recognized as a 'DNA ladder' on conventional agarose gel electrophoresis. Exposure of hepatocytes to cadmium led clearly to the DEVD-dependent protease activation, acting upstream from the endonucleases and considered as central mediators of apoptosis. DNA strand breaks in cadmium-treated trout hepatocytes was assessed using the comet assay, a rapid and sensitive single-cell gel electrophoresis technique used to detect DNA primary damage in individual cells. Simultaneous treatment of trout hepatocytes with cadmium and the nitroxide radical TEMPO used as a ROS scavenger, reduced significantly DNA fragmentation, DEVD-related protease activity and DNA strand breaks formation. These results lead to a working hypothesis that cadmium-induced apoptosis and DNA strand breaks in trout hepatocytes are partially triggered by the generation of ROS. Additional studies are required for proposing a mechanistic model of cadmium-induced apoptosis and genotoxicity in trout liver cells, in underlying the balance between DNA damage and cellular defence systems in fish.     

Genotoxicity of marine sediments in the fish hepatoma cell line PLHC-1 as assessed by the Comet assay

The main goal of this study was to test the usefulness of the Comet assay in the PLHC-1 hepatoma fish cell line as a tool for detecting the presence of genotoxic compounds in contaminated marine sediments. The system has been tested using both model chemicals (benzo[a]pyrene (B[a]P) and ethyl methanesulfonate (EMS)) and extracts of sediment samples obtained with solvent dichloromethane/methanol. For all of the analysed sediment extracts as well as for the model chemicals a concentration dependent genotoxic effect was observed. The sediment with the highest observed genotoxic potential was additionally extracted using various solvents in order to test which class of compounds, according to their polarity, is most responsible for the observed genotoxic effect. Non-polar solvents (cyclohexane and dichloromethane) yielded stronger genotoxic effect but the highest level of DNA damage was determined after exposure to sediment extract obtained with the solvent mixture dichloromethane/methanol which extracts a wide range of contaminants. Our results indicate that the PLHC-1 cell line is a suitable in vitro model in sediment genotoxicity assessment and encourage the use of fish cell lines as versatile tools in ecogenotoxicology.     

The use of cultured primary bovine colon epithelial cells as a screening model to detect genotoxic effects of heterocyclic aromatic amines in the comet assay

Isolated epithelial cells from the bovine colon were maintained in dividing monolayer cultures and used as a model system for colon tissue in in vitro toxicological studies. The cytotoxic effects of the heterocyclic aromatic amines 2-amino-3-methylimidazo [4,5-f]quinoline (IQ) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhiP) were investigated in these cells and IC(50) values were determined by inhibition of neutral red uptake into the cultured cells. Although PhiP was not cytotoxic up to concentrations of 500 microM, IQ was cytotoxic above 300 microM. The induction of DNA strand breaks in cultured bovine epithelial colon cells was determined using the alkaline single-cell gel electrophoresis (Comet assay) technique, and subsequently, the DNA damage was used as a determinant of genotoxic effects of the heterocyclic aromatic amines in order to establish this system for detection of adverse effects of chemicals in a model system for the colon. In the absence of an external enzymatic metabolizing system (S9 mix) both amines did not induce DNA strand breaks. When S9 mix was used, PhiP induced DNA strand breaks above 10 microM whereas IQ did not show any significant effect at 300 microM. This cell culture system was found to be a useful screening system for testing of compounds that are considered to affect colonic tissue.     

Genotoxic effects of myosmine in a human esophageal adenocarcinoma cell line

The incidence of esophageal adenocarcinoma is rapidly rising in Western populations. Gastroesophageal reflux disease (GERD) is thought to be one of the most important risk factors. However, the mechanisms by which GERD enhances tumor formation at the gastroesophageal junction are not well understood. Myosmine is a tobacco alkaloid which has also a wide spread occurrence in human diet. It is readily activated by nitrosation and peroxidation giving rise to the same hydroxypyridylbutanone-releasing DNA adducts as the esophageal carcinogen N'-nitrosonornicotine. Therefore, the genotoxicity of myosmine was tested in a human esophageal adenocarcinoma cell line (OE33). DNA damage was assessed by single-cell gel electrophoresis (Comet assay). DNA strand breaks, alkali labile sites and incomplete excision repair were expressed using the Olive tail moment (OTM). The Fapy glycosylase (Fpg) enzyme was incorporated into the assay to reveal additional oxidative DNA damage. DNA migration was determined after incubation of the cells for 1-24h. Under neutral conditions high myosmine concentrations of 25-50mM were necessary to elicit a weak genotoxic effect. At pH 6 genotoxicity was clearly enhanced giving a significant increase of OTM values at 5mM myosmine. Lower pH values could not be tested because of massive cytotoxicity even in the absence of myosmine. Co-incubation of 25 mM myosmine with 1mM H(2)O(2) for 1h significantly enhanced the genotoxicity of H(2)O(2) but not the oxidative lesions additionally detected with the Fpg enzyme. In the presence of the peroxynitrite donor 3-morpholinosydnonimine (SIN-1) a dose-dependent significant genotoxic effect was obtained with 1-10mM myosmine after 4h incubation. NS-398, a selective inhibitor of cyclooxygenase 2, did not affect the SIN-1 stimulated genotoxicity of myosmine. Finally, the 23 h repair of N-methyl-N'-nitro-N-nitrosoguanidine-induced DNA lesions was significantly inhibited in the presence of 10mM myosmine. In conclusion, myosmine exerts significant genotoxic effects in esophageal cells under conditions which may prevail in GERD such as increased oxidative and nitrosative stress resulting from chronic inflammation.     

Demonstration of Benzo(a)pyrene-Induced DNA Damage in Mice by Alkaline Single Cell Gel Electrophoresis: Evidence for Strand Breaks in Liver but Not in Lymphocytes and Bone Marrow

Abstract: Alkaline single cell gel electrophoresis (also known as the‘comet assay') was used to measure DNA strand breaks and alkali-labile sites in peripheral lymphocytes, bone marrow and liver cells of C57BL/6 mice orally exposed to benzo(a)pyrene. Although this polycyclic aromatic hydrocarbon is a well-known genotoxic agent, little is known about to what extent it actually induces DNA strand breaks in peripheral lymphocytes and other tissues after in vivo exposure. Significant and dose-related damage was observed in liver cells after three days of exposure (lowest observed effect level being 3×100 mg benzo(a)pyrene/kg b.wt.). No such damage could be observed in the lymphocytes and bone marrow cells even after administration of 3×150 mg benzo(a)pyrene/kg b.wt. The reference substance cyclophosphamide produced pronounced DNA damage in lymphocytes and bone marrow cells already in a single dose of 100 mg/kg b.wt. The present mouse study questions the usability of DNA strand breaks in peripheral lymphocytes as an indicator of benzo(a)pyrene-induced genotoxicity.     

The tobacco alkaloid nicotine demonstrates genotoxicity in human tonsillar tissue and lymphocytes.

Recent studies suggest a direct contribution of nicotine, the addictive component of tobacco and tobacco smoke, to human carcinogenesis. To assess the genotoxicity of nicotine, the DNA-damaging effect on human lymphocytes and target cells from lymphatic tissue of the palatine tonsils from 10 healthy patients was tested with the alkaline single-cell microgel electrophoresis (Comet) assay. The degree of DNA migration, a measure of possible DNA single strand breaks, alkali labile sites, and incomplete excision repair sites, was expressed as the Olive tail moment, the percentage of DNA in the tail, and the tail length. One hour exposure to nicotine at 0.125, 0.25, 0.5, 1, 2, and 4 mM induced a statistically significant dose-dependent increase of DNA migration up to 3.8-fold and 3.2-fold in tonsillar cells and lymphocytes, respectively. The lowest concentration eliciting significant DNA damage was 0.5 mM nicotine. The genotoxic effect was confirmed in a second series of experiments using nicotine of high purity from two different suppliers. There were no significant differences between the two series, excluding artifacts from the source of nicotine. Finally, DNA damage by nicotine was compared in cells incubated in medium strictly adjusted to neutral pH, with non-adjusted medium becoming alkaline with increasing nicotine concentrations. Again no differences in DNA migration were observed. The data indicate that nicotine expresses significant direct genotoxic effects in human target cells in vitro. However, no differences in DNA damage were observed in cells from smokers and nonsmokers incubated without nicotine. The lack of higher DNA damage in smokers compared to nonsmokers could be a question of nicotine dose, rapid DNA repair, or interactions with other smoke constituents. These results require further investigations on the contribution of nicotine to tobacco carcinogenesis.     

Genotoxic evaluation of titanium dioxide nanoparticles in vivo and in vitro

With the extensive application of titanium dioxide (TiO₂) nanoparticles (NPs) in food industry, there is a rising debate concerning the possible risk associated with exposure to TiO₂ NPs. The purpose of this study is to evaluate the genotoxicity of TiO₂ NPs using in vivo and in vitro test systems. In vivo study, the adult male Sprague-Dawley rats were exposed to anatase TiO₂ NPs (75 ± 15 nm) through intragastric administration at 0, 10, 50 and 200 mg/kg body weight every day for 30 days. The γ-H₂AX assay showed TiO₂ NPs could induce DNA double strand breaks in bone marrow cells after oral administration. However, the micronucleus test revealed that the oral-exposed TiO₂ NPs did not cause damage to chromosomes or mitotic apparatus observably in rat bone marrow cells. In vitro study, Chinese hamster lung fibroblasts (V79 cells) were exposed to TiO₂ NPs at the dose of 0, 5, 10, 20, 50 and 100 μg/mL. Significant decreases in cell viability were detected in all the treated groups after 24 h and 48 h exposure. Significant DNA damage was only observed at the concentration of 100 μg/mL after 24 h treatment using the comet assay. The obvious gene mutation was observed at the concentration of 20 and 100 μg/mL after 2 h treatment using hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene mutation assay. This study presented a comprehensive genotoxic evaluation of TiO₂ NPs, and TiO₂ NPs were shown to be genotoxic both in vivo and in vitro tests. The gene mutation and DNA strand breaks seem to be more sensitive genetic endpoints for the detection of TiO₂ NPs induced genotoxic effects.     

A method for the assessment of DNA damage in individual,one day old,zebrafish embryo (Danio rerio), without prior cell isolation

Zebrafish (Danio rerio) embryos are increasingly used as an experimental model in toxicology for the detection of lethal and sub-lethal effects of diverse chemicals. DNA damage, an early biomarker of long-term effects such as mutagenesis and carcinogenesis, is commonly assessed in vitro and in vivo using the comet assay – single cell gel electrophoresis. Here we describe a new rapid method for the detection of DNA strand breaks in individual, one day old, zebrafish embryos, without the need for prior cell isolation. After the completed spawning, the embryos were exposed to non-toxic concentrations of model genotoxic compounds for 24 h. The embryos were then treated with Pronase E, embedded on microscope slides and squashed to release the cells. After alkaline electrophoresis, the nuclei were stained with ethydium bromide and analyzed by fluorescence microscopy. Preparation of slides by the described method resulted in well separated cell nuclei with low background DNA damage. A significant increase in DNA damage was detected after exposure to the model genotoxic compounds, methylmethan sulphonate (MMS) and benzo(a)pyrene (BaP), while no DNA damage was induced by NaCl. Our method proved to be sensitive and suitable for the detection of DNA damage in one day old zebrafish embryos, suggesting it could serve as a useful tool for monitoring the genotoxic potential of chemicals and environmental pollutants.     

Assessment of evidence for nanosized titanium dioxide-generated DNA strand breaks and oxidatively damaged DNA in cells and animal models

Nanosized titanium dioxide (TiO₂) has been investigated in numerous studies on genotoxicity, including comet assay endpoints and oxidatively damaged DNA in cell cultures and animal models. The results have been surprisingly mixed, which might be attributed to physico-chemical differences of the tested TiO₂. In the present review, we assess the role of certain methodological issues and publication bias. The analysis shows that studies on DNA strand breaks without proper assay controls or very low intra-group variation tend to show statistically significant effects. Levels of oxidatively damaged DNA, measured by the enzyme-modified comet assay, tend to show no effect in studies that have not included proper assay controls or they have uncertainty about the measurement. In addition, there are indications of publication and reporting bias. Nevertheless, the analysis shows that Aeroxide P25 generates DNA strand breaks in a concentration-dependent manner, which is not dependent on the duration of exposure. The standard comet assay seems to be able to discriminate between the genotoxicity of different types of TiO₂, where anatase TiO₂ seems to be the form with strongest genotoxic potential. Cell culture studies also demonstrate increased levels of oxidatively damaged DNA after exposure to TiO₂. There are relatively few studies on animal models where DNA strand breaks and oxidatively damaged DNA have been tested with reliable methods. Collectively, this review shows that exposure to nanosized TiO₂ is associated with genotoxicity in cells, whereas there are still too few reliable studies to assess the genotoxic potential in animal models.     

Comparative genotoxicity investigation using comet and gammaH2AX assays for screening of genotoxicants in HepG2 human hepatoma cells

As public interest in safety has increased the toxicity evaluation of chemicals become more important. In this study, the DNA-damaging effect of genotoxicants was examined in HepG2 cell line originated from human hepatocellular carcinoma by widely used genotoxicity assays: the comet assay and gammaH2AX immunostaining. Four different direct/indirect genotoxicants were tested in dose-/time-dependent manner. The comet assay and the gammaH2AX immunostaining enables detection of DNA damages in the form of DNA strand breaks with different sensitivity. Therefore, the combination of comet assay and gammaH2AX immunostaining will be complementary tool for evaluation of various forms and degree of DNA damage. Our result also suggested that HepG2 cells could be a suitable model for assessing the genotoxicity of various mutagens and for determining the lowest genotoxic concentration. Further analysis using a larger number of chemicals is warranted to determine the sensitivity and the specificity of HepG2 with in vitro genotoxicity test.     

Genotoxic pressure of vineyard pesticides in fish: field and mesocosm surveys

The present study deals with the genotoxicity assessment of vineyard pesticides in fish exposed in the field or in mesocosm conditions. Primary DNA damage was quantified as strand breaks using the single cell gel electrophoresis assay (Comet assay) applied to fish erythrocytes. In a first experiment, a significant genotoxic effect was observed following an upstream-downstream gradient in early life stages of brown trout (Salmo trutta fario) exposed in the Morcille River contaminated by a mixture of vineyard pesticides during three consecutive years. The pronounced response in terms of DNA damage reported in the present study could argue for a high sensitivity of fish early life stage and/or a high level of exposure to genotoxic compounds in the Morcille River. This stresses the interest in using trout larvae incubated in sediment bed to assess genotoxic compounds in the field. In a second experiment, adult European topminnow (Phoxinus phoxinus) were exposed in water running through artificial channels to a mixture of diuron and azoxystrobin, two of the main pesticides detected in the Morcille watershed. As compared with the unexposed channel, a 3-5-fold increase in the DNA damage was observed in fish exposed to chronic environmental pesticide concentrations (1-2mugL(-1) for diuron and 0.5-1mugL(-1) for axoxystrobin). A single 6h pulse of pesticide (14mugL(-1) of diuron and 7mugL(-1) of azoxystrobin) was applied to simulate transiently elevated chemical concentrations in the river following storm conditions. It did not increase genotoxicity. After a 1-month recovery period, DNA damage in exposed fish erythrocytes recovered to unexposed level, suggesting possible involvement of both repair mechanisms and cellular turnover in this transient response. This work highlights that vineyard treatment by pesticides and in particular diuron and azoxystrobin can represent a genotoxic threat to fish from contaminated watershed rivers.     

What is Comet assay not telling us: AFLP reveals wider aspects of genotoxicity

DNA damage detected by genotoxicity biomarkers such as the Comet assay is not always a reliable indicator of the consequences that genotoxic agents can have on the genome integrity of the exposed organisms. Therefore, to reveal the existence of more permanent alterations of DNA structure after genotoxic stress, the RTG-2 rainbow trout cell line was exposed for 3 days to benzo[a]pyrene (B[a]P, 0.1–10 μM) and ethyl methanesulfonate (EMS, 0.1–1 mM) followed by 3 days of recovery period. Primary DNA damage was evaluated by the Comet assay and DNA alterations were assessed using AFLP (amplified fragment length polymorphism). Qualitative and quantitative modifications in AFLP profiles were analyzed in order to detect genetic alterations arising from mutation events and/or DNA damage. Significant induction in DNA damage measured by the Comet assay was noticed after B[a]P treatment at all concentrations but values returned to the control level after recovery. Exposure to EMS induced significant DNA damage only at the highest concentration and damage persisted after the recovery period. AFLP profiles detected DNA alterations even when Comet assay indicated complete DNA repair, revealing more persistent damage. Since such DNA damage can impair its structure and function, Comet assay results should preferably be supplemented with other methods in order to predict the consequences of genotoxic insult more accurately.     

Genotoxic effects of the alkaloids harman and harmine assessed by comet assay and chromosome aberration test in mammalian cells in vitro

Harman and harmine are beta-carboline alkaloids which are present in plants widely used in medical practice, in beverages used for religious purposes in Brazil, as well as in tobacco smoke and over cooked food. In view of the controversial results observed in the literature about the mutagenic effects of these alkaloids, we studied their cytotoxic and genotoxic effects in V79 Chinese hamster lung fibroblasts in vitro using single-cell gel assay, Comet assay, either in the presence or in absence of an exogenous metabolic activation system (S9-mix), and by the chromosome aberration test without S9-mix. Harmine was more cytotoxic than harman. Both harman and harmine increased aberrant cell frequency and induced DNA damage by the Comet assay. These results suggest that harman and harmine are genotoxic in V79 cells, probably as a consequence of their ability to induce DNA strand breaks.