Comet assay responses in human lymphocytes are not influenced by the menstrual cycle: a study in healthy Indian females

The single-cell gel electrophoresis or Comet assay measures qualitative and quantitative DNA damage in single cells. Its simplicity and non-invasive nature has made it widely accepted for the monitoring of human genotoxicity, employing peripheral blood lymphocytes. Factors, such as gender, age, and dietary and smoking habits are known to affect the Comet assay responses in lymphocytes. However, there is no information regarding the influence of the menstrual cycle on the results of the assay in lymphocytes of females.A study was therefore undertaken among 18 healthy Indian female volunteers to assess the effect of the menstrual cycle on Comet assay responses. During a complete menstrual cycle, only minor changes were observed in the basal levels of DNA damage in the lymphocytes as evident by Comet parameters, such as tail length (μm), tail DNA (%) and Olive tail moment (arbitrary units).To assess the effect of the estrogen 17β-estradiol (at physiological concentrations of 0.5, 1.0 and 2.0 nM) on the Comet assay responses, an in vitro study was conducted in the human lymphocyte cell line JM-1 and the breast cancer cell line MCF-7. As was evident from the Comet parameters, a significant (p < 0.01) concentration-dependent increase in the level of DNA damage was observed in the MCF-7 cells while no significant change was found in the JM-1 cells.The results indicate that the menstrual cycle does not influence the Comet assay responses in lymphocytes; hence, these can serve as a model for monitoring genotoxicity in females.     

Comet assay responses in human lymphocytes are not influenced by the menstrual cycle: a study in healthy Indian females

The single-cell gel electrophoresis or Comet assay measures qualitative and quantitative DNA damage in single cells. Its simplicity and non-invasive nature has made it widely accepted for the monitoring of human genotoxicity, employing peripheral blood lymphocytes. Factors, such as gender, age, and dietary and smoking habits are known to affect the Comet assay responses in lymphocytes. However, there is no information regarding the influence of the menstrual cycle on the results of the assay in lymphocytes of females. A study was therefore undertaken among 18 healthy Indian female volunteers to assess the effect of the menstrual cycle on Comet assay responses. During a complete menstrual cycle, only minor changes were observed in the basal levels of DNA damage in the lymphocytes as evident by Comet parameters, such as tail length (microm), tail DNA (%) and Olive tail moment (arbitrary units). To assess the effect of the estrogen 17beta-estradiol (at physiological concentrations of 0.5, 1.0 and 2.0 nM) on the Comet assay responses, an in vitro study was conducted in the human lymphocyte cell line JM-1 and the breast cancer cell line MCF-7. As was evident from the Comet parameters, a significant (p < 0.01) concentration-dependent increase in the level of DNA damage was observed in the MCF-7 cells while no significant change was found in the JM-1 cells. The results indicate that the menstrual cycle does not influence the Comet assay responses in lymphocytes; hence, these can serve as a model for monitoring genotoxicity in females.     

In-vitro cytotoxic and genotoxic effects of arsenic trioxide on human leukemia (HL-60) cells using the MTT and alkaline single cell gel electrophoresis (Comet) assays

Although arsenic trioxide (ATO) has been the subject of toxicological research, in vitro cytotoxicity and genotoxicity studies using relevant cell models and uniform methodology are not well elucidated. Hence, the aim of the present study was to evaluate the cytotoxicity and genotoxicity induced by ATO in a human leukemia (HL-60) cell line using the MTT [3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] and alkaline single cell gel electrophoresis (Comet) assays, respectively. HL-60 cells were treated with different doses of ATO for 24 h prior to cytogenetic assessment. Data obtained from the MTT assay indicated that ATO significantly (P < 0.05) reduced the viability of HL-60 cells in a dose-dependent manner, showing a LD₅₀ value of 6.4 ± 0.6 μg/mL. Data generated from the comet assay also indicated a significant dose-dependent increase in DNA damage in HL-60 cells associated with ATO exposure. We observed a significant increase (P < 0.05) in comet tail-length, tail arm and tail moment, as well as in percentages of DNA cleavage at all doses tested, showing an evidence of ATO-induced genotoxic damage in HL-60 cells. This study confirms that the comet assay is a sensitive and effective method to detect DNA damage caused by heavy metals like arsenic. Taken together, our findings suggest that ATO exposure significantly (P < 0.05) reduces cellular viability and induces DNA damage in HL-60 cells as assessed by MTT and alkaline single cell gel electrophoresis assays, respectively.     

In vivo genotoxicity of heterocyclic amines detected by a modified alkaline single cell gel electrophoresis assay in a multiple organ study in the mouse

We used a modification of the alkaline single cell gel electrophoresis (SCG) (Comet) assay to test the in vivo genotoxicity of 6 heterocyclic amines, Trp-P-1 (25 mg/kg), Trp-P-2 (13 mg/kg), IQ (13 mg/kg), MeIQ (13 mg/kg), MeIQx (13 mg/kg) and PhIP (40 mg/kg), in mouse liver, lung, kidney, brain, spleen, bone marrow and stomach mucosa. Mice were sacrificed 1, 3, and 24 h after intraperitoneal injection. Trp-P-2, IQ, MeIQ, and MeIQx yielded statistically significant DNA damage in the stomach, liver, kidney, lung and brain; Trp-P-1 in the stomach, liver and lung; and PhIP in the liver, kidney and brain. None of the heterocyclic amines induced DNA damage in the spleen and bone marrow. Our results suggest that the alkaline SCG assay applied to multiple organs is a good way to detect organ-specific genotoxicity of heterocyclic amines in mammals.     

Evaluation of genotoxicity of combined soil pollution by cadmium and imidacloprid

Cadmium (Cd) is one of the important pollutants of soil and the genotoxicity of Cd-contaminated soil was studied in combination with imidacloprid. The single cell gel electrophoresis or comet assay was used to quantify DNA strand breaks as a measure of DNA damage induced by Cd and imidacloprid contamination in soil. The soil was artificially contaminated by Cd (0.0, 0.2, 0.5, 1.0, 2.0 mg· kg^?1 dry soil) or Cd (0.0, 0.2, 0.5, 1.0, 2.0 mg · kg^?1 dry soil) and imidacloprid (0.5 mg · kg^?1 dry soil). Roots ofVicia faba were exposed to the contaminated soil for 2 h at 25°C and were used in the comet assay. DNA damage was measured as the values of percentage of nuclei with tails, tail length, tail DNA, tail moment (TM), and Olive tail moment (OTM). DNA damages of root tips ofVicia faba increased after Cd treatment and there were dose-related increases in DNA damage measured as these parameters. However, the addition of imidacloprid further increased the DNA damage. These data confirmed the genotoxic effect of Cd to plants, and that the combined pollution with imidacloprid can enhance the genotoxicity of Cd.     

Panax ginseng extract modulates oxidative stress,DNA fragmentation and up-regulate gene expression in rats sub chronically treated with aflatoxin B1 and fumonisin B1

Aflatoxins and fumonisins are important food-borne mycotoxins implicated in human health and have cytotoxic effects. The aims of the current study were to evaluate the protective role of Panax ginseng extract (PGE) against the synergistic effect of subchronic administration of aflatoxin B₁ (AFB₁) and fumonisin B₁ (FB₁) on DNA and gene expression in rat. Female Sprague–Dawley rats were divided into eight groups (ten rats/group) and treated for 12 weeks including the control group, the group having received AFB₁ (80 µg/kg bw), the group having received FB₁ (100 µg/kg bw), the group having received AFB₁ plus FB₁ and the groups having received PGE (20 mg/kg bw) alone or with AFB₁ and/or FB₁. At the end of experiment, liver and kidney were collected for the determination of DNA fragmentation, lipid peroxidation (LP), glutathione (GSH) contents and alterations in gene expression. The results indicated that these mycotoxins increased DNA fragmentation, LP and decreased GSH content in liver and kidney and down-regulated gene expression of antioxidants enzymes. The combined treatments with AFB₁ and/or FB₁ plus PGE suppressed DNA fragmentation only in the liver, normalized LP and increased GSH in the liver and kidney as well as up-regulated the expression of GPx, SOD1 and CAT mRNA. It could be concluded that AFB₁ and FB₁ have synergistic genotoxic effects. PGE induced protective effects against their oxidative stress and genotoxicity through its antioxidant properties.     

Aflatoxins in aquatic species: metabolism,toxicity and perspectives

Among all known mycotoxins, aflatoxins represent the most investigated, widespread and worrisome source of contamination of foods and feed worldwide. In the early 1960s, soon after the finding of aflatoxin B₁ (AFB₁) in the feedstuffs of aquacultured rainbow trout that had died in an epizootic of hepatomas, great scientific discoveries were made in several areas by a number of researchers under the direction of scientists like J. Halver, R. 0. Sinnhuber, G. S. Bailey, J. D. Hendricks and colleagues. Since that time, several studies have focused on the identification of new isoenzymes involved in AFB₁ metabolism and on the discovery of new modulators in AFB₁-induced cancer initiation and progression. However, metabolic and toxicological studies on aflatoxins in marine aquacultured species are fragmented and restricted to a limited number of fish species. Aflatoxins exert a substantial impact on the fish farming production, causing disease with high mortality and a gradual decline of reared fish stock quality, thus representing a significant problem in aquaculture systems. Based on these considerations, the goals of this review article are: (1) to gather the currently available scientific information, summarising existing data on aflatoxin contamination on feeds and fishmeals, and toxicological effects induced in reared aquatic species; (2) to make a comparative analysis of AFB₁ metabolism in the most representative species studied; (3) to gain new insights on the risk of DNA damage caused by aflatoxins on fish genomes and their role in cancer development.     

Cypermethrin-induced DNA damage in organs and tissues of the mouse: evidence from the comet assay

Cypermethrin is the most widely used Type II pyrethroid pesticide because of its high effectiveness against target species and its low mammalian toxicity reported so far. It is a fast-acting neurotoxin and is known to cause free radical-mediated tissue damage. The present study investigates the genotoxic effects of cypermethrin in multiple organs (brain, kidney, liver, spleen) and tissues (bone marrow, lymphocytes) of the mouse, using the alkaline comet assay. Male Swiss albino mice were given 12.5, 25, 50, 100, 200 mg/kg BW of cypermethrin intraperitoneally, daily for 5 consecutive days. A statistically significant (p<0.05) dose-dependent increase in DNA damage was observed in all the organs assessed, as evident from the comet-assay parameters, viz., Olive tail moment (OTM; arbitrary unit), tail DNA (%) and tail length (microm). Brain showed maximum DNA damage followed by spleen>kidney>bone marrow>liver>lymphocytes, as evident by the OTM. Our data demonstrate that cypermethrin induces systemic genotoxicity in mammals as it causes DNA damage in vital organs like brain, liver, kidney, apart from that in the hematopoietic system.     

DNA Damage Measured by the Comet Assay in Eight Agronomic Plants

For most crops growing in polluted areas or treated with agricultural chemicals, no genotoxicity assays are available. We have studied the possibility of using the alkaline protocol of the plant-based molecular assay — the Single Cell Gel Electrophoresis (SCGE) assay (also called Comet assay) as a method for detecting induced DNA damage in 8 agronomic important plants (ordered according to the diameter of the nuclei): sugar beet, alfalfa, tobacco, lentil, maize, potato, hard wheat, and bread wheat. The monofunctional alkylating agent ethyl methanesulphonate (EMS) was applied as a model genotoxic agent on young excised leaves of the tested crops for 18 h at 26 °C in the dark. With increasing concentrations of 2 to 10 mM EMS, the DNA damage, expressed by the averaged median tail moment values, significantly increased in nuclei of all crops studied. No correlation between the diameter of nuclei and sensitivity to EMS treatment was observed. The data obtained demonstrate the feasibility of using the Comet assay for detecting induced DNA damage in crops. This revised version was published online in July 2006 with corrections to the Cover Date.     

In vivo genotoxicity of ortho-phenylphenol, biphenyl, and thiabendazole detected in multiple mouse organs by the alkaline single cell gel electrophoresis assay

In Japan, ortho-phenylphenol (OPP), biphenyl (BP), and thiabendazole (2-(4'-thiazolyl)benzimidazole, TBZ) are commonly used as a postharvest treatment to preserve imported citrus fruits during transport and storage. We used a modification of the alkaline single cell gel electrophoresis (SCG) (Comet) assay to test the in vivo genotoxicity of those agents in mouse stomach, liver, kidney, bladder, lung, brain, and bone marrow. CD-1 male mice were sacrificed 3, 8, and 24 h after oral administration of the test compounds. OPP (2000 mg/kg) induced DNA damage in the stomach, liver, kidney, bladder, and lung, BP (2000 mg/kg) and TBZ (200 mg/kg) induced DNA damage in all the organs studied. For OPP, increased DNA damage peaked at 3–8 h and tended to decrease at 24 h. For BP, on the contrary, increased DNA migration peaked at 24 h. That delay may have been due to the fact that OPP is metabolized by cytochrome 450 and prostaglandin H synthase to phenylbenzoquinone (PBQ), a DNA binding metabolite, and BP is metabolized to PBQ via OPP and m-phenylphenol. The positive response to TBZ, an aneugen, supports the in vivo DNA-damaging action of TBZ.     

Protective effects of cyclosativene on H2O2-induced injury in cultured rat primary cerebral cortex cells

Sesquiterpenes have attracted much interest with respect to their protective effect against oxidative damage that may be the cause of many diseases including several neurodegenerative disorders and cancer. Our previous unpublished work suggested that cyclosativene (CSV), a tetracyclic sesquiterpene, has antioxidant and anticarcinogenic features. However, little is known about the effects of CSV on oxidative stress induced neurotoxicity. We used hydrogen peroxide (H₂O₂) exposure for 6 h to model oxidative stress. Therefore, this experimental design allowed us to explore the neuroprotective potential of CSV in H₂O₂-induced toxicity in new-born rat cerebral cortex cell cultures for the first time. For this aim, MTT and lactate dehydrogenase release assays were carried out to evaluate cytotoxicity. Total antioxidant capacity (TAC) and total oxidative stress (TOS) parameters were used to evaluate oxidative changes. In addition to determining of 8-hydroxy-2-deoxyguanosine (8-OH-dG) levels, the single cell gel electrophoresis (or Comet assay) was also performed for measuring the resistance of neuronal DNA to H₂O₂-induced challenge. Our results showed that survival and TAC levels of the cells decreased, while TOS, 8-OH-dG levels and the mean values of the total scores of cells showing DNA damage (Comet assay) increased in the H₂O₂ alone treated cultures. But pre-treatment of CSV suppressed the cytotoxicity, genotoxicity and oxidative stress which were increased by H₂O₂. On the basis of these observations, it is suggested that CSV as a natural product with an antioxidant capacity in mitigating oxidative injuries in the field of neurodegenerative disorders.     

Environmental and occupational biomonitoring using the Comet assay

Biomonitoring of human populations exposed to potential mutagens or carcinogens can provide an early detection system for the initiation of cell disregulation in the development of cancer. In recent years, the Comet assay, also known as a “single cell gel” (SCG) electrophoresis assay, has become an important tool for assessing DNA damage in exposed populations. This is the method of choice for population-based studies of environmental and occupational exposure to air pollutants, metals, pesticides, radiation, and other xenobiotics as we show in this review. To appreciate the role of the Comet assay in the field of biomonitoring, we review data from 122 studies that employed the assay. These studies evaluated environmental versus occupational exposures and the levels of DNA damage in cells of individuals exposed in each case. Our review of the literature reveals the importance of the need to establish standard methodological conditions that affect unwinding and electrophoresis times and tail values (tail length, tail DNA, tail moment), with the goal of being able to compare data collected in different laboratories throughout the world. The Comet assay is susceptible to subtle artifacts of manipulation depending on the type and timing of sampling performed. Therefore, in the reporting of DNA damage detected by the Comet assay, the context of how the DNA damage was created also needs to be reported and considered in the interpretation of Comet assay results. The success of the Comet assay is reflected by its use over the past 20 years in the field of biomonitoring, and by the increasing number of studies that continue to report its use. As the shortcomings of the assay are identified and considered in the interpretation of DNA damage detection, the Comet assay will continue to provide improved reliability as a biomarker in human biomonitoring studies.     

Comet assay using mullet (Mugil sp.) and sea catfish (Netuma sp.) erythrocytes for the detection of genotoxic pollutants in aquatic environment

The development of comet assay for aquatic organisms is of particular relevance in light of the importance of coastal fisheries to several countries around the world. Two of the most common fish species native to southern Brazil are the gray mullet (Mugil sp.) and sea catfish (Netuma sp.) for which we have produced a standardized comet assay using whole erythrocytes taken from samples of these fish. We investigated the potential of the comet assay for monitoring genotoxicity in mullet and sea catfish and made a preliminary investigation of the baseline levels of DNA damage in the erythrocytes of samples of these fish from non-polluted areas as well as assessing the in vitro sensitivity of erythrocyte exposed to 2, 4 and 8 x 10(-5) M of methyl methanesulfonate (MMS) for 1, 2, 6 and 24h at 25 and 37 degrees C. Our results show that there was an increase in baseline DNA damage at higher temperatures and that the amount of MMS-induced DNA damage also increased at higher temperatures and that there was a clear dose/time response to treatment with MMS. To assess the possibility of using fish for environmental biomonitoring we also used the comet assay to investigate the in vitro genotoxic effect of MMS on whole blood cells from human donors and found a clear concentration-related effect at all exposure times, findings which agree with those of other workers. This study demonstrates the potential application of the comet assay to erythrocytes of mullets and sea catfish. However, these findings also suggest that temperature could alter both baseline DNA damage in untreated animals and in vitro cell sensitivity towards genotoxic pollutants.     

Profenofos induced DNA damage in freshwater fish, Channa punctatus (Bloch) using alkaline single cell gel electrophoresis

The aim of the present study was to evaluate the induced genotoxicity (DNA damage) due to organophosphate pesticide profenofos (PFF) in gill cells of freshwater fish Channa punctatus using single cell gel electrophoresis (SCGE)/Comet assay. The 96h LC(50) value of PFF (50% EC) was estimated for the fish species in a semistatic system and then three sub-lethal of LC(50) concentrations viz the sub-lethal 1, sub-lethal 2 and sub-lethal 3 concentrations were determined as 0.58ppb, 1.16ppb and 1.74ppb, respectively. The fish specimens were exposed to these concentrations of the pesticide and the gill tissue samplings were done on 24h, 48h, 72h and 96h post exposure for assessment of DNA damage in terms of percentage of DNA in comet tails. In general, a concentration dependent response was observed in the gill cells with induction of maximum DNA damage at the highest concentration of PFF. The results of the present investigation indicated that PFF could potentially induce genotoxic effect in fish, even in sub-lethal concentrations and SCGE as a sensitive and reliable tool for in vivo assessment of DNA damage caused by the genotoxic agents.     

Effects of Embryonic Neural Stem Cell Transplantation on DNA Damage in the Brain and Spinal Cord Following Spinal Cord Injury

Embryonic neural stem cell (ENSC) transplantation is used experimentally for the improvement of spinal cord repair following spinal cord injury (SCI). However, the effects of such intervention on oxidative stress and cell death remain unknown. We used in vivo Comet assay in the acute and chronic SCI groups compared with the SCI+ENSC transplantation groups of experimental rats in order to evaluate DNA damage in the spinal cord. Chronic SCI resulted in the generation of oxidative DNA damage in the spinal cord brain and kidneys, as indicated by high Comet assay parameters, including the percentage of DNA in the tail (T%, or TD), tail moment (TM), and tail length (TL). The DNA damage levels significantly decreased after ENSC transplantation in the spinal cords of acute and chronic SCI groups within the lesion site and rostrally and caudally to the injury, and in the brains and kidneys of the chronic SCI group. Thus, ENSC transplantation is found to be an effective tool for limitation of DNA damage following spinal cord injury.     

Induction and repair of DNA damage as measured by the Comet assay and the yield of somatic mutations in gamma-irradiated tobacco seedlings

The advantage of using the tobacco (Nicotiana tabacum var. xanthi) mutagenicity assay is the ability to analyze and compare on the same plants under identical treatment conditions both the induced acute DNA damage in somatic cells as measured by the Comet assay and the yield of induced leaf somatic mutations. Gamma-irradiation of tobacco seedlings induced a dose-dependent increase in somatic mutations from 0.5 (control) to 240 per leaf (10Gy). The increased yield of somatic mutations was highly correlated (r = 0.996) with the increased DNA damage measured by the Comet assay immediately after irradiation. With increased dose of gamma-irradiation, the averaged median tail moment values ( +/- S.E.) significantly increased from 1.08 +/- 0.10 (control) to 20.26 +/- 1.61 microm (10Gy). Nuclei isolated from leaves 24h after irradiation expressed tail moment values that were not significantly different from the control (2.08 +/- 0.11). Thus a complete repair of DNA damage induced by gamma-irradiation and measurable by the Comet assay was observed, whereas the yield of somatic mutations increased in relation to the radiation dose. Data on the kinetics of DNA repair and of DNA damage induced by gamma-radiation on isolated tobacco nuclei, and on nuclei isolated from irradiated leaves and roots are presented.     

Evaluation of genotoxicity of combined soil pollution by cadmium and imidacloprid

Cadmium (Cd) is one of the important pollutants of soil and the genotoxicity of Cd-contaminated soil was studied in combination with imidacloprid. The single cell gel electrophoresis or comet assay was used to quantify DNA strand breaks as a measure of DNA damage induced by Cd and imidacloprid contamination in soil. The soil was artificially contaminated by Cd 2 h at 25℃ and were used in the comet assay. DNA damage was measured as the values of percentage of nuclei with tails, tail length, tail DNA, tail moment (TM), and Olive tail moment (OTM). DNA damages of root tips of Vicia faba increased after Cd treatment and there were dose-related increases in DNA damage measured as these parameters. However, the addition of imidacloprid further increased the DNA damage. These data confirmed the genotoxic effect of Cd to plants, and that the combined pollution with imidacloprid can enhance the genotoxicity of Cd.     

Does toxoplasmosis cause DNA damage? An evaluation in isogenic mice under normal diet or dietary restriction

Toxoplasmosis is an anthropozoonotic widespread disease, caused by the coccidian protozoan parasite Toxoplasma gondii. Since there are no data regarding the genotoxicity of the parasite in vivo, this study was designed to evaluate the genotoxic potential of the toxoplasmosis on isogenic mice with normal diet or under dietary restriction and submitted to a treatment with sulfonamide (375 microg/kg per day). DNA damage was assessed in peripheral blood, liver and brain cells using the comet assay (tail moment). The results for leucocytes showed increases in the mean tail moment in mice under dietary restriction; in infected mice under normal diet; in infected, sulfonamide-treated mice under normal diet; in infected mice under dietary restriction and in infected sulfonamide-treated mice under dietary restriction. In liver and brain cells, no statistically significant difference was observed for the tail moment. These results indicated that dietary restriction and T. gondii were able to induce DNA damage in peripheral blood cells, as detected by the comet assay.     

In vivo covalent binding of aflatoxin B1 and aflatoxin M1 to liver DNA of rat,mouse and pig

[¹⁴C]Aflatoxin B₁ (AFB₁) was isolated from cultures of Aspergillus parasiticus grown on [1-¹⁴C]sodium acetate. Covalent binding of AFB₁ to liver DNA of rat and mouse was determined 6–8 h after oral administration. The effectiveness of covalent binding, expressed as DNA binding per dose in the units of a ‘Covalent Binding Index’ (CBI), (μmol aflatoxin/mol DNA nucleotides)/(mmol aflatoxin/kg animal), was found to be 10 400 for rats and 240 for mice. These CBI partly explain the different susceptibility of the two species for the incidence of hepatic tumors.The corresponding values for pig liver DNA, 24 and 48 h after oral administration, were found to be as high as 19 100 and 13 300. DNA-binding has not so far been reported for this species although it could represent an appropriate animal model for studies where a human-like gastrointestinal tract physiology is desirable.Aflatoxin M₁ (AFM₁) is a metabolite found in the milk of cows that have been fed AFB₁-contaminated diet. [¹⁴C]AFM₁ was also found to be produced by cultures of A. parasiticus giving a yield of about 0.3% of the total aflatoxins. A test for covalent binding to rat liver DNA revealed a CBI of 2100 showing that AFM₁ must also be regarded as a strong hepatocarcinogen. It is concluded that AFB₁ contaminations should be avoided in dairy feed.     

Safrole-2',3'-oxide induces cytotoxic and genotoxic effects in HepG2 cells and in mice

Safrole-2',3'-oxide (SAFO) is a reactive electrophilic metabolite of the hepatocarcinogen safrole, the main component of sassafras oil. Safrole occurs naturally in a variety of spices and herbs, including the commonly used Chinese medicine Xi xin (Asari Radix et Rhizoma) and Dong quai (Angelica sinensis). SAFO is the most mutagenic metabolite of safrole tested in the Ames test. However, little or no data are available on the genotoxicity of SAFO in mammalian systems. In this study, we investigated the cytotoxicity and genotoxicity of SAFO in human HepG2 cells and male FVB mice. Using MTT assay, SAFO exhibited a dose- and time-dependent cytotoxic effect in HepG2 cells with TC(50) values of 361.9μM and 193.2μM after 24 and 48h exposure, respectively. In addition, treatment with SAFO at doses of 125μM and higher for 24h in HepG2 cells resulted in a 5.1-79.6-fold increase in mean Comet tail moment by the alkaline Comet assay and a 2.6-7.8-fold increase in the frequency of micronucleated binucleated cells by the cytokinesis-block micronucleus assay. Furthermore, repeated intraperitoneal administration of SAFO (15, 30, 45, and 60mg/kg) to mice every other day for a total of twelve doses caused a significant dose-dependent increase in mean Comet tail moment in peripheral blood leukocytes (13.3-43.4-fold) and in the frequency of micronucleated reticulocytes (1.5-5.8-fold). Repeated administration of SAFO (60mg/kg) to mice caused liver lesions manifested as a rim of ballooning degeneration of hepatocytes immediately surrounding the central vein. Our data clearly demonstrate that SAFO significantly induced cytotoxicity, DNA strand breaks, micronuclei formation both in human cells in vitro and in mice. More studies are needed to explore the role SAFO plays in safrole-induced genotoxicity.