Microalga Euglena as a bioindicator for testing genotoxic potentials of organic pollutants in Taihu Lake,China

The microalga Euglena was selected as a bioindicator for determining genotoxicity potencies of organic pollutants in Meiliang Bay of Taihu Lake, Jiangsu, China among seasons in 2008. Several methods, including the comet assay to determine breaks in DNA and quantification of antioxidant enzymes were applied to characterize genotoxic effects of organic extracts of water from Taihu Lake on the flagellated, microalga Euglena gracilis. Contents of photosynthetic pigments, including Chl a, Chl b and carotenoid pigments were inversely proportion to concentrations of organic extracts to which E. gracilis was exposed. Organic extracts of Taihu Lake water also affected activities of superoxide dismutase (SOD) and peroxidase (POD) of E. gracilis. There were no statistically significant differences in SOD activities among seasons except in June but significant differences in POD activities were observed among all seasons. The metrics of DNA fragmentation in the alkaline unwinding assay (Comet assay), olive tail moment (OTM) and tail moment (TM), used as measurement endpoints during the genotoxicity assay were both greater when E. gracilis was exposed to organic of water collected from Taihu Lake among four seasons. It is indicated that the comet assay was useful for determining effects of constituents of organic extracts of water on E. gracilis and this assay was effective as an early warning to organic pollutants.     

Organic pollutants and ambient severity for the drinking water source of western Taihu Lake

Measurement of the organic compounds found in western Taihu Lake and evaluation of the ambient severity (AS) of the water using multimedia environmental goals (MEG) was conducted. The comet assay and the antioxidant enzyme approach were used to test the potential toxicity of water samples on the microalgae Euglena gracilis. Total concentrations of 25 organic pollutants in samples from two sites were 6.700 and 14.655 μg/l, respectively, with a calculated total ambient severity (TAS) of less than 1 and therefore minimal risk to human and ecological health. Organic extracts from the samples at these two sites was found to induce dose-dependent DNA damage on microalgae cells. DNA damage together with changes in superoxide dismutase (SOD) and peroxidase (POD) activities indicated that the potential pollutant toxicity was far higher at one of the two sites than at the other site. The comet assay combined with the activities of antioxidant enzymes may be of value as a biomarker for presence of organic pollutants in drinking water sources.     

Bioassays for evaluating the water-extractable genotoxic and toxic potential of soils polluted by metal smelters

Physicochemical analyses of polluted soils are limited in their ability to determine all hazardous compounds, their bioavailability, and their combined effects on living organisms. Bioassays, on the other hand, can evaluate environmental quality more accurately. This study assesses the genotoxic potential of water extracts from soil polluted with metals (Pb, Cd, and Zn) by the former lead smelter in zerjav, Slovenia using comet assay with Tetrahymena thermophila and human hepatoma cells (HepG2). In addition, the toxicity of soil samples and their extracts was evaluated using Vibrio fischeri and delayed fluorescence of Lemna minor. Chemical analyses of metals using atomic absorption spectrophotometry (AAS) was performed for comparison. Measurements of the total metal concentrations showed that four of five plots near the former lead smelter were highly contaminated with Pb, Cd, and Zn, but the amount of metals in water/soil extracts was low at all the sampling plots. Genotoxicity was demonstrated using T. thermophila for the majority of the extracts, and HepG2 cells for only some of the extracts. Whereas V. fischeri indicated a gradual decrease in soil toxicity with greater distance from the smelter, the toxicity of extracts did not correlate with proximity. Low concentrations of metals in water extracts stimulated L. minor growth. The results indicate that comet assay with T. thermophila and HepG2 cells and the BSPT with V. fischeri are suitable protocols for screening the genotoxic and toxic potential of water/soil extracts by comet assay, whereas chemical analyses of total metal concentrations in soil do not solely suffice for evaluating metal pollution in the environment. Biological assays are thus crucial for risk assessment.     

体内彗星试验在药物遗传毒性评价中的研究方法

体内彗星试验是采用单细胞凝胶电泳的方法检测体内DNA损伤的技术。具有灵敏度高、操作简便、经济快速等优点。随着遗传毒性研究的发展,体内彗星试验已经成为重要的药物遗传毒性评价方法。对动物试验阶段和彗星试验阶段各操作步骤进行了归纳总结,以期对相关方法学建立提供参考,并提出采用简化和标准化的研究方法将有利于体内彗星试验在药物遗传毒性评价的应用。     

Toxicity evaluation of water samples collected near a hospital waste landfill through bioassays of genotoxicity piscine micronucleus test and comet assay in fish Astyanax and ecotoxicity Vibrio fischeri and Daphnia magna

In this study, we analyzed samples of water from a river and a lake located near a hospital waste landfill with respect to physico-chemical parameters and conducted bioassays of ecotoxicity using Vibrio fischeri and Daphnia magna, which are species commonly used to evaluate the water toxicity. We also evaluated damage to the genetic material of fish (Astyanax sp. B) that were exposed (96 h) to water from these two sites that were located near the tank ditch, using the alkaline comet assay and the piscine micronucleus test. Parameters including aluminum, manganese, biochemical oxygen demand, sulfide, conductivity, phenol, total coliforms and Escherichia coli counts, were above acceptable levels that have been established in environmental legislation. However, the toxicity bioassays that we carried out in Vibrio fischeri and Daphnia magna and the piscine micronucleus test in fish showed no immediate risk due to acute effects. Based on the results of the comet assay, however, it was possible to detect damage to genetic material in fish that were acutely exposed in the laboratory to water samples from the river and lake that are located near the trench septic tank. Thus, our results suggest that tests beyond those usually employed to test water toxicity, such as the comet assay we used in the fish, are required to assess the toxicity of water with greater accuracy.     

Genotoxicity of several clinically used topoisomerase II inhibitors

DNA topoisomerase II is an essential nuclear enzyme that modulates DNA topology during multiple cellular processes such as DNA replication and chromosome segregation. Several important clinical antitumor drugs and antibiotics act through inhibition of topoisomerase II. There are a number of different steps in the action of topoisomerase II, all of which are potential targets for inhibition through drugs and also for cellular and genetic toxicity as well as for mutagenesis. We have investigated and compared the genotoxicity and mutagenicity of the mechanistically different topoisomerase II inhibitors m-amsacrine, mitoxantrone, etoposide, genistein, ICRF 193, and berenil using the in vitro micronucleus test, single cell gelelectrophoresis (comet assay) and the mutation assay (tk-locus) in L5178Y mouse lymphoma cells. All six compounds induced micronuclei and all except berenil were mutagenic. M-amsacrine, mitoxantrone, etopside and genistein induced DNA migration in the comet assay, whereas ICRF 193 was only weakly positive and berenil was negative in this test. Our results are in good agreement with the compounds' proposed mechanisms of interaction with topoisomerase II.     

ReProComet: a new in vitro method to assess DNA damage in mammalian sperm.

The increasing request of chemical safety assessment demands for the validation of alternative methods to reduce the resort to animal experimentation. Methods that evaluate reproductive toxicity are among those requiring the largest use of animals. Presently, no validated in vitro alternative exists for the assessment of reproductive toxicity. Mammalian sperm are sensitive targets of DNA-reactive chemicals, which form premutagenic adducts. Here, we propose a new method based on comet assay to detect DNA damage induced by potential germ cell mutagens in bull sperm available from assisted reproduction practices. In somatic cells, chemical-induced adducts can be revealed by comet assay that detects DNA breaks produced during adduct repair. Mature sperm, however, are devoid of repair enzymes, and adducts are processed only after fertilization. For this reason, comet assay is not sensitive to detect DNA lesions induced in sperm by most chemicals. To overcome such limitation, we developed a modified comet assay based on the addition of a protein extract from HeLa cells to agarose-embedded sperm on microscopic slides. To test the method, sperm were treated in vitro with methyl methanesulfonate (MMS) or melphalan (MLP) and comet assay was conducted both with and without protein supplementation. No effect of MMS or MLP was detected without protein supplementation; on the contrary, a clear-cut dose-dependent effect was measured after addition of the cell extract. These results represent a proof of concept of a novel in vitro mutagenicity test on sperm that could offer a promising approach to complement previously validated in vivo germ cell genotoxicity assays.     

The alkaline comet assay: towards validation in biomonitoring of DNA damaging exposures

Generation of DNA damage is considered to be an important initial event in carcinogenesis. The single cell gel electrophoresis (comet) assay is a technically simple and fast method that detects genotoxicity in virtually any mammalian cell type without requirement for cell culture. This review discusses the strength of the comet assay in biomonitoring at its present state of validation. The simple version of the alkaline comet assay detects DNA migration caused by strand breaks, alkaline labile sites, and transient repair sites. By incubation with bacterial glycosylase/endonuclease enzymes, broad classes of oxidative DNA damage, alkylations, and ultraviolet light-induced photoproducts are detected as additional DNA migration. The most widely measured enzyme sensitive sites have been those detected by formamidopyrimidine DNA glycosylase (FPG) and endonuclease III (ENDOIII). Reports from biomonitoring studies show that the basal level of DNA damage in leukocytes is influenced be a variety of lifestyle and environmental exposures, including exercise, air pollution, sunlight, and diet. Although not all types of carcinogenic exposures should be expected to damage DNA in leukocytes, the comet assay is a valuable method for detection of genotoxic exposure in humans. However, the predictive value of the comet assay is unknown because it has not been investigated in prospective cohort studies. Also, it is important that the performance of the assay is investigated in multi-laboratory validation trials. As a tool in risk assessment the comet assay can be used in characterization of hazards.     

Genotoxicity of environmental agents assessed by the alkaline comet assay

Generation of DNA damage is considered to be an important initial event in carcinogenesis. A considerable battery of assays exists for the detection of different genotoxic effects of compounds in experimental systems, or for investigations of exposure to genotoxic agents in environmental or occupational settings. Some of the tests may have limited use because of complicated technical setup or because they only are applicable to a few cell types. The single cell gel electrophoresis (comet) assay is technically simple, relatively fast, cheap, and DNA damage can be investigated in virtually all mammalian cell types without requirement for cell culture. The aim of this thesis was to evaluate the comet assay as a genotoxicity test in genetic toxicology of environmental agents, encompassing both experimental animal models and biomonitoring. The comet assay detects strand breaks (SB). The cells are embedded in agarose and lysed, generating nucleus-like structures in the gel (referred to as nucleoids). Following alkaline electrophoresis, the DNA strands migrate toward the anode, and the extent of migration depends on the number of SB in the nucleoid. The migration is visualized and scored in a fluorescence microscope after staining. Broad classes of oxidative DNA damage can be detected as additional SB if nucleoids are incubated with bacterial DNA glycosylase/endonuclease enzymes. Oxidized pyrimidines and purines can be detected by incubation with endonuclease III and formamidopyrimidine DNA glycosylase, respectively. The animal experimental studies indicated that the comet assay was able to detect genotoxic effects of diesel exhaust particles in lung tissue, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ)-induced DNA damage in colon epithelial cells and liver tissue, and benzene-induced damage in bone marrow and liver cells. The strength of the comet assay was further outlined by application of repair enzymes, indicating no oxidative DNA base damage following IQ treatment. High levels of oxidative DNA lesions were detected after exposure to benzene or X-ray irradiation. The comet assay did not detect DNA damage in colon or liver following ingestion of diets containing of high contents of animal fat or sucrose, although other indices of DNA damage were found. Determined from the results of a large Japanese study, the discrimination between carcinogens and non-carcinogens appears to be similar between the comet assay and alkaline elution, which also detects SB. This suggests that the comet assay is a reliable genotoxicity test in animal experimental systems. In the biomonitoring studies, we investigated the effect of common exposures and lifestyle factors (rather than effects of known carcinogens) on the level of oxidative DNA damage in mononuclear blood cells of humans. In the first study, based on repeated measurements, it was shown that interindividual variation and seasonal variation were major determinants for the basal level of SB, whereas no effect of age, exercise, or antioxidant intake could be detected. The effect of exercise was further investigated under both normoxic and hypoxic circumstances, showing a strong effect of hypoxia, and only effect of exercise in terms of SB in hypoxia. In a placebo-controlled parallel dietary fruit and vegetable (or the corresponding amount of antioxidants) intervention study, no effects of the level of oxidative DNA damage or sensitivity to hydrogen peroxide were observed. Although this may seem in contrast to other antioxidant intervention studies, a critical literature survey of antioxidant intervention studies on oxidative DNA damage suggested that well-controlled studies tended to show no effect of antioxidant supplementation. In summary, the aggregated data from the publications included in this thesis, and other publications encompassing the comet assay, indicate that the comet assay is a reliable method for detection of DNA damage in tissues of experimental animals. Although not all types of genotoxic exposures should be expected to result in DNA damage in mononuclear blood cells, the comet assay seems to be a valuable tool for detection of genotoxic exposure in humans. The comet assay indicates that DNA damage is abundant in mammalian cells and affected by lifestyle and many environmental exposures, including diet, exercise, hypoxia, and sunlight.     

Detection and quantification of genotoxicity in wastewater-treated Tetrahymena thermophila using the comet assay

In the present study, the comet, or single-cell, gel electrophoresis assay was adapted for use with the ubiquitous unicellular protozoan Tetrahymena thermophila, and the method was evaluated for its ability to detect DNA damage induced by known genotoxins and wastewater samples. The original comet assay protocol was substantially modified (e.g., lower concentrations of detergents were used in the lysis buffer; electrophoresis time was reduced). Using the modified method, T. thermophila were subjected to short exposures of phenol, hydrogen peroxide, and formaldehyde, leading to concentration-dependent increases in DNA damage. The genotoxic potential of influent and effluent water samples from a local municipal wastewater treatment plant was evaluated. The results indicated that the influent wastewater was genotoxic and that the genotoxicity in the effluent water was substantially reduced. We assume employing T. thermophila in the use of the comet assay may become a cost-effective and reliable tool for genotoxicity screening and monitoring of wastewater and similar systems.     

Biomonitoring of the genotoxic potential of draining water from dredged sediments, using the comet and micronucleus tests on amphibian (Xenopus laevis) larvae and bacterial assays (Mutatox and Ames tests)

Management of contaminated dredged sediments is a matter of great human concern. The present investigation evaluates the genotoxic potential of aqueous extracts of five sediments from French channels (draining water from dredged sediments), using larvae of the frog Xenopus laevis. Two genotoxic endpoints were analyzed in larvae: clastogenic and/or aneugenic effects (micronucleus induction after 12 d of exposure) and DNA-strand breaking potency (comet assay after 1 and 12 d of exposure) in the circulating blood. Additionally, in vitro bacterial assays (Microtox and Ames tests) were carried out and the results were compared with those obtained with larvae. Physicochemical analyses were also taken into account. Analytical analyses highlighted in the five draining waters a heavy load of contaminants such as metals and hydrocarbons. The results obtained with the micronucleus test established the genotoxicity of three draining waters. The comet assay showed that all 5 draining waters were genotoxic after 1 d of exposure. Although 3 of them were still genotoxic after 12 d of exposure, DNA damage globally decreased between d 1 and 12. The comet assay can be considered as a genotoxicity-screening tool. Data indicate that both tests should be used in conjunction in Xenopus. Bacterial tests (Ames) revealed genotoxicity for only one draining water. The results confirm the relevance of the amphibian model and the need to resort to bioassays in vivo such as the Xenopus micronucleus and comet assays for evaluation of the ecotoxicological impact, an essential complement to the physicochemical data.     

Mutagenic impact on fish of runoff events in agricultural areas in south-west France

When heavy rainfall follows herbicide application, the intense surface runoff causes stream water contamination. Aquatic organisms are then briefly exposed to a complex mixture of contaminants. The aim of the present study is to investigate the genotoxic impact of such events on fish. A model fish, the Crucian carp (Carassius carassius) was exposed in controlled conditions, for 4 days, to water sampled daily in the Save River (France). The watershed of this stream is representative of agricultural areas in south-west France. Three hydrological conditions were compared: basal flow, winter flood, and spring flood. Chemical analysis of the water samples confirmed the higher contamination of the spring flood water, mainly explained by a peak of metolachlor. Genotoxicity was evaluated by micronucleus (MN) test and comet assay in peripheral erythrocytes. A significant increase in DNA breakdowns compared to controls was detected by the comet assay for all conditions. Exposure to spring flood water resulted in the highest damage induction. Moreover, induced chromosomal damage was only detected in this condition. In addition, fish were exposed, for 4 days, to an experimental mixture of 5 herbicides representative of the spring flood water contamination. Fish exhibited moderate DNA damage induction and no significant chromosomal damage. The mutagenicity induced by field-collected water is then suspected to be the result of numerous interactions between contaminants themselves and environmental factors, stressing the use of realistic exposure conditions. The results revealed a mutagenic impact of water contamination during the spring flood, emphasizing the need to consider these transient events in water quality monitoring programs.     

Use of the alkaline comet assay for industrial genotoxicity screening: comparative investigation with the micronucleus test.

We evaluated the suitability of the alkaline comet assay as a screening test in industrial routine testing of new chemicals. Thirty-six pharmaceutical compounds with unknown genotoxic potential were tested comparatively in the comet assay and micronucleus test (MNT) using V79 Chinese hamster cells. The comparison of results is generally based on at least two independent experiments, each with two replicate cultures at a minimum of three concentrations. We found a high degree of concordance between results of the comet assay and MNT. All compounds with negative MNT results were also negative in the comet assay. All positive compounds in the comet assay were also positive in the MNT. However, 16 of 38 positive MNT results were negative in the comet assay. Some of the contrary findings may be due to aneugenic effects, which are detected in the MNT but not in the comet assay. However, the majority of the contrary results may be a consequence of cytotoxicity, which can induce elevated micronucleus frequencies but may not lead to positive effects in the comet assay. Additional data of 39 compounds tested in the Ames test and the comet assay were compared. Four of these compounds that were Ames positive were also positive in the comet assay. However, the comet assay also detected 16 compounds that were negative in the Ames test. We believe that the comet assay in vitro is a useful, fast screening system in mammalian cells that can be used in a test battery during drug development.     

Microcystin contamination in fish from the Jacarepaguá Lagoon (Rio de Janeiro, Brazil): ecological implication and human health risk.

Chronic and subchronic toxicity from exposure to microcystins, cyclic peptide liver toxins from certain cyanobacteria, poses an important hazard, which has received little study. No in vivo information exists on accumulation and transfer of microcystin from the food chain to humans. This paper present results of a 3-year study that demonstrates bioaccumulation of microcystins by fish and potential rates of microcystin ingestion by humans. The study was carried out in a shallow coastal lagoon in the city of Rio de Janeiro (Jacarepaguá Lagoon). Fish (Tilapia rendalli) were collected every 2 weeks from August 1996 to November 1999. Microcystins were analyzed by HPLC in phytoplankton, fish liver and viscera while fish muscle tissue was analyzed by enzyme linked immunosorbant assay (ELISA). Phytoplankton samples, dominated by the genus Microcystis, were confirmed to contain microcystins as were fish livers, viscera and muscle tissue. During the entire study period, including times of low water bloom densities, fish muscle tissue contained concentrations of microcystins close to or above the recommended limit for human consumption (0.04 microg x kg(-1) day). Our findings demonstrate that microcystins can accumulate in fish tissue used for human consumption. Rates of ingestion routinely exceed the TDI guidelines as set by the WHO for drinking water. Appropriate epidemiology and risk assessment should be undertaken so that an acceptable TDI and appropriate risk management decisions can be made for human consumption of fish which are harvested from cyanobacterial blooms that contain cyanotoxins.     

Chicken nucleated blood cells as a cellular model for genotoxicity testing using the comet assay.

Mycotoxins can frequently occur in animal feed and human food. T-2 toxin, as the most toxic trichothecene, has been implicated as the causative agent in a variety of animal diseases and is associated with some human diseases. The comet assay was performed as a test for detection of DNA damage caused by T-2 toxin in peripheral blood cells of chicken. The suitability of the comet assay as a biomarker for genotoxic analysis has been applied in studies using human white blood cells. It can be applied to any tissue from which a single cell suspension can be obtained. The method has already been applied to chicken as a foodstuff for detection of irradiation of food containing DNA. However, application of the method on chicken blood cells has not been set up yet. The aim of this research was to develop a protocol for detection of DNA damage induced by T-2 toxin in chicken blood cells. Chickens were administered orally with T-2 toxin and the samples of whole blood were collected at 24 h post treatment. The DNA damage was determined by an increase in the comet parameters in tested animals. Our results show that T-2 toxin had induced significant DNA damage in treated chicken as compared with control animals, indicating that the assay can be used for the assessment of primary DNA damage caused by mycotoxins.     

Metallothionein-I/II null cardiomyocytes are sensitive to Fusarium mycotoxin butenolide-induced cytotoxicity and oxidative DNA damage

Previous studies revealed butenolide (BUT), a Fusarium mycotoxin distributes extensively, induced myocardial oxidative damage, which could be abated by antioxidants such as glutathione. Metallothionein (MT) has proved to attenuate several oxidative cardiomyopathies via its potent antioxidant property. The present study is therefore undertaken to investigate the protective potential of the endogenous expression of MT against BUT-induced myocardial toxicity. Primary cultures of neonatal cardiomyocytes from MT-I/II null mice along with the corresponding wild-type mice will be utilized to determine the possible mechanistic properties of MT. BUT treatment to the cardiomyocytes evoked significant cytotoxicity as evidenced by morphological changes and concentration- and time-dependent reductions in cell viability. Additionally, BUT treatment remarkably increased reactive oxygen species (ROS) production in the cardiomyocytes of both MT-I/II null and wild-type mice. As a result, noticeable DNA damage in both cardiomyocytes was detected by alkaline comet assay. Furthermore, the comparison between the MT-I/II null and wild-type cardiomyocytes indicated that ROS production in the cardiomyocytes from the MT-I/II null mice was higher than from wild-type mice. DNA damage as evaluated by percentage of comet tail DNA, tail length and tail moment was more severe in the MT-l/II null cardiomyocytes than in wild-type myocytes. And in agreement with those results mentioned above, the MT-l/II null cardiomyocytes were more sensitive to BUT-induced cytotoxicity than wild-type cardiomyocytes. Taken together, these findings clearly show that basal MT can efficiently attenuate BUT-induced cytotoxic injuries in cardiomyocytes via the inhibition of intracellular ROS production, and associated DNA damage.     

A comparative study of using comet assay and gammaH2AX foci formation in the detection of N-methyl-N'-nitro-N-nitrosoguanidine-induced DNA damage.

Comet assay is a useful technique in the detection of DNA damages, particularly DNA strand breaks; and it has been utilized to show that a potent carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), can induce such damages. Recently, gammaH2AX foci formation has been suggested as another sensitive way to detect DNA double strand breaks (DSBs). However, there is no systematic comparison being conducted to evaluate the consistency of these two methods. Using MNNG as a model chemical, the sensitivity of neutral comet assay and gammaH2AX foci formation in detecting MNNG-induced damage was studied. It was found that at concentrations of 0.1 and 1 microg/ml, both methods can detect MNNG-induced damage in human amnion FL cells. However, at 0.1 microg/ml, comet assay revealed more percentage of cells with DNA damage than gammaH2AX fluorescence revealed. On the other hand, while gammaH2AX foci were readily formed at very early times by 10 microg/ml MNNG treatment, neutral comet assay did not detect any significant DNA damage at the same time points. In addition, 10 microg/ml MNNG induced a distinct whole nuclei staining pattern of gammaH2AX, a type of DNA damage which was not detected by neutral comet assay but could be detected by alkaline comet assay. Therefore, gammaH2AX may be used as a sensitive indicator for DNA damage.     

Genotoxicity of organic pollutants in source of drinking water on microalga Euglena gracilis

The potential toxicities of organic pollutants in the drinking water source at Meiliang Bay of Lake Taihu were investigated by comet assay and antioxidant enzyme approach on microalgae Euglena gracilis. The organic extracts of the water samples could induce DNA damage on microalgae cells. Statistically significant differences (P < 0.05) were observed at groups of 0.3×, 3× and 10× concentrations compared with the control and a solvent control (DMSO). The organic extracts also affected antioxidant enzyme activity and induced lipid peroxidation in the microalga. In the high dose group, there was an obvious increase in SOD content (P < 0.05). The results suggest that the concentrated organics from water sample extracts have adversary effects on E. gracilis and could possibly damage the ecosystem.     

Evaluation of multi-organ DNA damage by comet assay from 28 days repeated dose oral toxicity test in mice: A practical approach for test integration in regulatory toxicity testing

The use of comet assay is not new in the evaluation of genotoxic potential of different agents; however, its broad use in product safety for regulatory testing is a relatively new approach. The present study was aimed to integrate genotoxicity tests (micronucleus and comet assay) in 28 days repeated dose oral toxicity of methotrexate (MTX) in mice. MTX was administered at the dose of 0.5, 1 and 2 mg/kg per oral repeatedly for 28 days in mice. The endpoints of evaluation for routine toxicity testing included body weight, organ weight, food intake, water intake, hematology and histology, while for the genotoxicity testing micronucleus and comet assay were used. There were no significant changes in food intake, water intake and organ weight; however, the body weight significantly decreased at the highest dose of MTX treatment as compared to control group. Histological data revealed the morphological alterations in the liver and lung cells at the highest dose of MTX treatment. Micronucleus assay results indicated that the highest dose of MTX led to significant increase in MNERTs/1000ERTs (P < 0.001) as compared to control group. Further, percentage of reticulocytes (% RETs) was significantly decreased at the highest dose of MTX as compared to control group. Comet assay results indicate significant DNA damage in different organs induced by MTX as compared to control group. The results of the present study successfully demonstrates the integration of genotoxicity tests using comet and micronucleus assay in 28 days repeated dose oral toxicity test. Integration of genotoxicity test with routine toxicity test would reduce the cost of additional animals, test item and provide further information at an early stage of product development.