Simplified mouse peripheral reticulocyte micronucleus test with dimethylnitrosamine.

The induction of micronuclei by treatment with dimethylnitrosamine was evaluated and compared in peripheral blood and bone marrow cells of male CD-1 mice. Peripheral blood preparations were made on acridine orange (AO)-coated slides and scanned by fluorescence microscopy. A significant increase in micronuclei was observed 24 h after treatment in bone marrow polychromatic erythrocytes, and 24-48 h after treatment in peripheral reticulocytes. The peak frequency of micronuclei in peripheral reticulocytes was delayed by about 24 h relative to bone marrow polychromatic erythrocytes. This micronucleus test using peripheral blood was shown to be easy to do and as sensitive as the test using bone marrow cells. From this result, it is concluded that the method with AO-coated slides and peripheral blood is as suitable as bone marrow cells for the micronucleus assay.     

The micronucleus assay using peripheral blood reticulocytes from mitomycin C- and cyclophosphamide-treated rats.

It used to be believed that the use of rat peripheral blood for the micronucleus assay would be difficult because micronucleated erythrocytes are captured and destroyed by the spleen quickly. We have applied an acridine orange (AO) supravital staining method to rat peripheral blood using AO-coated glass slides. Normal and splenectomized SD rats were treated once with mitomycin C (i.p.) or cyclophosphamide (p.o.), and 5 microliters of blood was collected at intervals from the tail vein between 0 and 72 h after treatment. For comparison, bone marrow cells were smeared conventionally 30 h after treatment. Although the frequencies of spontaneous and chemically induced micronucleated reticulocytes (MNRETs) from normal rats were lower on average in the highest dose group than those of splenectomized rats, the incidence of micronuclei among type I and II reticulocytes in normal rats at 48 h was almost identical to the incidence of RNA-containing erythrocytes with micronucleus in bone marrow. Thus, we suggest that rat peripheral reticulocytes can be used as target cells for the micronucleus assay.     

The micronucleus assay using peripheral blood reticulocytes from mitomycin C- and cyclophosphamide-treated rats

It used to be believed that the use of rat peripheral blood for the micronucleus assay would be difficult because micronucleated erythrocytes are captured and destroyed by the spleen quickly. We have applied an acridine orange (AO) supravital staining method to rat peripheral blood using AO-coated glass slides. Normal and splenectomized SD rats were treated once with mitomycin C (i.p.) or cyclophosphamide (p.o.), and 5 μl of blood was collected at intervals from the tail vein between 0 and 72 h after treatment. For comparison, bone marrow cells were smeared conventionally 30 h after treatment. Although the frequencies of spontaneous and chemically induced micronucleated reticulocytes (MNRETs) from normal rats were lower on average in the highest dose group than those of splenectomized rats, the incidence of micronuclei among type I and II reticulocytes in normal rats at 48 h was almost identical to the incidence of RNA-containing erythrocytes with micronucleus in bone marrow. Thus, we suggest that rat peripheral reticulocytes can be used as target cells for the micronucleus assay.     

The micronucleus test of methyl methanesulfonate with mouse peripheral blood reticulocytes using acridine orange-coated slides.

The usefulness of the micronucleus assay using mouse peripheral blood erythrocytes and acridine orange (AO)-coated slides was evaluated with methyl methanesulfonate (MMS). The micronucleus test was carried out at doses ranging from 20 to 80 mg/kg body weight in CD-1 mice by intraperitoneal injection. Peripheral blood cells were examined from 0 to 72 h after treatment at 12- or 24-h intervals. Bone marrow cells from other mice treated with 80 mg/kg MMS were also sampled at the same times. The frequency of micronucleated reticulocytes (MNRETs) increased dose-dependently at every sampling time except 72 h, and the maximum frequency of MNRETs was observed at about 36 h after treatment. Micronucleated polychromatic erythrocytes (MNPCEs) in bone marrow after a dose of 80 mg/kg were significantly induced at 12 h to 36 h, and the maximum frequency of MNPCEs was observed at 24 h after treatment. The induction of MNRETs was delayed by about 12 h compared to that of MNPCEs in bone marrow, and the maximum frequencies of MNRETs were lower than those of MNPCEs, but the induction of MNRETs by MMS was significant and dose-dependent. It is concluded, therefore, that bone marrow cells could be replaced by peripheral blood cells as material for the micronucleus assay using AO-coated slides.     

Evaluation of nongenotoxic and genotoxic factors modulating the frequency of micronucleated erythrocytes in the peripheral blood of mice

The hematological micronucleus test is regarded as an indicator of the clastogenic effect of chemicals and acute cytogenetic damage. The test can be carried out in red blood cells of the bone marrow and of the spleen, as well as in peripheral erythrocytes. We have determined the precise background values of micronucleated red blood cells for the peripheral blood of BALB/c, DBA/2, and NMRI mice. Bleeding, phenylhydrazine-induced hemolysis, and splenectomy generated an increase of micronucleated erythrocytes in the peripheral blood of mice. Our data thus demonstrate that such factors should be taken into consideration when the micronucleus test is used for screening the genotoxic potential of chemicals. Furthermore, the micronucleus-inducing effect of cyclophosphamide was studied in normal and splenectomized mice and, in addition, a comparison of the sensitivity of the micronucleus test was carried out in peripheral blood and bone marrow after cyclophosphamide treatment. Our data demonstrate that the kinetics of micronucleus formation were similar in normal and in splenectomized mice in which the micronucleus levels had returned to normal. The comparison of micronucleus formation in bone marrow and peripheral blood after cyclophosphamide treatment revealed the generation of similar quantities of micronucleated red blood cells in both tissues. The physiological mechanisms of micronucleus formation and removal and the potential role of chemically induced spleen damage during this process are discussed; the usefulness of the peripheral micronucleus test as a simple, rapid, and animal-saving modification of the standard bone marrow test is evaluated.Abbreviations CP cyclophosphamide - MN micronuclei - MNCE micronucleated normochromatic erythrocytes - MNPCE micronucleated polychromatic erythrocytes - MNRBC micronucleated red blood cells - NCE normochromatic erythrocytes - PCE polychromatic erythrocytes     

Evaluation of nongenotoxic and genotoxic factors modulating the frequency of micronucleated erythrocytes in the peripheral blood of mice

The hematological micronucleus test is regarded as an indicator of the clastogenic effect of chemicals and acute cytogenetic damage. The test can be carried out in red blood cells of the bone marrow and of the spleen, as well as in peripheral erythrocytes. We have determined the precise background values of micronucleated red blood cells for the peripheral blood of BALB/c DBA/2, and NMRI mice. Bleeding, phenylhydrazine-induced hemolysis, and splenectomy generated an increase of micronucleated erythrocytes in the peripheral blood of mice. Our data thus demonstrate that such factors should be taken into consideration when the micronucleus test is used for screening the genotoxic potential of chemicals. Furthermore, the micronucleus-inducing effect of cyclophosphamide was studied in normal and splenectomized mice and, in addition, a comparison of the sensitivity of the micronucleus test was carried out in peripheral blood and bone marrow after cyclophosphamide treatment. Our data demonstrate that the kinetics of micronucleus formation were similar in normal and in splenectomized mice in which the micronucleus levels had returned to normal. The comparison of micronucleus formation in bone marrow and peripheral blood after cyclophosphamide treatment revealed the generation of similar quantities of micronucleated red blood cells in both tissues. The physiological mechanisms of micronucleus formation and removal and the potential role of chemically induced spleen damage during this process are discussed; the usefulness of the peripheral micronucleus test as a simple, rapid, and animal-saving modification of the standard bone marrow test is evaluated.Abbreviations CP cyclophosphamide - MN micronuclei - MNCE micronucleated normochromatic erythrocytes - MNPCE micronucleated polychromatic erythrocytes - MNRBC micronucleated red blood cells - NCE normochromatic erythrocytes - PCE polychromatic erythrocytes     

Technical aspects of automatic micronucleus analysis in rodent bone marrow assays

The mouse bone marrow micronucleus assay is anin vivo test commonly used in the pharmaceutical industry to evaluate the genotoxic potential of new compounds. The test detects agent-induced chromosomal damage or damage of the mitotic spindle apparatus. In this paper the state-of-the-art in automated rodent micronucleus evaluation using computerized image analyis in combination with high-quality slides obtained by the cellulose column fractionation technique is reviewed. The latter allows the effective removal of nucleated cells from rodent bone marrow. It has been found that automatic micronucleus scoring with the Leitz MIAC image analyzer is substantially faster than labor-intensive manual analysis. Automatic scoring can be performed overnight for up to 16 slides. We have been successfully using automatic micronucleus analysis for the testing of new pharmaceutical drugs for more than 3 years.Abbreviations MNE NCE containing micronuclei - MPE PCE containing micronuclei - NCE normochromatic erythrocyte - PCE polychromatic erythrocyte deceased on 25 May 1994     

In vivo erythrocyte micronucleus assay III. Validation and regulatory acceptance of automated scoring and the use of rat peripheral blood reticulocytes, with discussion of non-hematopoietic target cells and a single dose-level limit test

The in vivo micronucleus assay working group of the International Workshop on Genotoxicity Testing (IWGT) discussed new aspects in the in vivo micronucleus (MN) test, including the regulatory acceptance of data derived from automated scoring, especially with regard to the use of flow cytometry, the suitability of rat peripheral blood reticulocytes to serve as the principal cell population for analysis, the establishment of in vivo MN assays in tissues other than bone marrow and blood (for example liver, skin, colon, germ cells), and the biological relevance of the single-dose-level test. Our group members agreed that flow cytometric systems to detect induction of micronucleated immature erythrocytes have advantages based on the presented data, e.g., they give good reproducibility compared to manual scoring, are rapid, and require only small quantities of peripheral blood. Flow cytometric analysis of peripheral blood reticulocytes has the potential to allow monitoring of chromosome damage in rodents and also other species as part of routine toxicology studies. It appears that it will be applicable to humans as well, although in this case the possible confounding effects of splenic activity will need to be considered closely. Also, the consensus of the group was that any system that meets the validation criteria recommended by the IWGT (2000) should be acceptable. A number of different flow cytometric-based micronucleus assays have been developed, but at the present time the validation data are most extensive for the flow cytometric method using anti-CD71 fluorescent staining especially in terms of inter-laboratory collaborative data. Whichever method is chosen, it is desirable that each laboratory should determine the minimum sample size required to ensure that scoring error is maintained below the level of animal-to-animal variation. In the second IWGT, the potential to use rat peripheral blood reticulocytes as target cells for the micronucleus assay was discussed, but a consensus regarding acceptability for regulatory purposes could not be reached at that time. Subsequent validation efforts, combined with accumulated published data, demonstrate that blood-derived reticulocytes from rats as well as mice are acceptable when young reticulocytes are analyzed under proper assay protocol and sample size. The working group reviewed the results of micronucleus assays using target cells/tissues other than hematopoietic cells. We also discussed the relevance of the liver micronucleus assay using young rats, and the importance of understanding the maturation of enzyme systems involved in the processes of metabolic activation in the liver of young rats. Although the consensus of the group was that the more information with regard to the metabolic capabilities of young rats would be useful, the published literature shows that young rats have sufficient metabolic capacity for the purposes of this assay. The use of young rats as a model for detecting MN induction in the liver offers a good alternative methodology to the use of partial hepatectomy or mitogenic stimulation. Additional data obtained from colon and skin MN models have been integrated into the data bases, enhancing confidence in the utility of these models. A fourth topic discussed by the working group was the regulatory acceptance of the single-dose-level assay. There was no consensus regarding the acceptability of a single dose level protocol when dose-limiting toxicity occurs. The use of a single dose level can lead to problems in data interpretation or to the loss of animals due to unexpected toxicity, making it necessary to repeat the study with additional doses. A limit test at a single dose level is currently accepted when toxicity is not dose-limiting.     

Evaluation of the micronucleus assay in bone marrow and peripheral blood of rats for the determination of cigarette mainstream-smoke activity

The mammalian in vivo micronucleus assay is widely used as part of the genotoxicity testing battery required during the development of new drugs. As such, the in vivo micronucleus assay has been used in a battery of assays for the assessment of cigarette ingredients or design modifications to help ensure that there is no increase in risk or any new risk introduced by these additions or modifications. The present series of studies was conducted to optimize and evaluate this assay for the assessment of the effects of mainstream smoke on the micronucleus frequency in the bone marrow and peripheral blood of rats. In a first experiment, the optimal conditions for performing the micronucleus assay in these tissues were determined. This was done by use of two compounds known for their micronucleus-inducing activity, i.e., the clastogen cyclophosphamide and the aneugen colchicine. In a second experiment, the effects of tube restraint on untreated control rats were investigated. In a third experiment, the optimal conditions were used to assess the clastogenic/aneugenic activity of cigarette smoke in Sprague-Dawley rats. The rat micronucleus assay in both bone marrow and peripheral blood is able to detect clastogenic and aneugenic activity. The flow cytometric determination of micronucleated cells in rat blood is at least as sensitive as determinations in bone marrow. No statistically significant differences were observed in micronucleus frequencies between rats with and without the additional stress of tube restraint; however, the cautious approach would be to use a fresh-air-exposed group (with tube restraint) as the negative control in inhalation experiments. Using the conditions identified as optimal in the above-mentioned experiments, the micronucleus assay was not able to detect effects induced by smoke from conventional cigarettes. Nevertheless, the micronucleus assay will remain a valuable tool as part of a testing battery used to investigate possible adverse effects related to product modifications.     

Evaluation of the micronucleus assay in bone marrow and peripheral blood of rats for the determination of cigarette mainstream-smoke activity

The mammalian in vivo micronucleus assay is widely used as part of the genotoxicity testing battery required during the development of new drugs. As such, the in vivo micronucleus assay has been used in a battery of assays for the assessment of cigarette ingredients or design modifications to help ensure that there is no increase in risk or any new risk introduced by these additions or modifications. The present series of studies was conducted to optimize and evaluate this assay for the assessment of the effects of mainstream smoke on the micronucleus frequency in the bone marrow and peripheral blood of rats. In a first experiment, the optimal conditions for performing the micronucleus assay in these tissues were determined. This was done by use of two compounds known for their micronucleus-inducing activity, i.e., the clastogen cyclophosphamide and the aneugen colchicine. In a second experiment, the effects of tube restraint on untreated control rats were investigated. In a third experiment, the optimal conditions were used to assess the clastogenic/aneugenic activity of cigarette smoke in Sprague-Dawley rats. The rat micronucleus assay in both bone marrow and peripheral blood is able to detect clastogenic and aneugenic activity. The flow cytometric determination of micronucleated cells in rat blood is at least as sensitive as determinations in bone marrow. No statistically significant differences were observed in micronucleus frequencies between rats with and without the additional stress of tube restraint; however, the cautious approach would be to use a fresh-air-exposed group (with tube restraint) as the negative control in inhalation experiments. Using the conditions identified as optimal in the above-mentioned experiments, the micronucleus assay was not able to detect effects induced by smoke from conventional cigarettes. Nevertheless, the micronucleus assay will remain a valuable tool as part of a testing battery used to investigate possible adverse effects related to product modifications.     

The mouse peripheral blood micronucleus test with 2-acetylaminofluorene using the acridine orange supravital staining method.

The peripheral blood micronucleus test using the acridine orange (AO) supravital staining method was validated with the potent bone marrow clastogen 2-acetylaminofluorene (2-AAF). 2-AAF induced micronuclei in peripheral blood reticulocytes dose-dependently as well as in bone marrow polychromatic erythrocytes. The incidence of micronucleated reticulocytes (MNRETs) peaked 48 h after a single treatment in both CD-1 and BDF1 mice, and the incidence of micronucleated polychromatic erythrocytes (MNPCEs) peaked 24 or 48 h after treatment. The maximum incidences of MNRETs were always higher than those of MNPCEs in both mouse strains treated once. In the double-treatment regime, the maximum incidence of MNRETs was observed at 24 h after the second treatment in each strain. The incidences of MNRETs in BDF1 mice were higher than in CD-1 mice after a single treatment but were comparable after double treatment. These results indicate that the peripheral blood micronucleus test using AO supravital staining is as sensitive as the conventional bone marrow assay. The new staining method can be performed more easily than the original smear method using either bone marrow or peripheral blood cells. Thus, the peripheral blood method using AO supravital staining is a possible alternative to the conventional bone marrow assay.     

The mouse peripheral blood micronucleus test with 2-acetylaminofluorene using the acridine orange supravital staining method

The peripheral blood micronucleus test using the acridine orange (AO) supravital staining method was validated with the potent bone marrow clastogen 2-acetylaminofluorene (2-AAF). 2-AAF induced micronuclei in peripheral blood reticuiocytes dose-dependently as well as in bone marrow polychromatic erythrocytes. The incidence of micronucleated reticuiocytes (MNRETs) peaked 48 h after a single treatment in both CD-1 and BDF₁ mice, and the incidence of micronucleated polychromatic erythrocytes (MNPCEs) peaked 24 or 48 h after treatment. The maximum incidences of MNRETs were always higher than those of MNPCEs in both mouse strains treated once. In the double-treatment regime, the maximum incidence of MNRETs was observed at 24 h after the second treatment in each strain. The incidences of MNRETs in BDF₁ mice were higher than in CD-1 mice after a single treatment but were comparable after double treatment.These results indicate that the peripheral blood micronucleus test using AO supravital staining is as sensitive as the conventional bone marrow assay. The new staining method can be performed more easily than the original smear method using either bone marrow or peripheral blood cells. Thus, the peripheral blood method using AO supravital staining is a possible alternative to the conventional bone marrow assay.     

转基因抗矮花叶玉米对大白鼠骨髓细胞遗传毒性的影响

以SD大鼠为研究对象,研究了转基因抗矮花叶玉米和常规玉米对大白鼠骨髓细胞微核率与染色体畸变率的影响,以观察该转基因玉米对大白鼠可能产生的遗传毒理效应。实验结果表明：饲喂30%和50%转基因玉米日粮组的大白鼠骨髓细胞微核率和染色体畸变率与饲喂常规玉米相比均没有显著差异,而与阳性对照组之间存在极显著差异,这说明转基因玉米与常规玉米对大白鼠骨髓细胞均无遗传毒性。     

The mouse bone marrow micronucleus test: evaluation of 21 drug candidates

The mouse bone-marrow micronucleus test is one of the most widely used genetic toxicology assays. In this report the results of testing 21 compounds in the micronucleus test are presented. Of the 21 compounds tested, 3 potential chemotherapeutic agents were identified as strongly clastogenic. In addition, one compound was identified as a weak inducer of micronuclei in the assay. Further testing of this compound in an in vivo bone marrow metaphase analysis failed to confirm this material as clastogenic. The remaining 17 compounds were classified as negative in the assay. In general the results of the micronucleus test agreed with the results of other genetic toxicology assays on this group of compounds.     

Flow cytometric analysis of micronucleated reticulocytes in mouse bone marrow

This laboratory has previously reported a flow cytometric procedure for quantitatively analyzing mouse peripheral blood reticulocytes for micronucleus content. The current study extends this line of investigation by evaluating whether these same flow cytometric scoring procedures can be applied to the analysis of mouse bone marrow samples. To validate the method, three groups of male BALB/c mice were treated with 100 mg/kg b.wt. methyl methanesulfonate. Bone marrow samples were collected 20, 40 or 60 h after administration. A set of 5 untreated animals was included to provide an indication of spontaneous micronucleus frequencies. The cells were fixed with ultracold methanol, treated with ribonuclease, and labeled with anti-CD71 antibody (FITC conjugate) and propidium iodide. This fixing and labeling procedure resulted in the resolution of the micronucleated reticulocyte population and facilitated high-speed acquisition and enumeration via flow cytometry. The number of micronucleated reticulocytes was determined flow cytometrically by the analysis of 10?000 total reticulocytes per bone marrow sample. In addition to these automated measurements, slides stained with acridine orange were prepared and the number of micronuclei per 1000 reticulocytes was determined microscopically for each sample. The resulting data demonstrate that flow cytometry can effectively enumerate micronucleated reticulocytes in mouse bone marrow. The advantages associated with an objective, high throughput scoring methodology are also clearly indicated.     

Genotoxicity and Cytotoxicity Evaluation of the Neolignan Analogue 2-(4-Nitrophenoxy)-1Phenylethanone and its Protective Effect Against DNA Damage

Neolignans are secondary metabolites found in various groups of Angiosperms. They belong to a class of natural compounds with great diversity of chemical structures and pharmacological activities. These compounds are formed by linking two phenylpropanoid units. Several compounds that have ability to prevent genetic damage have been isolated from plants, and can be used to prevent or delay the development of tumor cells. Genetic toxicology evaluation is widely used in risk assessment of new drugs in preclinical screening tests. In this study, we evaluated the genotoxicity and cytotoxicity of the neolignan analogue 2-(4-nitrophenoxy)-1-phenylethanone (4NF) and its protective effect against DNA damage using the mouse bone marrow micronucleus test and the comet assay in mouse peripheral blood. Our results showed that this neolignan analogue had no genotoxic activity and was able to reduce induced damage both in mouse bone marrow and peripheral blood. Although the neolignan analogue 4NF was cytotoxic, it reduced cyclophosphamide-induced cytotoxicity. In conclusion, it showed no genotoxic action, but exhibited cytotoxic, antigenotoxic, and anticytotoxic activities.     

Micronucleus test with ethyl methanesulfonate in mouse peripheral blood reticulocytes stained supravitally using acridine orange-coated slides.

A new method for the micronucleus test using peripheral blood reticulocytes stained supravitally using acridine orange-coated slides was evaluated in male CD-1 mice treated with ethyl methanesulfonate (EMS) at doses of 100, 200, 300, and 400 mg/kg. Peripheral blood samples were taken 0, 24, 48, 72, and 96 h after treatment from each mouse without killing. The frequencies of micronucleated reticulocytes increased dose-dependently with the peak at 48 h after treatment. These results indicate that, at least for EMS, the new method used here can be an alternative to the conventional method using bone marrow polychromatic erythrocytes.     

Micronucleus test with ethyl methanesulfonate in mouse peripheral blood reticulocytes stained supravitally using acridine orange-coated slides

A new method for the micronucleus test using peripheral blood reticulocytes stained supravitally using acridine orange-coated slides was evaluated in male CD-1 mice treated with ethyl methanesulfonate (EMS) at doses of 100, 200, 300, and 400 mg/kg. Peripheral blood samples were taken 0, 24, 48, 72, and 96 h after treatment from each mouse without killing. The frequencies of micronucleated reticulocytes increased dose-dependently with the peak at 48 h after treatment. These results indicate that, at least for EMS, the new method used here can be an alternative to the conventional method using bone marrow polychromatic erythrocytes.     

Micronucleus assays on 5-fluorouracil and 6-mercaptopurine with mouse peripheral blood reticulocytes.

As part of the 5th collaborative study of the Collaborative Study Group for the Micronucleus Test (CSGMT), the sensitivity and advantages of the micronucleus assay using mouse peripheral blood cells were evaluated using 5-fluorouracil (5-FU) and 6-mercaptopurine (6-MP). The peripheral blood cells were collected from a tail vein of CD-1 male mice just before and 24-120 h after intraperitoneal injection. At 24-h intervals. The maximum incidence of micronucleated reticulocytes (MNRETs) at 50 mg/kg 5-FU was observed 96 h after injection; at 100 mg/kg, the peak was delayed to 120 h, and followed severe bone marrow depression. With 6-MP, maximum MNRETs were observed 48 h after treatment at all doses tested. At dose levels higher than 50 mg/kg, severe bone marrow depression was observed after maximum MNRETs. Though the appearance patterns of MNRETs and the bone marrow depression were different between 5-FU and 6-MP, the positive response of both chemical could be detected with this assay system as well as with the micronucleus test using femoral bone marrow cells.     

Intervention of astaxanthin against cyclophosphamide-induced oxidative stress and DNA damage: A study in mice

Astaxanthin, a natural and nutritional red carotenoid pigment, is used as a dietary supplement. The intention of the present study was to investigate the beneficial effects of dietary pigment astaxanthin, against cyclophosphamide-induced oxidative stress and DNA damage. The end points of evaluation of the study included: (a) malondialdehyde, glutathione and superoxide dismutase concentration in liver to detect oxidative stress; (b) normal and modified alkaline comet assays (the latter includes lesion-specific enzymes formamidopyrimidine-DNA glycosylase and endonuclease-III) to detect normal and oxidative stress-induced DNA damage by cyclophosphamide in the mouse bone marrow and the peripheral blood lymphocytes. In addition, micronucleus assay and chromosomal aberration test capable of detecting the DNA damage were also carried out in peripheral blood and bone marrow of mice. Cyclophosphamide (100 mg/kg intra-peritoneal) treatment led to significant increase in liver malondialdehyde and decreased the antioxidant enzymes glutathione and superoxide dismutase. Further, cyclophosphamide also significantly increased the DNA damage as observed from normal and modified comet assays as well as micronucleus and chromosomal aberration assay. Pre-treatment with astaxanthin (12.5, 25 and 50 mg/kg/day for 5 days per oral) resulted in the restoration of oxidative stress markers such as malondialdehyde, glutathione and superoxide dismutase in liver. The amelioration of oxidative stress with astaxanthin pre-treatment correlated well with the decreased DNA damage as evident from normal and modified alkaline comet assays of bone marrow cells and peripheral blood lymphocytes. Further astaxanthin pre-treatment also reduced the frequency of chromosomal breakage and micronucleus formation in the mouse bone marrow cells and peripheral blood reticulocytes. It is thus concluded that pre-treatment with astaxanthin attenuates cyclophosphamide-induced oxidative stress and subsequent DNA damage in mice and it can be used as a chemoprotective agent against the toxicity of anticancer drug cyclophosphamide.