Municipal sludge leachate-induced genotoxicity in mice--a subacute study

Inappropriate disposal of municipal sludge (MS) results in the leaching of toxic metals and organic chemicals, which can contaminate the surface and ground water leading to the serious health hazards. In this study, the genotoxic potential of the leachate prepared from MS sample was examined in mouse bone marrow cells through chromosomal aberrations (CA), micronucleus test (MT) and comet assay. Analysis of metals and physicochemical parameters of the leachate was also carried out to correlate the genotoxic results. The dried sludge showed high concentrations of heavy metals, viz. Cr, Cu, Pb and Ni. However, in 10% leachate, concentrations of these metals were manifold lower than that of obtained in dried sludge. Male mice orally gavaged to leachates (0.1-0.4 ml/mouse/day) for 15 days revealed significant (P<0.01, P<0.001) inhibition of mitotic index (MI) and induction of chromatid/chromosome fragments and breaks in all the treatment groups. The effect was observed to be dose-dependent. Treatment of mice with leachates also induced significant (P<0.001) frequencies of micronucleated polychromatic erythrocytes (MNPCE). The results of comet assay revealed a statistically significant (P<0.05 and <0.01) DNA damage in bone marrow cells exposed to 0.2-0.4 ml/mouse/day. Findings of the present study indicate that the constant exposure of MS leachate can cause genotoxic effects in mammals and suggest risks in human population.     

Genotoxicity of 2-[2-(acetylamino)-4-[bis(2-hydroxyethyl)amino]-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-6) and 4-amino-3,3'-dichloro-5,4'-dinitro-biphenyl (ADDB) in goldfish (Carassius auratus) using the micronucleus test and the comet assay

2-[2-(Acetylamino)-4-[bis(2-hydroxyethyl)amino]-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-6) and 4-amino-3,3'-dichloro-5,4'-dinitrobiphenyl (ADDB) are two compounds, which show strong mutagenicity toward bacteria, that have been identified as major mutagens in river water in Japan. In the present study, we examined the genotoxicity of PBTA-6 and ADDB in goldfish (Carassius auratus) by the micronucleus test and single-cell gel electrophoresis (comet assay). The frequencies of micronuclei in gill cells gradually increased until 96h after i.p. injection of PBTA-6 and ADDB at doses of 50mg/kg body weight, and then decreased 144h after injection. PBTA-6 induced micronuclei in gill cells dose-dependently at a dose range of 1-100mg/kg body weight, giving significantly high frequencies at doses of 50 and 100mg/kg body weight. On the other hand, no significant increase was observed in the peripheral erythrocytes of goldfish exposed to PBTA-6 or ADDB. In the comet assay, values of DNA tail moment and tail length in peripheral erythrocytes increased significantly until 6h after the i.p. injection of PBTA-6 (50mg/kg body weight), only to decrease by 9h after injection. Both the DNA tail moment and tail length were dose-dependently increased by injections of PBTA-6 at doses ranging from 1 to 50mg/kg. Significantly high values for tail moment and tail length were found in peripheral erythrocytes 3h after an i.p. injection of ADDB and persisted for up to 6h. These results show that both PBTA-6 and ADDB have genotoxic effects in goldfish.     

Mutagenicity and genotoxicity of isatin in mammalian cells in vivo

Isatin (1H-indole-2,3-dione) is a synthetically versatile substrate used for the synthesis of heterocyclic compounds and as a raw material for drug synthesis. Isatin and its derivatives demonstrate anticonvulsant, antibacterial, antifungal, antiviral, and anticancer properties. We evaluated the genotoxic and mutagenic effects of acute (24h) and repeated (14d) exposure to isatin in vivo, using the comet assay and the micronucleus test. Three doses (50, 100, and 150mg/kgb.w.) were administered to mice via gavage. Doses were selected according to the LD(50) of isatin, estimated in a preliminary test to be 1g/kgb.w. To evaluate the results, parametric (ANOVA/Tukey) and non-parametric (Kruskal-Wallis/Dunn's post hoc test) tests were used, according to the nature of the data distribution. At all doses (50, 100 and 150mg/kgb.w.), after acute treatment with isatin, alterations in DNA migration (comet assay) were not observed and mutagenic effects were not seen (micronucleus test on peripheral blood cells). After repeated doses, only the highest dose of isatin (150mg/kgb.w.) induced alterations in the DNA that gave rise to micronuclei in the bone marrow and peripheral blood cells of the mice. Our results show that the mutagenic and genotoxic effects of isatin depend on dose and on period of exposure.     

Antimutagenic effects of the mushroom Agaricus blazei Murrill extracts on V79 cells

Agaricus blazei Murrill, a native mushroom in Brazil, has been widely consumed in different parts of the world due to its medicinal power. Its anticarcinogenic activity has been shown in experimental animals, and antimutagenic activity has been demonstrated only in Salmonella. In this work, the mutagenic and antimutagenic activities of mushroom teas of strains AB96/07, AB96/09 and AB97/11 were evaluated in Chinese hamster V79 cells, using the comet assay and the micronucleus test. The cells were treated with three different concentrations (0.05, 0.1 and 0.15) of teas prepared from a 2.5% aqueous solution, under three different temperatures: (1) room (20-25 degrees C); (2) ice-cold (2-8 degrees C); and (3) warm (60 degrees C). The teas were applied in co-, pre- and post-treatments in combination with the mutagen methyl methanesulfonate (MMS; 1.6x10(-4) and 4x10(-4)M). The duration of the treatment was 1h in the comet assay and 2h in the micronucleus test. The results showed that the mushroom was not mutagenic itself. Nevertheless, the mushroom is an efficient antimutagen against the induction of micronuclei by MMS in all concentrations and preparations tested. The observed reductions in the frequencies of micronuclei ranged from 61.5 (room temperature 0.1% tea in post-treatment) to 110.3% (co-treatment with warm and ice-cold 0.15% tea). In the comet assay, the antimutagenic activity was detected only when the cells were pre-treated with the following teas: warm 0.1 and 0.15%, room temperature 0.05% and ice-cold 0.1%. The results indicate that the mushroom A. blazei extracts are antimutagenic when tested in V79 cells.     

Mutagenic effect of pesticides fastac 10 EK and durs ban 4E studied in a micronucleus test iin mouse bone marrow cells

The mutagenic activity of vastak and durs ban pesticides was studied by the micronucleus test in mouse bone marrow. The frequency of micronuclei in polychromatic erythrocytes was tested at 24, 36 and 42 h after oral administration of 50% LD50 dose of vastak (14 mg/kg) and durs ban (30.5 mg/kg). Significantly different increase in micronucleated polychromatic erythrocytes was established at 24, 36 and 48 h after vastak administration, and at 24 and 36 h after durs ban treatment. Doses of 25% LD50 for both pesticides showed no mutagenic activity, as judged by the induction of micronuclei in polychromatic erythrocytes.     

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.     

Genotoxicity and mutagenicity of Rosmarinus officinalis (Labiatae) essential oil in mammalian cells in vivo

Rosmarinus officinalis (rosemary) oil is widely used by the cosmetic, food, and pharmaceutical industries as a fragrance component of soaps, creams, lotions, and perfumes. Although it is popular, potential harmful side-effects of the oil have been described. We investigated the genotoxic and mutagenic potential of essential oil of R. officinalis in rodents, using comet, micronucleus and chromosome aberration assays. The animals were treated by gavage with one of three dosages of rosemary oil (300, 1000 or 2000 mg/kg). Liver and peripheral blood cells were collected from Swiss mice 24 h after treatment for the comet assay (genotoxicity endpoint), along with bone marrow cells for the micronucleus test (mutagenicity endpoint). Bone marrow cells were collected from Wistar rats 24 h after oil treatment for the micronucleus and chromosome aberration assays. Based on the comet assay, all three doses of rosemary oil induced significant increases in DNA damage in the mouse cells. There was a significant increase in micronucleated cells and chromosome aberrations only at the two higher doses. We conclude that rosemary essential oil provokes genotoxic and mutagenic effects when administered orally.     

Cytogenetic effects of ribavirin on mouse bone marrow

The micronucleus test and mitotic chromosome analysis were used to study the in vivo mutagenic activity of ribavirin on bone marrow cells of Swiss albino mice. To determine the incidence of micronuclei, mice were injected i.p. twice, at an interval of 24 h. with the drug at doses of 20, 100 and 200 mg/kg. Animals were killed 6 h after the second dose and bone marrow was examined for the presence of micronuclei in developing erythrocytes. Ribavirin significantly (P less than 0.05) induced micronuclei in polychromatic erythrocytes at all doses. A study was conducted to investigate the cytogenetic effect of the drug on mitotic chromosomes. Ribavirin at 200 mg/kg/day was administered to mice for 3 and 5 days. Repeated treatment with the high dose of ribavirin produced a highly significant (P less than 0.02) increase in abnormal metaphase spreads. The results indicate that ribavirin is mutagenic to bone marrow cells of mice as evaluated by the micronucleus test and by chromosome analysis.     

The micronucleus test. Methodological aspects

Changes in the cellular compositiion of bone marrow were studied in relation to dose of Trenimon® and time after treatment. Strong mutagenic effects caused a partial depletion of the marrow cavities of nucleated blood cell precursors, with subsequent retention of newly formed erythrocytes and inundation with peripheral blood. The influence of these changes on the results of micronucleus scoring was investigated in a time-effect and a dose-effect study using three different methods of evaluation, relating the incidence of micronuclei to (a) nucleated cells, (b) all erythrocytes, and (c) polychromatic erythrocytes. Conclusions are drawn on the practical use of the micronucleus test system. The simplest scoring procedure, namely relating micronucleated polychromatic erythrocytes to a given total number of erythrocytes, was very efficient in the range of low mutagenic effects and therefore well suited for safety screening wherease dose-effect studies comprising very high mutagenic effects required the application of a modified method of scoring.     

Mutagenic activity of the leachate of municipal solid waste landfill.

Organic concentrates were recovered using XAD-2/8 resin adsorption from the leachates of municipal solid waste landfills and their mutagenic activities were tested for 8 months using the Ames Salmonella/microsome assay. Highly polluted leachates (COD and BOD > or = 40 mg/l) generally had equal or higher mutagenic activities than lightly polluted leachates (COD and BOD < 40 mg/l). But there was no clear difference in mutagenicity per amount of concentrate between the two leachates. These results suggest that the mutagenic activity of landfill leachate is decided to some degree by the organic concentration in the leachate. The mutagenic activities detected even in lightly polluted leachates were not so low as those of various kind of surface waters ever reported. It is suggested that it is important to investigate the mutagenic activity of the leachate for evaluation of the impact of landfill leachate on the environment.     

Gamma ray induced genetic changes in different organs of chick embryo using peripheral blood micronucleus test and comet assay

Ionizing radiation is known to produce a variety of cellular and sub cellular damage in both prokaryotic and eukaryotic cells. Present studies were undertaken to assess gamma ray induced DNA damage in different organs of the chick embryo using alkaline comet assay and peripheral blood micronucleus test. Further the suitability of chick embryo, as an alternative model for genotoxicity evaluation of environmental agents was assessed. Fertilized eggs of Rhode island red strain were exposed to 0.5, 1 and 2 Gy of gamma rays delivered at a dose rate of 0.316 Gy/min using a ⁶⁰Co teletherapy machine. Peripheral blood smears were prepared from 8- to 11-day-old chick embryos for micronucleus test. Alkaline comet assay was performed on 11-day-old chick embryos in different organs such as the heart, liver, lung, blood, bone marrow, brain and kidney.Analysis of the data revealed a significant increase in the frequency of micronucleated polychromatic erythrocytes, micronucleated normochromatic erythrocytes and total micronucleated erythrocytes in the peripheral blood of gamma irradiated chick embryos at all the doses tested as compared to the respective controls. The polychromatic to normochromatic erythrocytes ratio which is an indicator of proliferation rate of hematopoetic tissue, decreased in the irradiated groups as compared to the controls. Data obtained from comet assay, clearly demonstrated a significant increase in DNA strand breaks in all the organs of irradiated chick embryos as compared to the respective controls. However, maximum damage was observed in the heart tissue on all the doses tested, followed by kidney, brain, lung, blood and liver. The lowest damage was observed in the bone marrow tissue. Both micronucleus test and comet assay were found to be suitable biomarkers for the evaluation of genotoxicity of gamma radiation in the chick embryo.     

Antimutagenicity of rosmarinic acid in Swiss mice evaluated by the micronucleus assay

Rosmarinic acid (RA) is a natural phenolic compound which presents different biological activities such as antitumor, antibacterial, anti-inflammatory, hepatoprotective and cardioprotective properties. In view of its important biological activities, the study of the effects of RA on genetic material becomes relevant. Thus, the objective of the present study was to evaluate the mutagenic and/or antimutagenic potential of RA on peripheral blood cells of Swiss mice using the micronucleus assay. Three doses of RA (50, 100 and 200 mg/kg body weight, b.w.) were used for the evaluation of its mutagenic potential. In the antimutagenicity assays, the different concentrations of RA were combined with the chemotherapeutic agent doxorubicin (DXR, 15 mg/kg b.w.). Peripheral blood samples were collected 24, 48 and 72 h after treatment for the evaluation of micronucleated polychromatic erythrocytes (MNPCEs). The results of the mutagenicity assays showed no increase in the frequency of micronuclei in animals treated with different concentrations of RA when compared to the negative controls. Treatment with different concentrations of RA combined with DXR revealed a significant reduction in the frequency of micronuclei compared to animals treated with DXR only. Although the mechanisms underlying the protective effect of RA are not completely understood, the putative antioxidant activity of RA might explain its effect on DXR mutagenicity.     

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.     

Assessment of DNA damage in peripheral blood of heavy smokers with the comet assay and the micronucleus test

The comet assay (single-cell gel electrophoresis, SCG) is being increasingly used in human biomonitoring for the detection of genotoxic exposures. Cigarette smoking is a well-documented source of a variety of potentially mutagenic and carcinogenic compounds. Therefore, smoking should represent a relevant mutagenic exposure and lead to genotoxic effects in exposed cells. However, our previous investigations as well as several other published studies on human biomonitoring failed to show an effect of smoking on DNA migration in the comet assay, while some other studies did indicate such an effect. Although many factors can contribute to the generation of discrepant results in such studies, clear effects should be obtained after high exposure. We therefore performed a comparative study with healthy male heavy smokers (>20 cigarettes per day) and non-smokers (n=12 in each group). We measured the baseline comet assay effects in fresh whole blood samples and isolated lymphocytes. In addition, the amount of 'formamidopyrimidine DNA-glycosylase (FPG)-sensitive sites' was determined by a combination of the standard comet assay with the bacterial FPG protein. Furthermore, the influence of a repair inhibitor (aphidicolin, APC) on baseline DNA damage was comparatively analysed. Duplicate slides from each sample were processed and analysed separately. In all experiments, a reference standard (untreated V79 cells) was included to correct for assay variability. Finally, to compare the comet assay results with another genetic endpoint, all blood samples were investigated in parallel by the micronucleus test (MNT). Baseline and gamma radiation-induced micronucleus frequencies were determined. None of these approaches revealed a significant difference between heavy smokers and non-smokers with regard to a genotoxic effect in peripheral blood cells.     

Studies on the in vivo and in vitro mutagenicity and the lipid peroxidation of chlorinated surface (drinking) water in rats and metabolically competent human cells.

In the present study, DNA damaging and mutagenic effects of chlorinated drinking water (CDW) extracts obtained from polluted raw water resources were examined in metabolically competent human Hep G2 hepatoma cells using the in vitro micronucleus assay and the single cell gel electrophoresis (SCGE, comet assay). Additionally, the in vivo induction of micronuclei (MN) was studied in polychromatic erythrocytes (PCEs) derived from bone marrow of CDW-treated Wistar rats. Furthermore, we examined the influence of CDW on the lipid peroxidation (LpO) in blood, liver, kidney and testicle of rats. The results demonstrated significant increases of micronucleated PCEs in the bone marrow of rats fed with relatively low CDW doses (33.3ml/kg body weight per day). Similar effects, i.e. increases of MN frequencies, were found in Hep G2 hepatoma cells after CDW treatment (41 MN/1000 binucleated cells (BNCs) for 167ml CDW) in comparison to the vehicle control (24 MN/1000 BNC). Additionally, DNA damages caused by CDW were observed in the comet assay. As a product of LpO, the levels of malondialdehyde (MDA) were significantly enhanced almost in all animals and organs tested after CDW treatment. In livers and serum of rats dose-dependent increases of MDA were observed. The data indicated that extracts from CDW obtained from polluted raw water were able to cause oxidative damages and to induce various biological effects in mammalian cells in vivo and in vitro, i.e. clastogenicity and/or aneugenicity, DNA strand breaks and/or alkali-labile damages. The consistency of the results among the various biological systems and endpoints led to the conclusion that the consumption of chlorinated drinking water obtained from polluted raw water may enhance the body burden with mutagenic and/or carcinogenic substances and therefore, means a potential genetic hazard for human health.     

Studies on the in vivo and in vitro mutagenicity and the lipid peroxidation of chlorinated surface (drinking) water in rats and metabolically competent human cells

In the present study, DNA damaging and mutagenic effects of chlorinated drinking water (CDW) extracts obtained from polluted raw water resources were examined in metabolically competent human Hep G2 hepatoma cells using the in vitro micronucleus assay and the single cell gel electrophoresis (SCGE, comet assay). Additionally, the in vivo induction of micronuclei (MN) was studied in polychromatic erythrocytes (PCEs) derived from bone marrow of CDW-treated Wistar rats. Furthermore, we examined the influence of CDW on the lipid peroxidation (LpO) in blood, liver, kidney and testicle of rats. The results demonstrated significant increases of micronucleated PCEs in the bone marrow of rats fed with relatively low CDW doses (33.3 ml/kg body weight per day). Similar effects, i.e. increases of MN frequencies, were found in Hep G2 hepatoma cells after CDW treatment (41 MN/1000 binucleated cells (BNCs) for 167 ml CDW) in comparison to the vehicle control (24 MN/1000 BNC). Additionally, DNA damages caused by CDW were observed in the comet assay. As a product of LpO, the levels of malondialdehyde (MDA) were significantly enhanced almost in all animals and organs tested after CDW treatment. In livers and serum of rats dose-dependent increases of MDA were observed. The data indicated that extracts from CDW obtained from polluted raw water were able to cause oxidative damages and to induce various biological effects in mammalian cells in vivo and in vitro, i.e. clastogenicity and/or aneugenicity, DNA strand breaks and/or alkali-labile damages. The consistency of the results among the various biological systems and endpoints led to the conclusion that the consumption of chlorinated drinking water obtained from polluted raw water may enhance the body burden with mutagenic and/or carcinogenic substances and therefore, means a potential genetic hazard for human health.     

Antimutagenicity of rosmarinic acid in Swiss mice evaluated by the micronucleus assay

Rosmarinic acid (RA) is a natural phenolic compound which presents different biological activities such as antitumor, antibacterial, anti-inflammatory, hepatoprotective and cardioprotective properties. In view of its important biological activities, the study of the effects of RA on genetic material becomes relevant. Thus, the objective of the present study was to evaluate the mutagenic and/or antimutagenic potential of RA on peripheral blood cells of Swiss mice using the micronucleus assay. Three doses of RA (50, 100 and 200 mg/kg body weight, b.w.) were used for the evaluation of its mutagenic potential. In the antimutagenicity assays, the different concentrations of RA were combined with the chemotherapeutic agent doxorubicin (DXR, 15 mg/kg b.w.). Peripheral blood samples were collected 24, 48 and 72 h after treatment for the evaluation of micronucleated polychromatic erythrocytes (MNPCEs). The results of the mutagenicity assays showed no increase in the frequency of micronuclei in animals treated with different concentrations of RA when compared to the negative controls. Treatment with different concentrations of RA combined with DXR revealed a significant reduction in the frequency of micronuclei compared to animals treated with DXR only. Although the mechanisms underlying the protective effect of RA are not completely understood, the putative antioxidant activity of RA might explain its effect on DXR mutagenicity.     

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.     

DNA damage and repair after exposure to sevoflurane in vivo, evaluated in Swiss albino mice by the alkaline comet assay and micronucleus test

The relationship between DNA damage and repair of peripheral blood leukocytes, liver, kidney and brain cells was investigated in Swiss albino mice (Mus musculus L.) after exposure to sevoflurane (2.4 vol% for 2 h daily, for 3 days). Genetic damage of mouse cells was investigated by the comet assay and micronucleus test. To perform the comet assay, mice were divided into a control group and 4 groups of exposed mice sacrificed on day 3 of the experiment, at 0, 2, 6 or 24 h after the last exposure to sevoflurane. Mean tail length (TL), tail moment (TM), and tail intensity (TI) values were significantly higher in exposed mice (all examined organs) than in the control group. Significant DNA damage immediately after exposure to sevoflurane was observed in leukocytes. Damage induction in the liver, kidney, and brain occurred 6 h later than in leukocytes, as expected according to the toxicokinetics of the drug, where blood is the first compartment to absorb sevoflurane. However, none of the tested tissues revealed signs of repair until 24 h after the exposure. To distinguish the unrepaired genome damage in vivo, the micronucleus test was applied. Number of micronuclei in reticulocytes showed a statistically significant increase, as compared with the control group at all observed times after the treatment.     

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.