Attenuation of N-methyl-N'-nitro-N-nitrosoguanidine induced genotoxicity and oxidative stress by tomato and garlic combination

The protective effect of pretreatment with tomato and garlic against N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced genotoxicity and oxidative stress was investigated in male Swiss mice. In vivo bone marrow micronucleus test was performed to assess the antigenotoxic effect of tomato and garlic. Oxidative stress was monitored by estimating the extent of lipid peroxidation and the status of the glutathione redox cycle antioxidants. Increased frequency of bone marrow micronuclei with enhanced lipid peroxidation was associated with compromised antioxidant defenses in MNNG treated animals. Although pretreatment with tomato and garlic significantly reduced the frequencies of MNNG-induced bone marrow micronuclei, the combination of tomato and garlic exerted a greater protective effect. This was associated with modulation of lipid peroxidation as well as reduced glutathione (GSH) and the GSH-dependent enzymes glutathione peroxidase (GPx) and glutathione-S-transferase (GST). These findings suggest that a diet containing even low levels of different naturally occurring compounds is effective in exerting antigenotoxic effects by modulating oxidative stress.     

Antimutagenic effects of stobadine: review of results

The paper summarizes the results of our previously published studies testifying the hypothesis of the antimutagenic effect of stobadine (STB) in vivo and in vitro. The micronucleus test was used in in vivo experiments with ICR mice. Oral pretreatment with STB significantly decreased the mutagenic effect of cyclophosphamide (CP) in a concentration-dependent way. The protective effect of STB was confirmed in fetuses of CP-treated mice. STB pretreatment exerted also a radioprotective effect in Co60-irradiated mice. The ineffectiveness of STB posttreatment is indicative of its effect operative in the initiation of mutagenesis and of its radical-scavenging mechanism. The ability of STB to reduce N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)induced gene mutations and MNNG-induced calcinosis/Raynaud's phenomenon/esophageal dysmotility/sclerodactyly/telangiectasia variant of scleroderma (CREST)-positive and CREST-negative micronuclei in V79 cells was tested in in vitro experiments. We found that this drug reduced the level of both gene mutations and CREST-negative micronuclei mainly if given as pretreatment before exposure of cells to MNNG. We conclude that STB may have inhibited mutagenesis not only by scavenging reactive oxygen species, but also as a result of induction of metabolic enzymes, which reduced the level of DNA lesions.     

Involvement of mismatch repair proteins in adaptive responses induced by N-methyl-N'-nitro-N-nitrosoguanidine against γ-induced genotoxicity in human cells

As humans are exposed to a variety of chemical agents as well as radiation, health effects of radiation should be evaluated in combination with chemicals. To explore combined genotoxic effects of radiation and chemicals, we examined modulating effects of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), a direct-acting methylating agent, against genotoxicity of γ-radiation. Human lymphoblastoid TK6 cells and its mismatch-deficient derivative, i.e., MT1 cells, were treated with MNNG for 24h before they were exposed to γ-irradiation at a dose of 1.0 Gy, and the resulting genotoxicity was examined. In TK6 cells, the pretreatments with MNNG at low doses suppressed frequencies of the thymidine kinase (TK) gene mutation and micronucleus (MN) formation induced by γ-irradiation and thus the dose responses of TK and MN assays were U-shaped along with the pretreatment doses of MNNG. In contrast, the genotoxic effects of MNNG and γ-irradiation were additive in MT1 cells and the frequencies of TK mutations and MN induction increased along with the doses of MNNG. Apoptosis induced by γ-radiation was suppressed by the pretreatments in TK6 cells, but not in MT1 cells. The expression of p53 was induced and cell cycle was delayed at G2/M phase in TK6, but not in MT1 cells, by the treatments with MNNG. These results suggest that pretreatments of MNNG at low doses suppress genotoxicity of γ-radiation in human cells and also that mismatch repair proteins are involved in the apparent adaptive responses.     

FISH in analysis of gamma ray-induced micronuclei formation in barley

A micronucleus test in combination with fluorescent in situ hybridization (FISH) using telomere-, centromere-specific probes and 5S and 25S rDNA was used for a detailed analysis of the effects of gamma ray irradiation on the root tip meristem cells of barley, Hordeum vulgare (2n = 14). FISH with four DNA probes was used to examine the involvement of specific chromosomes or chromosome fragments in gamma ray-induced micronuclei formation and then to explain their origin. Additionally, a comparison of the possible origin of the micronuclei induced by physical and chemical treatment: maleic hydrazide (MH) and N-nitroso-N-methylurea (MNU) was done. The micronuclei induced by gamma ray could originate from acentric fragments after chromosome breakage or from whole lagging chromosomes as a result of a dysfunction of the mitotic apparatus. No micronuclei containing only centromeric signals were found. An application of rDNA as probes allowed it to be stated that 5S rDNA–bearing chromosomes are involved in micronuclei formation more often than NOR chromosomes. This work allowed the origin of physically- and chemically-induced micronuclei in barley cells to be compared: the origin of micronuclei was most often from terminal fragments. FISH confirmed its usefulness in the characterization of micronuclei content, as well as in understanding and comparing the mechanisms of the actions of mutagens applied in plant genotoxicity.     

Sex differences in micronucleus induction with hycanthone methanesulfonate in bone marrow cells of mice.

The clastogenic effect of the antischistosomal drug hycanthone methanesulfonate was studied with the micronucleus test in mouse bone marrow cells. Male and female (102/El x C3H/El)F1 mice were treated with single i.p. injections. Bone marrow was sampled 18, 24 and 30 h after treatment with 100 mg/kg. The highest micronucleus yield occurred at 24 h. The dose response for micronucleus induction at 24 h after treatment was non-linear for doses between 5 and 300 mg/kg. The lowest effective dose was 5 mg/kg for females and 10 mg/kg for males. The experiments revealed a significantly higher sensitivity of female mice for the induction of micronuclei in polychromatic erythrocytes by hycanthone methanesulfonate. This result supports the recommendation to use both sexes for quantitative assessment of genotoxicity in the micronucleus test.     

Gene expression in E. coli after treatment with streptozotocin

Gene induction by the methylating agents streptozotocin (STZ), N-methyl-N-nitrosourea (MNU), and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was evaluated in E. coli fusion mutants. These mutants have fusions of the lac operon to genes induced by treatment with sublethal levels of alkylating agents and were previously selected from random insertions of the Mu-dl (Apr lac) phage by screening for induction of beta-galactosidase activity in the presence of methyl methanesulfonate or MNNG. The results demonstrate that STZ differs from MNNG and MNU in failing to induce aidC expression. Further, expression of aidC after exposure to MNU and MNNG occurs only in nonaerated cultures; aeration blocks the induction. Induction of aidD, alkA, aidB, and sfiA expression occurs with all 3 agents although at markedly lower concentrations of MNNG and STZ compared to MNU. alkA and to a lesser extent aidD mutants of E. coli strains were more sensitive to these agents, while no differences were evident between wild-type and aidB or aidC fusion mutants.     

Induction of micronuclei in mouse fetal liver after exposure in utero to N-methyl-N′-nitro-N-nitrosoguanidine

The effects of N-ethyl-N′-nitro-N-nitrosoguanidine (MNNG) on the induction of micronuclei were examined in mouse fetuses exposed in utero. By this study, MNNG was proved to be mutagenic in vivo. The frequency of micronucleated erythrocytes (MNEs) in fetal liver peaked at 18 h after a single intraperitonela injection into pregnant mice on day 13 of gestation. Then, to examine the effects of administration routes on the induction of micronuclei, the chemical was given by various routes, and the percentage of MNEs (%MNEs) in fetuses were examined 18 h after treatment. The %MNEs after administration of MNNG intraperitoneally, subcutaneously, intravenously, and orally was 4.7, 1.9, 0.8 and 0.3, respectively. The control value was 0.3. In the intraperitoneally treated mice, %MNEs for fetuses in the uterine horn olcated nearer the injection site was higher than that in the other. In addition, in the intraperitoneally treated mice, there was a tendency for the higher %MNEs to occur in the fetuses located near the injection site. Together with the results on the distribution of MNNG in mice (Frei and Lawley, 1976), these findings suggest that MNNG might be inactivated in the maternal systemic circulation and that the agent which induces micronuclei might be distributed to the fetuses by diffusion.     

Mutagenicity and cytotoxicity of congeners of two classes of nitroso compounds in Chinese hamster ovary cells

The induction of mutation by certain nitrosamidines and nitrosamides has been quantitated utilizing the hypoxanthine--guanine phosphoribosyl transferase (HGPRT) locus in Chinese hamster ovary cells. Dose--response relationships for cytotoxicity and mutagenicity are presented for N-methyl-N-nitrosourea (MNU), N-ethyl-N-nitrosourea (ENU), N-butyl-N-nitrosourea (BNU), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), and N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG). Based on the concentration of each agent required to kill 90% of the cells, the following order of cytotoxicity was observed: MNNG greater than ENNG greater than MNU greater than ENU greater than BNU. This is the same order of potency as observed for mutation induction per unit concentration of mutagen.     

Multicolour FISH in an analysis of chromosome aberrations induced by N-nitroso-N-methylurea and maleic hydrazide in barley cells

The present study is a rare example of a detailed characterization of chromosomal aberrations by identification of individual chromosomes (or chromosome arms) involved in their formation in plant cells by using fluorescent in situ hybridization (FISH). In addition, the first application of more than 2 DNA probes in FISH experiments in order to analyse chromosomal aberrations in plant cells is presented. Simultaneous FISH with 5S and 25S rDNA and, after reprobing of preparations, telomeric and centromeric DNA sequences as probes, were used to compare the cytogenetic effects of 2 chemical mutagens: N-nitroso-N-methylurea (MNU) and maleic hydrazide (MH) on root tip meristem cells of Hordeum vulgare (2n=14). The micronucleus (MN) test combined with FISH allowed the quantitative analysis of the involvement of specific chromosome fragments in micronuclei formation and thus enabled the possible origin of mutagen-induced micronuclei to be explained. Terminal deletions were most frequently caused by MH and MNU. The analysis of the frequency of micronuclei with signals of the investigated DNA probes showed differences between the frequency of MH- and MNU-induced micronuclei with specific signals. The micronuclei with 2 signals, telomeric DNA and rDNA (5S and/or 25S rDNA), were the most frequently observed in the case of both mutagens, but with a higher frequency after treatment with MH (46%) than MNU (37%). Also, 10% of MH-induced micronuclei were characterized by the presence of only telomere DNA sequences, whereas there were almost 3-fold more in the case of MNU-induced micronuclei (28%). Additionally, by using FISH with the same probes, an attempt was made to identify the origin of chromosome fragments in mitotic anaphase.     

Genotoxicity and cell proliferative activity of omeprazole in rat stomach mucosa.

Induction of unscheduled DNA synthesis (UDS) as a marker of genotoxicity and induction of ornithine decarboxylase (ODC) activity as a marker of cell proliferative activity by omeprazole were determined in the glandular stomach mucosa of male F344 rats after oral administration. Commercial enteric-coated omeprazole (Losec) at doses of 30 and 100 mg/kg body weight induced a dose-dependent increase in UDS but not replicative DNA synthesis in the pyloric mucosa of rat stomach 4 h after its administration. Dose-dependent significant induction of ODC activity was observed in fundic and pyloric mucosa with a maximum 8 h after administration of omeprazole at doses of 37.5-100 mg/kg body weight. These results show that omeprazole has genotoxicity and cell proliferative activity in the rat glandular stomach mucosa.     

Comparison of the effects of 1,2-dimethylhydrazine and cyclophosphamide on micronucleus incidence in bone marrow and colon

An in vivo micronucleus assay has been developed that utilizes colonic epithelial cells. The genotoxic effects of 1,2-dimethylhydrazine (54-07-3), a colon carcinogen, and of the nitrogen mustard, cyclophosphamide (50-18-0), on the bone-marrow polychromatic erythrocytes and on colonic epithelium from mice were compared using micronucleus induction in each organ as the end point. In the bone marrow, cyclophosphamide was a potent inducer of micronuclei, while 1,2-dimethylhydrazine administration had little effect on the micronucleus incidence. In the colon, 1,2-diemthylhydrazine was an effective inducer of micronuclei. Thus, the colonic micronucleus assay appears to be a potentially useful test for the detection of colon carcinogens.     

Polyozellus multiplex, a Korean wild mushroom, as a potent chemopreventive agent against stomach cancer

Polyozellus multiplex, a Korean wild mushroom, was extracted using methanol, and the extract was further fractionated with water and ethylacetate. Assay of each fraction with MTT revealed significant tumoristatic effects of the water fraction of Polyozellus multiplex against human gastric and other cancer cells but not normal human lymphocytes. Modifying effects of the water fraction on glandular stomach mucosa were investigated in male Wistar rats treated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). The dietary 0.5% or 1% water fraction of Polyozellus multiplex significantly increased glutathione S-transferase (GST) and superoxide dismutase (SOD) activities, and showed a tendency for increase in glutathione (GSH) levels, compared to the MNNG alone group. It also caused a significant reduction in proliferating cell nuclear antigen (PCNA)-labeling index of the glandular stomach epithelium, along with increase in p53 tumor suppressor gene expression. These results suggest that Polyozellus multiplex is a candidate for chemoprevention against gastric cancer.     

Comutagenic and coclastogenic effects of selenium in vitro and in vivo

The comutagenic activity of selenium was investigated using in vitro and in vivo techniques, including the liquid suspension modification of the standard Salmonella/microsome mutagenicity assay, the metaphase analysis of chromosome aberrations in CHO cells and in mouse bone marrow as well as the micronucleus assay in mouse bone marrow. 4 h growth of S. typhimurium TA1535 in a nutrient broth containing 2.9 x 10(-5) M but not 1.16 x 10(-5) M Na2SeO3 caused an up to 10-fold increase of the number of N-methylnitrosourea (MNU, 2.0-2.5 mM)-induced his+ revertants and an up to 2-fold elevation of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG, 1.48 x 10(-5))-induced mutation rate. Pretreatment of bacteria with Na2SeO3 alone had no effect on the spontaneous mutation level. The combined treatment of CHO cells with MNNG (1.25 x 10(-5) M) or tobacco smoke (TS, 2-3 puffs generated by a cigarette inhalation machine) plus Na2SeO3 (0.58-1.16 x 10(-5) M) starting 2 h and 4 h before the MNNG or TS treatment respectively resulted in a 2-3-fold increase in the percent of metaphases with chromosome aberrations. Furthermore, treatment for 7-14 days of male BDF1 (C57Bl x DBA2) or CC57W mice with Na2SeO3, added to the drinking water at a concentration of 10 ppm, potentiated by 2-3 times the chromosome-damaging activity of urethane (0.5-1.0 g/kg, i.p.) in mouse bone marrow, as measured by the formation of micronuclei or chromosome aberrations. In addition, Na2SeO3 increased up to 43.8% the number of micronucleated polychromatic erythrocytes (MNPCE) induced by mitomycin C (MMC, 1.5 mg/kg, i.p.) in BDF1 mouse bone marrow. Treatment of mice with Na2SeO3 alone had no effect on the spontaneous level of MNPCE. All these findings are consistent with a comutagenic and coclastogenic activity of selenium both in prokaryotes and in eukaryotes, in vitro as well as in vivo after pretreatment of target cells with the trace element.     

The in vitro micronucleus assay for detection of cytogenetic effects induced by mutagen-carcinogens: comparison with the in vitro sister-chromatid exchange assay

The sensitivity of a cytogenetic assay, as expressed by the in vitro induction of micronuclei (MN), was compared to the in vitro induction of sister-chromatid exchanges (SCEs). Chinese hamster lung (V79) cells were exposed to 3 known alkylating agents: methyl methanesulphonate (MMS), ethyl methanesulphonate (EMS) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and to 5 newly synthesized naphthofurans: 2-nitro-7-methoxynaphtho[2,1-b]furan (A), 2-nitro-8-methoxynaphtho[2,1-b]furan (B), 2-nitronaphtho[2,1-b]furan (C), 2-nitro-7-bromonaphtho[2,1-b]furan (D) and 7-methoxynaphtho[2,1-b]furan (E). The induction of MN only was also analysed after exposure of the cells to 4 alcohols: ethanol, methanol, butanol and propanol. The lowest dose at which a significant effect could be observed was determined. In both assays, MNNG, MMS and EMS were equally active with the following order of potency: MNNG greater than MMS greater than EMS, the latter being a very weak inducer of MN and SCE. Compounds A and B were also very effective in both assays. Compound C was a more active inducer of SCE than MN. Compounds D and E were not active in either assay. None of the 4 alcohols induced MN. Our results are compared with the previously published data on in vitro and in vivo induction of SCE and MN. We conclude that the MN in vitro assay which detects clastogens as well as agents affecting the spindle apparatus, is a good indicator of genotoxicity, though slightly less sensitive than the in vitro SCE test. It could provide a rapid, simple and inexpensive complementary short-term test for the evaluation of potentially mutagenic chemicals.     

Genotoxicity of paraquat: micronuclei induced in bone marrow and peripheral blood are inhibited by melatonin

The ability of melatonin to influence paraquat-induced genotoxicity was tested using micronucleated polychromatic erythrocytes as an index of damage in both bone marrow and peripheral blood cells of mice. Melatonin (10 mg/kg) or an equal volume of saline were administered intraperitoneally (ip) to mice 30 min prior to an ip injection of paraquat (20 mg/kgx2), and thereafter at 6-h intervals until the conclusion of the study (72 h). The number of the micronucleated polychromatic erythrocytes increased after paraquat administration both in peripheral blood and bone marrow cells. Melatonin administration to paraquat-treated mice significantly reduced micronuclei formation in both peripheral blood and bone marrow cells; these differences were apparent at 24, 48 and 72 h after paraquat administration. The induction of micronuclei was time-dependent with peak values occurring at 24 and 48 h. The reduction in paraquat-related genotoxicity by melatonin is likely due in part to the antioxidant activity of the indole. We did not observe effects of melatonin over paraquat in paraquat+melatonin groups incubated at 0, 60 and 120 min. Mitomycin C, which was used as a positive control, also caused the expected large rises in micronuclei in both bone marrow and peripheral blood cells at 24, 48 and 72 h after its administration.     

Cell killing by various monofunctional alkylating agents in Chinese hamster ovary cells

Cell killing by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), N-methyl-N-nitrosourea (MNU), N-ethyl-N-nitrosourea (ENU), and methyl methanesulfonate (MMS) was measured in Chinese hamster ovary (CHO) cells using the colony-formation assay. Cell killing by these agents was determined in exponentially growing asynchronous cells, in synchronous cells as a function of cell-cycle position and in nondividing cells. Distinct differences in the cytotoxic effect of the 4 alkylating agents were found in respect to dose-response, cell cycle phase-sensitivity and growth state. MNNG and MNU showed the same biphasic dose-survival relationship in exponentially growing cells, with an initial steep decline followed by a shallow component. The shallow component disappeared in growth-arrested cells. MNNG and MNU differed, however, in the cell-cycle age response. No cell-cycle phase difference was seen with MNNG, whereas cells in G1 seemed more sensitive to MNU than cells in S phase. MMS and ENU both showed shouldered dose-response curves for exponentially growing asynchronous cells, and the same cell-cycle pattern for synchronous cultures with cells in early S phase being the most sensitive. However, survival of nondividing cells versus dividing cells was reduced much more by MMS than by ENU. Caffeine, which interferes with the regulation of DNA synthesis and is known to modify cell killing by DNA-damaging agents, enhanced cell killing by all agents. It is concluded that there must be a number of factors which contribute to cell killing by monofunctional alkylating agents, and that besides alkylation of DNA reaction with other cellular macromolecules should be considered.     

2-Hydroxy-1,4-naphthoquinone, the natural dye of Henna, is non-genotoxic in the mouse bone marrow micronucleus test and does not produce oxidative DNA damage in Chinese hamster ovary cells

2-Hydroxy-1,4-naphthoquinone (HNQ) has been found positive in a previous chromosome aberration test in Chinese hamster ovary (CHO) cells and in a mouse bone marrow micronucleus test at 72h after oral administration (vehicle: DMSO). However it was negative at 24 and 48h sampling times, and in subsequent micronucleus tests that used 0.5% aqueous methyl cellulose (MC) as vehicle. We performed a bone marrow micronucleus test in male and female NMRI BRL/BR mice at oral doses of 75, 150 and 300mg/kg in two vehicles (DMSO and 0.5% aqueous MC), evaluated micronuclei at 24, 48 and 72h, plasma levels of HNQ at 0.5, 1 and 4h, and haematology parameters at 72h after administration. The mechanism of in vitro clastogenic activity of HNQ was investigated by evaluation of the potential of HNQ to produce oxidative DNA damage after treatment of CHO with 10mM HNQ, followed by quantification of DNA fragments using the comet assay. In the micronucleus test, HNQ at 300mg/kg produced mortality and clinical signs at similar incidence and severity for both vehicles. Levels of HNQ in the plasma of treated mice were dose-related, of similar magnitude for both vehicles, but higher in females than in males. Maximum concentrations were found at 0.5 or 1h. At 300mg/kg, HNQ slightly affected RBC parameters suggesting haematotoxicity. No increase in the frequency of micronuclei was observed for any dose, vehicle or time point, whereas the positive control substance (CPA) produced a clear positive response. No evidence of HNQ-induced oxidative DNA damage was found at clastogenic concentrations in vitro, whereas the positive control substance (H(2)O(2)) produced a clear increase. In conclusion, HNQ was negative for induction of bone marrow micronuclei in mice up to 72h after administration in two different vehicles, and its in vitro clastogenicity was not due to oxidative damage. These results confirm that HNQ poses no or negligible genotoxic risk.     

Influence of anoxia and respiratory deficiency on the genotoxicity of some direct-acting alkylating agents in yeast.

We have studied the influence of anoxia and respiratory deficiency (RD) in yeast on the cytotoxic and recombinogenic effects of 5 direct-acting alkylating agents, namely N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), methylnitrosourea (MNU), ethylnitrosourea (ENU), methyl methanesulphonate (MMS) and ethyl methanesulphonate (EMS). We found that the effects of both conditions parallel each other for MMS, MNNG, MNU and ENU. Both anoxia and RD did not modify the effects of MMS to any significant extent. On the other hand, anoxic and respiratory-deficient cells were found to be more resistant than euoxic and respiratory-proficient cells respectively for MNNG, MNU and ENU. In the case of EMS, which is similar to MMS in its chemical reaction with DNA, the respiratory-deficient cells were found to be more sensitive than the respiratory-proficient ones. These studies indicate that the response of anoxic and respiratory-deficient cells cannot be predicted solely on the basis of the chemical reactivity pattern of the alkylating agents. The physiological state which exists under these conditions may exert considerable influence on the cellular response.     

The micronucleus test with mouse peripheral blood on N-methyl-N'-nitro-N-nitrosoguanidine and mitomycin C.

The micronucleus test using mouse peripheral blood was conducted with N-methyl-N'-nitro-N-nitro-soguanidine (MNNG) and mitomycin C (MMC) as part of the 5th collaborative study supported by the Environmental Mutagen Society of Japan (CSGMT/MMS.JEMS). Male CD-1 mice were intraperitoneally injected once with 12.5-100 mg/kg of MMC. Peripheral blood was drawn at different intervals after treatment, placed on slides previously coated with acridine orange and the numbers of reticulocytes with micronuclei (MNRETs) were scored. The experiments indicated that the maximum effect of both MNNG and MMC was found about 48 h after treatment, and that the micronucleus test using peripheral blood is useful for the screening of chemicals throughout the experimental period in a single animal.     

Mutagenicity of 4-nitroquinoline 1-oxide in the MutaMouse.

As part of a collaborative study, the Mammalian Mutagenesis Study Group (MMS), a sub-organization of the Environmental Mutagen Society of Japan (JEMS) conducted mutagenicity tests in MutaMouse. Using a positive selection method, we studied the organ-specificity and time dependence of mutation induction by 4-nitroquinoline 1-oxide (4NQO). A single dose of 4NQO was administered intraperitoneally (7.5 or 15 mg/kg) or orally (200 mg/kg) to groups of male mice. On days 7, 14 and 28 after treatment, we isolated the liver, kidney, lung, spleen, bone marrow, testis and stomach in the intraperitoneal administration experiment and the liver, lung, bone marrow, testis and stomach in the oral administration experiment. In addition, we performed the peripheral blood micronucleus test to evaluate clastogenicity. In the mice treated intraperitoneally at 7.5 mg/kg, we found increased mutant frequency (MF) only in the lung, where the MF did not vary with expression time. In the mice treated at 15 mg/kg, we found increased MF in the liver, bone marrow and lung. In orally treated mice, the MF was high in the lung and liver and very high in the bone marrow and stomach while the increase in the testis was negligible. As the expression time was prolonged, the MF tended to increase in the liver, decrease in the bone marrow, and remain stable in the lung, testis and stomach. The incidence of micronucleus induction in peripheral blood cells was significantly increased (p<0.01) in the 4NQO groups when compared with the vehicle control group by intraperitoneal treatment. Thus, these assay systems appeared to be of use in detecting not only genetic mutation but also chromosomal aberration.