Mouse Micronucleus Research Articles

Positive responses in the MS mouse micronucleus test of otherwise quasi-positive or inconclusive chemicals: effectiveness of MS/Ae mice in screening tests: II

Positive responses in the MS mouse micronucleus test of otherwise quasi-positive or inconclusive chemicals: effectiveness of MS/Ae mice in screening tests: II

The genotoxicity profile of atorvastatin, a new drug in the treatment of hypercholesterolemia

While HMG-CoA reductase inhibitors such as fluvastatin, lovastatin, pravastatin and simvastatin demonstrate lack of in vitro and in vivo mutagenicity and clastogenicity in bacterial and mammalian cells, long term rodent carcinogenicity studies resulted in an increased incidence in neoplasms at high doses. These effects may be attributable to an exaggeration of the desired biochemical effect of the drug and/or a tumor promoting effect. The genotoxicity of atorvastatin, a newly developed HMG-CoA reductase inhibitor, was evaluated in a variety of test systems. In bacterial mutagenicity tests, the E. coli tester strain WP2(uvrA) and S. typhimurium strains TA98, TA100, TA1535, TA1537, and TA1538 were exposed to concentrations of atorvasatin as high as 5000 μg/plate both in the absence (S9-) and presence (S9+) of metabolic activation. Atorvastatin was not mutagenic in either E. coli or S. typhimurium. Chinese hamster lung V79 cell cultures were exposed to atorvastatin at concentrations of 50–300 μg/ml (S9-) and 100–300 μg/ml (S9+) and structural chromosome aberrations were assessed. Mutation at the hgprt locus was assessed at concentrations of 100–300 μg/ml (S9-) and 150–275 μg/ml (S9+). Atorvastatin was neither mutagenic nor clastogenic in the absence or presence of S9. The lack of in vitro genotoxicity was corroborated in vivo in a mouse micronucleus study in which single oral doses of atorvastatin were administered to male and female CD-1 mice at 1, 2500, or 5000 mg/kg. No biologically significant increases in the frequency of micronucleated polychromatic erythrocytes in bone marrow at 24, 48, or 72 h postdosing were observed. Thus, atorvastatin, as with the other tested HMG-CoA reductase inhibitors, is not genotoxic.

Evaluation of the genotoxicity potential and chronic inhalation toxicity of 1,1-dichloro-1-fluoroethane (HCFC-141 b)

A battery of in vitro and in vivo tests were conducted on HCFC-141b as a vapour. Bacterial gene mutation assays with Escherichia coli and Salmonella typhimurium were negative in all tester strains. In vitro chromosomal aberration assays were positive on CHO cells but negative on human lymphocytes. Moreover, HCFC-141b was negative in vivo in a mouse micronucleus inhalation assay. On the basis of these data and previously reported genotoxicity testing, HCFC-141b is considered non-genotoxic. Groups of 80 male and 80 female Sprague-Dawley rats were exposed, by inhalation (6 hr/day, 5 days/wk) to vapours of HCFC-141b for 104 wk at target concentrations of 0 (control), 1500, 5000 and 20,000 ppm (increased from 15,000 ppm after 17 wk of exposure). No exposure-related effects of toxicological significance were noted with respect to survival, clinical signs, ophthalmoscopy, haematology, clinical chemistry, urinalysis or organ weight analysis. Reduced food intake and body weight gain were noted in both sexes of the 15,000 ppm group during the first 16 wk; thereafter, body weight gains in all groups were similar although the intergroup differences in body weight remained evident. Reduced food intake persisted in both sexes through wk 52 and in females during the second year of exposure. Treatment-related effects on macroscopic pathology were confined to increased incidences of testicular masses and altered appearance. Microscopic pathology examinations confirmed the testes as the target organ with findings of increased incidences of benign interstitial cell tumours and hyperplasia at 5000 and 20,000 ppm. The no-observable-adverse-effect level (NOAEL) was 1500 ppm. The testicular changes at high exposure levels were considered to be due to a change of the senile hormonal imbalance in geriatric rats and of little significance for the assessment of human health effects.

Mammalian genotoxicity assessment of methylene blue in plasma: implications for virus inactivation

The risk of adverse consequences of virus-inactivation procedures for plasma and cellular blood components must be less than the risk of transfusion-associated viral disease. Previous studies demonstrated that methylene blue, which is currently used in Europe for virus inactivation in fresh-frozen plasma, can elicit mutations in bacterial test systems. This study investigates the potential for methylene blue genotoxicity in two mammalian test systems. Different concentrations of methylene blue were prepared in plasma (heat-treated at 56 degrees C for 1 hour to reduce cytotoxicity) and used, without illumination, in an in vitro mouse lymphoma cell assay designed to detect forward mutations in the gene encoding thymidine kinase. The assay was performed in the presence or absence of rat liver S9 microsomal fraction. Similarly prepared samples of methylene blue in heat-treated plasma were used in an in vivo mouse micronucleus assay. Each system included a negative vehicle control (heat-treated plasma without methylene blue) and a positive control consisting of a known genotoxic agent. Intravenous administration to mice of 62 mg per kg of methylene blue did not increase the frequency of micronuclei in polychromatic red cells harvested from bone marrow. However, methylene blue concentrations of 10 micrograms per mL (with S9 activation) and 30 micrograms per mL (without S9 activation) significantly increased the thymidine kinase mutation frequency of mouse lymphoma cells to approximately 110 x 10(-6), from a spontaneous frequency of 28 x 10(-6). Methylene blue is mutagenic in cultured mammalian cells. In contrast, results from the mouse micronucleus assay suggest that the genotoxicity is not expressed in vivo. Considerably more investigation will be required to assess the genotoxic potential of intravenously administered methylene blue used in virus-inactivation procedures, because of the likelihood of the formation of methylene blue photoproducts or the impact of metabolic conversion of methylene blue to leukomethylene blue in vivo.

Suppressing effect of WR-2721 on micronuclei induced by cyclophosphamide in mice.

The effect of WR-2721 against cyclophosphamide-induced clastogenicity was studied using the in vivo micronucleus assay. The frequency of micronuclei in polychromatic erythrocytes in the peripheral blood of mice treated with WR-2721 and cyclophosphamide (CP), each of the compounds at a dose of 200 mg/kg body weight, was evaluated during the 15-day period. The suppressing effect of WR-2721, given 30 min prior to cyclophosphamide administration, on micronuclei induced by the alkylating agent was demonstrated. The number of micronuclei was increased on day 1 after CP application and declined thereafter with the frequency of micronucleated polychromatic erythrocytes remaining lower in WR-2721 pre-treated mice. The modulatory effect of WR-2721 on the clastogenic activity of cyclophosphamide in the erythropoietic system by the mouse micronucleus test was shown.

Mutagenicity studies of iodixanol, a new non-ionic isotonic contrast medium

A new non-ionic isotonic contrast medium, iodixanol (a diastereomeric mixture of 5,5'-[(2-hydroxytrimethylene) bis (acetylimino)] bis [N, N'-bis (2,3-dihydroxypropyl)-2,4,6-triiodo-1,3-benzene-dicarboxamide]) was studied for mutagenicity by using the Ames method, in vitro cytogenetics and micronucleus test. Iodixanol had no mutagenic effect on S. typhimurium (TA1535, TA1537, TA1538, TA98 and TA100) or E. coli (WP2uvrA) in the reverse mutation assay with or without metabolic activations. In the cytogenetic study, iodixanol had no effect on the Chinese hamster cells with or without metabolic activations. Intravenous injections of iodixanol for two times at a dose of 800, 1,600 or 3,200 mgI/kg in the mouse micronucleus test did not increase the incidence of micronucleated polychromatic erythrocytes. These results show that iodixanol has no mutagenic potential.

Radiation-induced point mutations, deletions and micronuclei in lacI transgenic mice

Ionizing radiation induces gene mutations (point mutations, deletions and insertions) as well as chromosome damage in mammalian cells. Although these effects have been studied extensively in cells in culture, until recently it has not been possible to analyze the mutagenic potential of ionizing radiation in vivo, especially at the molecular level. The development of transgenic mutagenesis has now made it possible to study the effects of ionizing radiation at both the molecular and chromosomal levels in the same animal. In this report we present preliminary data on the response of Big Blue TM lacI transgenic mice to ionizing radiation as measured by lacI mutations and micronuclei: C57B1/6 transgenic mice were irradiated with 137Cs γ-rays at doses ranging from 0.1 to 14 Gy, and expression times ranging from 2 to 14 days. Dose-related increases in the mutant frequency were observed after irradiations with longer expression times. Mutant plaques were analyzed by restriction enzyme digestion to detect large structural changes in the target sequence. Of 34 γ-ray-induced mutations analyzed, 4 were large-scale rearrangements. 3 of these rearrangements were deletions within the lacI gene characterized by the presence of short regions of homology at the breakpoint junctions. The fourth rearrangement was a deletion that extended from within the αlacZ gene into downstream sequences and that had 43 bp of homology at the junction. These data indicate that the Big Blue TM lacI transgenic mouse system is sensitive to the types of mutations induced by ionizing radiation. To determine whether the presence of the transgene effects micronucleus induction we compared the response of nontransgenic to hemizygous transgenic B6C3F1 mice and the response of nontransgenic to hemizygous and homozygous transgenic C57B1/6 mice. The presence or absence of the lacI transgene had no effect on spontaneous micronucleus frequencies for either strain. However, radiation-induced micronucleus frequencies were significantly higher in hemizygous lacI B6C3F1 mice than in nontransgenic litter mates; the converse was true in C57B1/6 mice. These data suggest that the lacI transgene does not cause chromosome instability as measured by spontaneous micronucleus levels. However, the response of these transgenic mice to a variety of clastogenic agents needs to be investigated before they are integrated into standard in vivo assays for chromosome damage.

Radioprotective effect of stobadine in the mouse micronucleus test

Induction of micronuclei was studied 40 h post irradiation in peripheral blood reticulocytes of male mice treated or not with stobadine dipalmitate (70.07 mg/kg body weight) at two time intervals (2 h or 1 h) prior to and immediately after 6.5 Gy 60Co exposure. A significant decrease of micronucleated reticulocytes was observed in the group of mice pretreated 2 h ( P < 0.05) or 1 h ( P < 0.01) before irradiation. 60Co irradiation followed by treatment with stobadine did not lead to the same protective effect in the micronucleus assay. It is therefore assumed that a radical-scavenging mechanism must be involved in radioprotection by stobadine.

Clastogenicity of trophosphamide in somatic and germinal cells of mice

Trophosphamide, a chemotherapeutic agent structurally related to cyclophosphamide, was tested in the micronucleus and heritable translocation assays in mice. It induced a linear increase of micronuclei in polychromatic erythrocytes 24 h after treatment with 1, 5, 25 or 50 mg/kg. In spermatids and spermatozoa of mice heritable translocations were induced by 150 mg/kg with an average frequency of 6%. The doubling doses calculated for micronucleus induction and heritable translocation induction were 5.0 and 1.3 mg/kg, respectively. These values are in the same order of magnitude and suggest that somatic and germinal cells are similarly sensitive to the clastogenic action of trophosphamide.

Stobadine-inhibitor of cyclophosphamide-induced micronuclei in mice.

The potential antimutagenic effect of stobadine dipalmitate (STB) on the frequency of micronuclei in reticulocytes of peripheral blood in female ICR mice was studied. The cyclophosphamide model was used to verify this effect. Stobadine dipalmitate was administered orally in three concentrations: STB I, 7.07; STB II, 23.6; STB III, 70.07 mg/kg body wt 2 h prior to or 4 h after (STB II only) cyclophosphamide administration (intraperitoneally, twice 80 mg/kg body wt with a 24 h interval). The method designed by Hayashi et al. [(1990) Mutat. Res., 245, 245-249] was used to prepare and to stain the slides. The results of the experiment show that pretreatment with stobadine 2 h prior to cyclophosphamide administration significantly decreased its mutagenic effect, as manifested by the reduced frequency of micronucleated reticulocytes. This protective effect of stobadine was concentration-dependent with the highest concentration of stobadine inducing the most pronounced decrease of micronuclei. Analysis and identification of the exact mechanism of the protective effects of stobadine is the aim of our further studies.

Comparative mouse micronucleus evaluation in bone marrow and spleen using immunofluorescence and Wright's Giemsa

Bone marrow and spleen toxicity, clastogenicity and aneugenicity were analyzed in the CD 1 mouse using an antikinetochore antibody (AKA) procedure (Krishna et al., Mutation Res., 282, 159–169, 1992). Further, to verify the fluorescence micronucleus (MN) analysis, additional slides were stained with Wright's Giemsa and results were compared. 5 mice per sex were treated with cyclophosphamide (CP) (40 mg/kg) or vincristine (VC) (0.1 or 0.2 mg/kg). Slides were prepared 24 h postdose using a column fractionation procedure. Per animal, 400 total erythrocytes (TEs) for toxicity and 2000 polychromatic erythrocytes (PCEs) for MN per tissue were analyzed. In the fluorescent method, the clastogen, CP, produced MNPCEs predominantly devoid of kinetochores (K) and the aneugen, VC, produced mostly MNPCEs containing K. The MNP CE frequency did not differ significantly between tissues; however, it differed statistically between sexes. On an overall basis, spleen had significantly lower PCE to TE ratios compared to bone marrow. In general, CP and VC caused a small, but statistically significant decrease in PCE frequencies compared to controls, suggesting possible toxicity to these tissues at the given doses. The data on Wright's stain indicated that the proportion of PCEs and MNPCEs in general, were comparable to those using fluorescent stain. This study further confirms the usefulness of an AKA-staining technique in a multiple genetic endpoint evaluation under a single set of microscope conditions.

The effect of combinations of clastogens in the mouse micronucleus assay

The effect of combinations of clastogens in the mouse micronucleus assay

Positive responses in the MS mouse micronucleus assay of otherwise quasi-positive or inconclusive chemicals: effectiveness of MS/Ae mice in screening tests

Positive responses in the MS mouse micronucleus assay of otherwise quasi-positive or inconclusive chemicals: effectiveness of MS/Ae mice in screening tests

The mouse micronucleus test of five possible human carcinogens (ethylene thiourea (ETU), methylthiouracil (MTU), metronidazole (MTZ), 2-nitropropane (2NP) and sodium phenobarbital (PB))

The mouse micronucleus test of five possible human carcinogens (ethylene thiourea (ETU), methylthiouracil (MTU), metronidazole (MTZ), 2-nitropropane (2NP) and sodium phenobarbital (PB))

Evaluation of the mouse micronucleus assay as a screen for carcinogens: summary report of the 6th collaborative study by CSGMT/JEMS·MMS

Evaluation of the mouse micronucleus assay as a screen for carcinogens: summary report of the 6th collaborative study by CSGMT/JEMS·MMS

The mouse micronucleus test with 2-acetylaminofluorene: Sex difference and strain difference

The mouse micronucleus test with 2-acetylaminofluorene: Sex difference and strain difference

Evaluation of the genotoxic potential of zinc pyrithione in the Salmonella mutagenicity (Ames) assay, CHO/HGPRT gene mutation assay and mouse micronucleus assay.

The mutagenic potential of zinc pyrithione (Znpt) was evaluated in vitro in the Salmonella/mammalian microsome plate incorporation mutagenicity (Ames) assay and the CHO/HGPRT gene mutation assay. The clastogenic potential of Znpt was evaluated in vivo using the mouse micronucleus test. Znpt was negative in the Ames test in five tester strains in the presence and absence of rat liver microsomal enzymes when assayed at concentrations ranging between 10 and 333 micrograms per plate and between 0.03 and 33 micrograms per plate, respectively. Znpt also produced negative results in the CHO/HGPRT assay. No significant increases in mutant frequencies were seen in the presence and absence of rat liver microsomal enzymes. In each case, the highest concentrations reduced cellular viability by 83% and 85%, respectively. Znpt also did not induce increased frequencies of micronuclei in mouse bone marrow cells when tested at the maximally tolerated dose (MTD) (44 mg kg-1). These data support the conclusion that Znpt lacks genotoxic activity under the conditions of these tests.

Effect of ascorbic acid in the detoxification of the insecticide dimethoate in the bone marrow erythrocytes of mice

The antimutagenic potential of ascorbic acid in the detoxification of the organophosphorus insecticide dimethoate was evaluated in female Swiss albino mice using the in vivo bone marrow micronucleus test. Groups of three mice were treated with distilled water (control), 1% dimethyl sulphoxide (solvent control), or 86, 129.5 or 259 mg ascorbic acid/kg body weight, with or without the concurrent administration of 150 mg dimethoate/kg. There was a statistically significant increase in the frequency of micronuclei in dimethoate-treated mice. However, in mice that were given dimethoate and ascorbic acid simultaneously, the numbers of micronuclei did not differ significantly from control values, thus indicating the protective role of ascorbic acid.

Mouse micronucleus assays of sporidesmin, the toxin associated with facial eczema in ruminants

Sporidesmin, a fungal toxin with widespread distribution within New Zealand, was previously shown to be a potent clastogen in Chinese hamster cells in vitro, but not in peripheral blood lymphocytes of sheep in vivo. In mice, massive oral doses led only to slight increases in micronucleus levels in the bone marrow, despite highly significant changes to other toxicological parameters. It would appear that the intact animal is protected in some fashion from the clastogenic effects of sporidesmin, although this substance must still be considered a potential human genotoxic agent.

Radioprotective efficiency of a combination of hydroxytryptophan and AET on mouse bone marrow micronuclei.

Radioprotective effectiveness of a combination of HT and AET was evaluated by radiation induced micronucleus screening in mouse bone marrow. Swiss albino mice were injected with 2-Aminoethylisothiouronium bromide hydrobromide (AET)+5-hydroxy-L-tryptophan (HT) 30 minutes prior to a single whole body exposure of 8 and 12 Gy Co60 gamma rays. Animals were sacrificed at different intervals viz., 24 hours, 40 hours, 72 hours and 7 days after irradiation. Slides of femur marrow were screened thoroughly for cells with micronuclei. Marked decreases in the frequency of micronuclei in irradiated mice receiving radioprotector suggested that the combination has rendered protection to bone marrow cells against 8 Gy and 12 Gy whole body gamma irradiation. Micronucleated cells count is not altered by administration of the combination alone.