The micronucleus test with mouse spleen cells

The results of this study show that the micronucleus test can be carried out with mouse spleen cells as well as with cells from bone marrow. Polychromatic erythrocytes occurred in the spleen at a frequency of about 9% of the whole spleen cells compared with about 13% in the bone marrow. 3 test compounds were used to compare the frequency of micronuclei in cells from the 2 tissues. Mitomycin C and cyclophosphamide induced micronucleated polychromatic erythrocytes in both spleen and bone marrow. Fosfomycin, an antibiotic having a broad spectrum of antimicrobial activities, did not induce micronucleated erythrocytes in either organ.     

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     

Cytogenetic effect of the thiocarbamate herbicides butylate, molinate and vernolate in the mouse bone marrow micronucleus test

Three thiocarbamate herbicides, butylate (S-ethyl-diisobutylthiocarbamate), vernolate (S-propyl dipropylthiocarbamate) and molinate (S-ethyl-N,N-hexamethylenethiocarbamate) were assayed for cytogenetic effect in the mouse bone marrow micronucleus test. Butylate was inactive in bone marrow, vernolate caused a marginal increase in the incidence of micronucleated polychromatic erythrocytes only at a high toxic dose level. Molinate, the N,N-hexamethylene derivative was, however, strongly active in the bone marrow, causing a high frequency of micronucleated erythrocytes, even at subtoxic concentrations.     

Clastogenic activity of urethane in mice.

Single intraperitoneal (i.p.) treatment of male and female BDF1 (C57B1 x DBA2) mice with urethane (0.5 or 1.0 g/kg) caused a significant increase in micronucleated polychromatic erythrocytes (MNPCE) in bone marrow after 24 h. The clastogenic effect observed was dose-, sex- and age-dependent, the male and younger (6-8 weeks old) animals being more susceptible than the female and older (6 months of age) mice. 3-week oral treatment of female Balb/c mice with urethane (3 g/l added to the drinking water) caused an up to 4-fold increase in the number of micronucleated normochromatic erythrocytes (MNNCE) in mouse peripheral blood. In a month after the carcinogen treatment was stopped, the number of MNNCE dropped to the control values. In addition, a single i.p. treatment of pregnant BDF1 mice on day 17 of gestation with urethane (1.0 g/kg) caused a 514.3% (p less than 0.001) elevation of MNPCE in mouse fetal liver after 24 h as well as a 154.4% (p less than 0.05) increase in MNPCE frequency in the fetal peripheral blood. At this time point, the clastogenic response in mouse fetal liver erythroblasts was less pronounced than that detected in the maternal bone marrow cells. Urethane is a strong clastogen in mice when administered either intraperitoneally or orally and the micronucleus test applied to adult and fetal erythroblasts is a convenient method of choice for studying the acute and subchronic clastogenicity of this carcinogen, its transplacental effects as well as the influence of modifying factors on these processes.     

Transplacental mutagenesis: The micronucleus test on fetal mouse blood

Summary The induction of cytogenetic damage (micronuclei) in mouse fetal blood was studied with four selected mutagens: cyclophosphamide, procarbazine, trenimon, and mitomycin-C. For comparison the standard micronucleus test on maternal bone marrow was also performed. In contrast to the results obtained from maternal bone marrow the changes in the cellular composition in fetal blood were only slight after treatment with mutagens. A significant and dosepdependent increase in the incidence of micronucleated fetal blood cells was found with all four mutagens. The inducibility of micronuclei by indirect mutagens was particularly interesting. The three mutagens other than mitomycin-C induced a higher frequency of micronucleated polychromatic erythrocytes in fetal blood cells than in maternal bone marrow. The results indicate that this modified micronucleus test is well suited and useful for mutagenicity screening of environmental chemicals and especially for assessment of risks to the fetus when pregnant females are exposed to environmental chemicals.Supported by the Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg     

The micronucleus test and erythropoiesis: effects of cyclic adenosine monophosphate (cAMP) on micronucleus formation

The aim of this study was to determine the mechanism of the rodent bone marrow micronucleus test in relation to erythropoiesis. We have previously reported that an acceleration of erythropoiesis increases the frequency of micronucleated polychromatic erythrocytes (MPCE) induced by mutagens. The blood plasma erythropoietin level increased after the injection of N6-2-O-dibutyladenosine-3',5'-cyclic monophosphate into adenosine 3',5'-cyclic monophosphate (cAMP) at a dose of 500 mg/kg. A peak of erythropoietin induction was observed 3 h after the injection of cAMP. cAMP itself did not induce any micronuclei in erythroblasts of BALB/c mice. So, the frequency of MPCE did not increase after injection of cAMP. The highest frequency of MPCE and the dose-response relationship between the cAMP doses and micronucleus frequency were observed 30 h after injection of mitomycin C (MMC) in mice which had been administered cAMP 24 h previously. The highest effect of cAMP on the increase of MPCE was observed when cAMP was given 24 h before MMC injection, thus indicating that accelerating the multiplication of erythroblasts increases the frequency of MPCE induced by mutagens. The induction of MPCE in the bone marrow by three other chemicals (carboquone, 5-fluorouracil, and vincristine) also increased after pretreatment with cAMP. Our results suggest that the increase of MPCE induced by mutagens can be amplified following the acceleration of erythropoiesis by pretreatment with cAMP.     

Clastogenic activity of urethane in mice

Single intraperitoneal (i.p.) treatment of male and female BDF1 (C57B1 × DBA2) mice with urethane (0.5 or 1.0 g/kg) caused a significant increase in micronucleated polychromatic erythrocytes (MNPCE) in bone marrow after 24 h. The clastogenic effect observed was dose-, sex- and age-dependent, the male and younger (6–8 weeks old) animals being more susceptible than the female and older (6 months of age) mice. 3-week oral treatment of female Balb/c mice with urethane (3 g/l added to the drinking water) caused an up to 4-fold increase in the number of micronucleated normochromatic erythrocytes (MNNCE) in mouse peripheral blood. In a month after the carcinogen treatment was stopped, the number of MNNCE dropped to the control values. In addition, a single i.p. treatment of pregnant BDF1 mice on day 17 of gestation with urethane (1.0 g/kg) caused a 514.3% (p < 0.001) elevation of MNPCE in mouse fetal liver after 24 h as well as a 154.4% (p < 0.05) increase in MNPCE frequency in the fetal peripheral blood. At this time point, the clastogenic response in mouse fetal liver erythroblasts was less pronounced than that detected in the maternal bone marrow cells. Urethane is a strong clastogen in mice when administered either intraperitoneally or orally and the micronucleus test applied to adult and fetal erythroblasts is a convenient method of choice for studying the acute and subchronic clastogenicity of this carcinogen, its transplacental effects as well as the influence of modifying factors on these processes.     

Fetal liver micronucleus assay in mice of 5-fluorouracil and related compounds

The cytogenetic effects of 5-fluorouracil (5-FU), 1-hexyl-carbamoyl-5-fluorouracil (HCFU) and 1-(2-tetrahydrofuryl)-5-fluorouracil (TF) were examined with the fetal liver micronucleus assay in mice. The frequencies of micronucleated polychromatic erythrocytes (MNPCEs) in fetal liver peaked at 27, 24 and 27 h, respectively, after single intraperitonealinjections into pregnant mice on day 13 of gestation. The highest frequency of MNPCEs by 5-FU treatment in fetal liver was 13.6%, whereas the frequency in maternal bone marrow was only 0.4%. The micronucleus frequency and the number of micronuclei per individual polychromatic erythrocyte were clearly dose-dependent.These results suggest that the micronucleus test in fetal liver has particular advantages compared to maternal bone marrow for evaluating the cytogenetic effects of 5-FU and related compounds after a single treatment. The cytogenetic effect was ranked 5-FU = HCFU > TF, in both a time-course study and a dose-response study of micronucleus distribution.     

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.     

4CMB: Investigation of activity in a mouse micronucleus test

To study possible clastogenic effects of 4-chloromethylbiphenyl (4CMB), a mouse micronucleus test was performed (as part of the UKEMS genetic toxicology trial). Mice were given 4CMB by intraperitoneal injection, once daily for 5 consecutive days. Although the range of dosages employed included highly toxic or lethal doses, the frequency of micronucleated polychromatic erythrocytes in the bone marrow was not significantly greater in 4CMB-treated groups than in control-group animals treated with the Tween 80 vehicle alone. Thus, in this test, 4CMB did not induce chromosomal damage resulting in micronucleus formation, in bone-marrow erythrocytes.     

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.     

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.     

Induction of micronuclei by vinyl acetate in mouse bone marrow cells and cultured human lymphocytes

A dose-dependent increase in micronucleated polychromatic erythrocytes was observed in the bone marrow of male C57B1/6 mice 30 h after a single intraperitoneal injection of vinyl acetate (250, 500, 1000 or 2000 mg/kg b.wt.; (9-14 animals per group). The effect was statistically significant at 1000 mg/kg (1.33 +/- 0.29% vs. 0.6 +/- 0.10% in olive oil-treated controls) and at 2000 mg/kg (1.57 +/- 0.19%) of vinyl acetate. These doses were fatal to 6 (1000 mg/kg) and 8 (2000 mg/kg) out of 14 animals in both groups. The ratio of polychromatic to normochromatic cells decreased as a function of vinyl acetate dose. Cyclophosphamide (20 mg/kg), used as a positive control chemical, induced a clear increase in micronucleated polychromatic erythrocytes (2.07 +/- 0.20%). None of the treatments affected the number of micronuclei in normochromatic erythrocytes. In human whole-blood lymphocyte cultures, micronucleus induction by a 48-h treatment with vinyl acetate (0.125, 0.25, 0.5, 1 and 2 mM; 24 h after culture initiation) was studied in lymphocytes with preserved cytoplasm from smear slides prepared by a method involving the removal of erythrocytes at harvest by sodium cyanide treatment to improve preparation quality. The frequency of micronucleated lymphocytes reached a peak at 0.5 mM (3.2 +/- 1.0% vs. 0.9 +/- 0.1% in control cultures) and 1 mM (3.1 +/- 0.7%), with a decline at 2 mM probably because of a toxic effect resulting in mitotic inhibition.     

Development of a method to increase the proportion of polychromatic erythrocytes from mouse bone marrow for more rapid evaluation of the micronucleus assay. Comparison with the conventional method with respect to micronucleus frequency and time required for preparation and evaluation

By treatment of bone marrow suspension in hypotonic (0.51%) saline, followed by purification on a cellulose column, the proportion of polychromatic erythrocytes in bone marrow smears can be increased 2.6-fold. Furthermore, practically all nucleated cells of erythropoietic or leukopoietic origin are removed. This kind of bone marrow preparation can lead to a considerable reduction in the time required for microscopical evaluation of the micronucleus assay without altering the rate of micronucleated polychromatic erythrocytes in a negative (isotonic saline) and a positive (mitomycin C) control group.     

Development of a method to increase the proportion of polychromatic erythrocytes from mouse bone marrow for more rapid evaluation of the micronucleus assay: Comparison with the conventional method with respect to micronucleus frequency and time required for preparation and evaluation

By treatment of bone marrow suspension in hypotonic (0.51%) saline, followed by purification on a cellulose column, the proportion of polychromatic erythrocytes in bone marrow smears can be increased 2.6-fold. Furthermore, practically all nucleated cells of erythropoietic or leukopoietic origin are removed. This kind of bone marrow preparation can lead to a considerable reduction in the time required for microscopical evaluation of the micronucleus assay without altering the rate of micronucleated polychromatic erythrocytes in a negative (isotonic saline) and a positive (mitomycin C) control group.     

Prior bleeding enhances the sensitivity of the in vivo micronucleus test

It has been reported that the sensitivity of the in vivo mouse bone marrow micronucleus test can be increased by inducing erythropoiesis with exogenous erythropoietin prior to treatment (Suzuki et al., 1989). In these studies we demonstrate that removing approximately 0.5 ml of blood from an adult male BDF1 mouse, another method for increasing the rate of erythropoiesis, synergistically increased the frequency of bone marrow micronucleated polychromatic erythrocytes induced by mitomycin C, with maximal enhancement occurring when the mutagen was given 24 h after bleeding. This enhancement response was also demonstrated for benzo[a]pyrene and dimethylnitrosamine but not for 2-acetylaminofluorene. These results indicate that bleeding mice prior to chemical treatment is a simple method for increasing the sensitivity of the micronucleus assay.     

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.     

1-Methyl-2-pyrrolidinone (NMP) does not induce structural and numerical chromosomal aberrations in vivo

1-Methyl-2-pyrrolidinone induces aneuploidy in yeast, but only under special treatment conditions. Other genotoxic effects have not been found in vitro, and in vivo no data are available in the literature. Therefore, NMP was investigated in the mouse micronucleus test and the Chinese hamster bone marrow test for structural and numerical chromosomal aberrations. These tests can detect both types of alterations as demonstrated by appropriate positive control substances (cyclophosphamide, vincristine sulfate and benomyl). NMP at single oral doses up to 3800 mg/kg body weight (∼ 80% of the LD₅₀) did not lead to an increase either in micronucleated erythrocytes or in structural or numerical chromosomal aberrations when bone marrow was sampled 16, 24 and 48 h after treatment in the micronucleus test or after 24 and 48 h for karyotype analysis.     

微核直径测试作为非整倍体诱发剂的分析手段

以小鼠骨髓红细胞微核直径测试,比较了秋水仙素（COL）与昆明山海棠（THH）、对苯二酚（HQ）在哺乳动物体细胞中的非整倍体诱发效应。丝裂霉素C（MMC）作为多功能染色体断裂剂引入实验,为诱发非整倍体的阴性对照。结果发现,COL组,71%的微核直径（d）大于或等于所在细胞直径（D）的五分之一（d≥D/5）;THH诱发微核中,54%的微核d≥D/5;HQ及MMC组,分别有47%及14%的微核相对直径达此阈值。暗示THH及HQ具有类似COL的某种非整倍体诱发效应。微核直径测试可作为非整倍体诱发剂检测的辅助手段。 The relative diameters of micronucleus induced by colchicines(COL),Triptergium hypoglaucum(Level)Hutch(THH)and hydroquinone(HQ)were compared to evaluate their aneugenic activities in mouse bone marrow erythrocytes.MitomycinC(MMC)was taken as the negative control in the experiment because it is a multifunctional clastogen without aneugenic potential.Diameters of the cytoplasm(D)and the micronucleus(d)of each micronucleated erythrocytes were measured with a micrometer in a microscope.The frequency of relatively large micronuclei(d≥D/5)was found(71%) in COL treated group.In the THH and HQ treated groups,the relatively large micronuclei were 54% and 47%,respectively.Such micronuclei were infrequent(14%)in the MMC treated group.The results implied that THH and HQ may possess some aneugenic potential like COL.