Density-gradient enrichment of newly-formed mouse erythrocytes: Application to the micronucleus test

To facilitate scoring micronuclei in peripheral blood erythrocytes, we have developed a centrifugation method to concentrate polychromatic and newly-formed normochromatic erythrocytes from microliter quantities of blood in a Percoll density gradient. Erythrocytes were separated into two discrete bands in a continuous gradient generated in situ in a microhematocrit capillary tube. The upper band contained white blood cells and a mixture of polychromatic and young normochromatic erythrocytes with a density of 1.080–1.082 g/ml. More than 75% of the polychromatic erythrocytes in samples of normal blood were recovered in the upper band. Older normochromatic erythrocytes migrated to the lower band. The frequency of polychromatic erythrocytes was increased from approximately 2% in whole blood to 60–80% in the upper band. After clastogen treatments, the elevated frequencies of micronuclei in the upper band polychromatic erythrocytes were similar to those in unfractionated blood. The frequencies of micronucleated normochromatic erythrocytes in the upper band were higher than those in whole blood at 48, 72 and 96 h after clastogen treatment, consistent with the expectation that the low-density normochromatic cells are newly derived from polychromatic erythrocytes. This density-gradient centrifugation technique enhances the efficiency of scoring micronuclei in the acute peripheral blood micronucleus test.     

Density-gradient enrichment of newly-formed mouse erythrocytes. Application to the micronucleus test

To facilitate scoring micronuclei in peripheral blood erythrocytes, we have developed a centrifugation method to concentrate polychromatic and newly-formed normochromatic erythrocytes from microliter quantities of blood in a Percoll density gradient. Erythrocytes were separated into two discrete bands in a continuous gradient generated in situ in a microhematocrit capillary tube. The upper band contained white blood cells and a mixture of polychromatic and young normochromatic erythrocytes with a density of 1.080-1.082 g/ml. More than 75% of the polychromatic erythrocytes in samples of normal blood were recovered in the upper band. Older normochromatic erythrocytes migrated to the lower band. The frequency of polychromatic erythrocytes was increased from approximately 2% in whole blood to 60-80% in the upper band. After clastogen treatments, the elevated frequencies of micronuclei in the upper band polychromatic erythrocytes were similar to those in unfractionated blood. The frequencies of micronucleated normochromatic erythrocytes in the upper band were higher than those in whole blood at 48, 72 and 96 h after clastogen treatment, consistent with the expectation that the low-density normochromatic cells are newly derived from polychromatic erythrocytes. This density-gradient centrifugation technique enhances the efficiency of scoring micronuclei in the acute peripheral blood micronucleus test.     

Induction of micronuclei by CsCl in vivo and in vitro

In the present study, we report the results of an investigation of the potential of nonradioactive CsCl for the induction of micronuclei in polychromatic erythrocytes of mouse bone marrow and in human lymphocytes cultured and blocked with cytochalasin-B. No significant increase in micronucleus frequency was observed in the polychromatic erythrocytes of mice which received 500 mg/kg of CsCl. In vitro experiments with human lymphocytes cultured in medium containing 250 and 500 μg/ml CsCl also showed no increase in micronucleus frequency compared to untreated controls. These same experiments, however, demonstrated a reduction in mitotic activity with increasing CsCl concentration in the culture medium. This report is the first to describe studies on the possible induction of micronuclei in vitro and in vivo by nonradioactive CsCl.     

Induction of micronuclei by CsCl in vivo and in vitro

In the present study, we report the results of an investigation of the potential of nonradioactive CsCl for the induction of micronuclei in polychromatic erythrocytes of mouse bone marrow and in human lymphocytes cultured and blocked with cytochalasin-B. No significant increase in micronucleus frequency was observed in the polychromatic erythrocytes of mice which received 500 mg/kg of CsCl. In vitro experiments with human lymphocytes cultured in medium containing 250 and 500 micrograms/ml CsCl also showed no increase in micronucleus frequency compared to untreated controls. These same experiments, however, demonstrated a reduction in mitotic activity with increasing CsCl concentration in the culture medium. This report is the first to describe studies on the possible induction of micronuclei in vitro and in vivo by nonradioactive CsCl.     

Cytogenetic effects of pesticides. III. Induction of micronuclei in mouse bone marrow by the insecticides cypermethrin and rotenone

The production of micronuclei in mouse bone marrow by the pyrethroid insecticide, cypermethrin and the botanical insecticide, rotenone was examined. Three routes of administration were used for the insecticides: intraperitoneal, oral and dermal. The different routes of treatment with cypermethrin and rotenone caused toxicity of marrow as indicated by a significant increase in the percentage of polychromatic erythrocytes (PEs) over that of the control. Cypermethrin showed mutagenic potential as evidenced by a positive response in the micronucleus assay. Oral administration of the insecticide at a dietary level of 900 ppm for 7 and 14 consecutive days as well as double and multiple (total 4) dermal treatments (360 mg/kg body wt.) induced a statistically significant increase in the frequency of PEs with micronuclei. The conducted intraperitoneal (i.p.) treatments with cypermethrin: single injection at 60 and 180 mg/kg body wt., double and multiple injections (total 3) at 60 mg/kg body wt. did not affect the percentage of PEs with micronuclei. The different treatments with rotenone: single, double and multiple (i.p.) injections (total 3) at 2 and 3 mg/kg body wt., oral administration for 14 consecutive days at dietary level of 225 ppm and multiple dermal treatments (total 4) with 135 mg/kg body wt. showed no effect on the frequency of micronuclei in PEs.     

Induction of micronuclei in rat bone marrow and peripheral blood following acute and subchronic administration of azathioprine

The frequency of micronuclei was assessed in polychromatic erythrocytes of bone marrow and in polychromatic and normochromatic erythrocytes in peripheral blood of rats following exposure to azathioprine for 28 days. This was compared with the incidence of micronuclei in bone-marrow following exposure to a single dose of azathioprine. The incidence of micronuclei in bone-marrow polychromatic erythrocytes at the maximum tolerated dose (10 mg/kg) following exposure for 28 days was 29.5%. The incidence of micronucleated polychromatic erythrocytes in the peripheral blood at this dose was 4.4%. At the maximum tolerated dose in the single-dose study (40 mg/kg) the incidence obtained at 48 h post-treatment was 15.7%. This supports the view that the use of animals in a subchronic toxicity study is at least as sensitive for assessing in vivo clastogenic activity as an acute study and could reduce animal usage in toxicology assessments.     

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.     

减毒麻疹活疫苗对小鼠的细胞遗传学效应

本文以C57BL/6J小鼠为材料,以骨髓细胞微核、染色体畸变和生殖细胞微核、染色体畸变为指标,对国产减毒麻疹活疫苗的遗传安全性进行评价。结果表明,麻疹疫苗可引起小鼠骨髓微核细胞率、染色体畸变率以及精细胞微核细胞率明显升高,与接种剂量和接种后的时间有关;生殖细胞染色体畸变和对照比无显著性差异。     

Cytogenetic effects of pesticides. I. Induction of micronuclei in mouse bone marrow by the insecticide Dursban

The induction of micronuclei in mouse bone marrow by the organophosphorous insecticide 'Dursban' was tested. 3 routes of administration were used for the pure insecticide: intraperitoneal, oral and dermal. The different routes of treatment with Dursban induced a statistically significant increase in the percentage of polychromatic erythrocytes over that of the control. Both intraperitoneal and oral treatments with the insecticide induced a high percentage of polychromatic erythrocytes with micronuclei, whereas dermal treatment did not induce micronuclei.     

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.     

Relative efficacy of short-term tests in detecting genotoxic effects of cadmium chloride in mice in vivo

The ability of intraperitoneally administered cadmium chloride (0.42-6.75 mg/kg) to induce genotoxic damage in somatic and germ cells of mice was evaluated using chromosomal aberrations, sister-chromatid exchanges (SCE), micronuclei and sperm-head abnormalities as end-points. A significant increase in the frequency of chromosomal aberrations and SCEs was observed in almost all treated series when compared to the negative control. Micronucleus formation in polychromatic erythrocytes was not affected significantly except at the highest concentration used (6.75 mg/kg). Significant differences were observed in the frequency of sperm with abnormal head morphology at all concentrations tested except the lowest one. The clastogenic effects of cadmium chloride in both somatic and germinal cells are found to depend directly on the concentrations used.     

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.     

A correlative study on the genetic damage induced by chemical mutagens in bone marrow and spermatogonia of mice : I. CNU-ethanol

Cytogenetic damage induced by a wide range of concentrations of CNU-ethanol in mice was evaluated by determining the frequencies of (a) micronuclei nuclei in polychromatic erythrocytes of the bone marrow, (b) chromatid aberrations in bone marrow, (c) chromatid aberrations in spermatogonia, and (d) reciprocal translocations induced in spermatogonia and scored in spermatocytes.For CNU-ethanol the following order of sensitivity was found between the tests performed: micronuclei > aberrations in bone marrow > aberrations in spermatogonia > translocations in spermatocytes.Correlation coefficients were calculated for the first three parameters. Positive correlations existed (a) between micronuclei in polychromatic erythrocytes and chromatid aberrations in bone marrow on the first day after treatment, and (b) between chromatid aberrations in bone marrow and spermatogonia at the first day after treatment.Three reciprocal translocations were induced in spermatogonia and recorded in primary spermatocytes; all were of a rare type, namely between an X chromosome and an autosome.     

Suppressing effects of vanillin,cinnamaldehyde, and anisaldehyde on chromosome aberrations induced by X-rays in mice

X-ray-induced chromosome aberrations were suppressed when vanillin, cinnamaldehyde, or p-anisaldehyde was given orally to mice after X-ray irradiation. Chromosome aberrations were monitored by the occurrence of polychromatic erythrocytes with micronuclei in bone marrow cells. The frequency of micronuclei was depressed about 55–60% without toxicity of the test compounds to the bone marrow.     

Chemoprotective effects of carnosine against genotoxicity induced by cyclophosphamide in mice bone marrow cells

The protective effects of carnosine as a natural dipeptide were investigated in mouse bone marrow cells against genotoxicity induced by cyclophosphamide. Mice were injected with solutions of carnosine at three different doses (10, 50 and 100?mg kg(-1) bw) for five consecutive days. On the fifth day of treatment, mice were injected cyclophosphamide and killed after 24?h. The frequency of micronuclei in polychromatic erythrocytes and the ratio of polychromatic erythrocyte/polychromatic erythrocyte?+?normochromatic erythrocyte [PCE/(PCE?+?NCE)] were evaluated by May-Grunwald/Giemsa staining. Histopathology of bone marrow was examined in mice treated with cyclophosphamide and carnosine. Carnosine significantly reduced micronucleated polychromatic erythrocytes (MnPCEs) induced by cyclophosphamide at all three doses. Carnosine at dose of 100?mg kg(-1) bw reduced MnPCEs 3.76-fold and completely normalized the PCE/(PCE?+?NCE) ratio. Administration of carnosine inhibited bone marrow toxicity induced by cyclophosphamide. It appeared that carnosine with protective activity reduced the oxidative stress and genotoxicity induced by cyclophosphamide in bone marrow cells of mice. Copyright ? 2012 John Wiley & Sons, Ltd.     

Micronucleus formation in cultured fetal liver blood cells using mitomycin C.

Mouse fetal-liver blood cells were cultured and used to investigate micronucleus formation after exposure to mitomycin C (MMC). The isolated fetal cells were incubated in a medium supplemented with erythropoietin (EPO), and the frequency of micronuclei formation was detected in polychromatic erythrocytes (PCE). The effects of four variables were investigated: (1) MMC exposure dose, (2) MMC exposure time, (3) incubation time, and (4) EPO concentration. PCE were formed by proliferation and differentiation of the erythroid cells in culture. Micronucleated PCE (MNPCE) were observed in a dose-dependent manner after exposing the cultured cells with up to 1.0 microgram/ml MMC. The optimum time of MMC exposure and post-exposure incubation was 3 h and 48 h, respectively, and the optimum EPO concentration was 0.25 U/ml. Mouse fetal-liver PCE are sensitive primordial cell targets that can be obtained in relatively large numbers from a single pregnant animal. The procedure is relatively simple and potentially useful in detecting mutagens and carcinogens capable of causing chromosomal damage.     

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.     

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.     

Induction of micronuclei by Zearalenone in Vero monkey kidney cells and in bone marrow cells of mice: protective effect of Vitamin E

Zearalenone (ZEN) is a non-steroidal estrogenic mycotoxin mainly produced by Fusarium graminaerum, found as a world-wide contaminant mainly of corn and wheat. Previous studies have demonstrated that among several other effects on animals and humans, ZEN also displays hepatotoxicity, immunotoxicity and nephrotoxicity. ZEN is mainly known as a hormonal disrupter due to its estrogenic activities and consequent toxicity for reproduction. Furthermore, mutagenic and genotoxic proprieties of ZEN were disclosed recently, the molecular mechanisms of which are not yet well understood. In the present study, the genotoxic potential of ZEN was evaluated using genotoxicity tests: the 'cytokinesis block micronucleus assay' in Vero monkey kidney cells and the 'in vivo mouse bone marrow micronucleus assay'. In cultured cells treated with 5, 10 and 20 microM ZEN, the frequency of binucleated micronucleated cells (BNMN) was assessed in 1000 binucleated cells and in mice given oral doses of 10, 20 and 40 mg/kg bw, the frequency of polychromatic erythrocytes micronucleated (PCEMN) in bone marrow cells was assessed in 2000 polychromatic erythrocytes (PCE). The potential prevention of ZEN-induced effects by 25 microM Vitamin E (Vit E) was also evaluated.In vivo, doses of 10, 20 and 40 mg/kg bw ZEN representing, respectively 2, 4 and 8% of the LD50 (LD50 of ZEN in mice is 500 mg/kg bw), were administered to animals either with or without pre-treatment with Vit E (216.6 mg/kg bw) in order to evaluate its preventive potential.ZEN was found to induce micronuclei (MN) in a dose-dependent manner in cultured Vero cells as well as in mouse bone marrow cells. The present data emphasise the likely clastogenic pathway among the molecular mechanisms that underlay the ZEN-induced genotoxicity. Vit E was found to prevent partially-from 30 to 50%-these toxic effects, most likely acting either as a structural analogue of ZEN or as an antioxidant.     

Administration-route-related differences in the micronucleus test with benzene

The effect of route of administration on the outcome of the micronucleus test was studied in 2 laboratories by administering the model chemical benzene intraperitoneally (i.p.) and orally (p.o.) to 2 strains of mice: MS/Ae and CD-1. On the basis of results obtained in a small-scale acute toxicity study and in a pilot micronucleus test, full-scale micronucleus tests were performed with a 24-h sampling time at doses of 250, 500, 1000, and 2000 mg/kg i.p. and 500, 1000, 2000, and 4000 mg/kg p.o. In both strains of mice, a higher incidence of micronucleated polychromatic erythrocytes (MNPCEs) was observed after p.o. administration. The ratio of polychromatic erythrocytes (PCEs) to total erythrocytes decreased more markedly at higher doses i.p. in both strains. Thus, benzene induced more micronuclei via the p.o. route, while inhibitory effects on bone marrow cells were stronger after i.p. administration.