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.     

Induction of micronuclei in mouse polychromatic erythrocytes by the administration of non-radioactive CsCl by the oral and intraperitoneal route

In the present study, we describe the effects of the concentration and route of administration of non-radioactive cesium chloride (CsCl) in inducing micronuclei in mouse bone marrow polychromatic erythrocytes (PCEs). When the dose of 500mg/kg body weight was administered perorally (p.o.), no significant incidence of micronuclei was detected. However, when the same dose was administered intraperitoneally (i.p.), a significant induction of micronuclei in PCEs was observed compared to control. At the dose of 1000mg/kg, both routes were efficient, with no significant difference in micronucleus frequencies. We conclude that both the p.o. and i.p. routes are efficient in inducing micronuclei, with the i.p. route being more efficient when lower CsCl doses are used.     

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.     

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.     

Assessment of the genotoxicity of imidacloprid and metalaxyl in cultured human lymphocytes and rat bone-marrow

Imidacloprid and metalaxyl are two pesticides that are widely used in agriculture, either separately, or in combination. These agents were studied for their possible genotoxic effects with respect to the following cytogenetic end-points: (1) in vitro micronucleus (MN) formation and sister-chromatid exchange (SCE) induction in human lymphocytes and (2) in vivo micronucleus induction in polychromatic erythrocytes (PCEs) of the rat bone-marrow. The results of the MN analysis indicate that MN frequencies after treatment with both pesticides, separately or as a mixture, do not significantly differ from those in the controls except after treatment with metalaxyl alone at 50 microg/ml (p<0.05). The results of the SCE analysis show that SCE frequencies after treatment with imidacloprid do not differ significantly from those in the controls. A statistically significant increase (p<0.05) in SCE frequency resulted from treatments with metalaxyl at 5, 10 and 100 microg/ml and with the combination of imidacloprid and metalaxyl at 100 and 200 microg/ml. Finally, the in vivo micronucleus assay with rat bone-marrow polychromatic erythrocytes showed a statistically significant effect upon separate treatments with imidacloprid and metalaxyl at doses of 300 mg/kg body weight (b.w.) (p<0.01) or upon combined treatment with 200 mg/Kg b.w. (p<0.001) and 400 mg/kg b.w. (p<0.05).     

Cypermethrin induction of micronuclei in mammalian somatic cells

The genotoxic effects of the preparative cypermethrin form on the induction of micronuclei in cultured Chinese hamster V-79 cells and polychromatic erythrocytes of mouse bone marrow have been studied. The cypermethrin has induced micronuclei in cultured cells without metabolic activation in toxic concentrations, similar effects being observed in polychromatic erythrocytes after treatment with subtoxic concentrations.     

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.     

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.     

Micronucleus formation in different lymphocyte subpopulations in peplomycin-treated and control cultures

Spontaneous micronucleus formation and micronucleus induction by peplomycin in B and T lymphocytes was studied by a recently developed MAC (Morphology, Antibody, Chromosomes) method allowing the immunologic identification of different cell lineages. Blood samples from 3 healthy donors were cultured in the presence of phytohaemagglutinin and pokeweed mitogen. An increased frequency of micronuclei was observed in peplomycin cultures compared with controls. B cells were found to be more sensitive to peplomycin induction than were T lymphocytes. In control cultures, pokeweed mitogen yielded a higher frequency of micronuclei than did phytohaemagglutinin. In both pokeweed- and phytohaemagglutinin-stimulated cultures, B cells showed a higher frequency of micronuclei than did T cells. The relative proportion of mitotic B cells was equal in pokeweed and phytohaemagglutinin cultures. In peplomycin cultures, the proportion of B cells decreased as compared with control cultures.     

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.     

Caffeic acid as an inhibitor of DMBA-induced chromosomal breakage in mice assessed by bone-marrow micronucleus test

Female mice of hybrid strain B6C3F1, 8-10 weeks old, were fed on powdered food with or without 2% caffeic acid. After one week on these diets, some of each group of mice were injected i.p., with 7,12-dimethyl benz[a]anthracene (25 mg/kg) dissolved in dimethyl disulfoxide. In the course of separate experiments, bone-marrow samples were taken at various intervals after injection for analysis in the micronucleus assay. From each mouse 500 polychromatic erythrocytes were scored to determine the frequency with micronuclei. At the time at which the maximum response was observed, which differed between experiments, the frequency of micronuclei induced by DMBA was reduced by 50% by the presence of caffeic acid. Caffeic acid (3,4-dihydroxy cinnamic acid) is widely distributed in plant materials in both free and combined forms and, as such, is a component of the human diet. Our results suggest that caffeic acid provides significant protection against the genotoxicity of DMBA.     

The prevention of benzene-induced genotoxicity in mice by indomethacin

Benzene is a myelotoxin which affects hemopoietic progenitor cells leading to bone-marrow depression as well as a genotoxin which causes chromosomal abnormalities including micronucleus formation. We have demonstrated previously that benzene administered to DBA/2 or C57B1/6 mice causes bone-marrow depression and increased prostaglandin E2 levels in bone marrow; both of these effects can be prevented by the coadministration of indomethacin, a selective inhibitor of prostaglandin synthase. We report, herein, that benzene (400-600 mg/kg body weight), under conditions where it depresses bone-marrow cellularity, also induces an increase in the frequency of micronucleus formation in polychromatic erythrocytes of C57B1/6 mice which is also prevented by the coadministration of indomethacin at levels that do not inhibit cytochrome P450 or myeloperoxidase. In Swiss Webster wild-type mice doses of benzene from 400 to 1000 mg/kg were without effect on marrow cellularity, but did induce the formation of micronucleated polychromatic erythrocytes which could be prevented by indomethacin. In contrast, DBA/2 mice, a strain highly sensitive to benzene, exhibited significant bone-marrow depression at a dose of benzene of 100 mg/kg body weight. Even at this low dose, benzene is too toxic toward developing erythrocytes to allow the evaluation of micronucleus formation. The frequency of induction of micronucleated polychromatic erythrocytes by benzene thus depends on the strain of mouse used. Furthermore, micronucleus formation appears to be an early and very sensitive indicator of benzene toxicity. A possible role for prostaglandin H synthase in the geno- and myelo-toxicity of benzene is discussed.     

Measurement of micronuclei in lymphocytes

The micronucleus technique has been proposed as a method for measurement of chromosomal damage in mitogen-stimulated human lymphocytes. Micronuclei require one cell division to be expressed and, consequently, the conventional micronucleus technique is very imprecise since the cells which have undergone only one division, and the micronuclei in them, cannot be identified separately from the total population of lymphocytes. To overcome this problem, two methods were developed to identify cells which have undergone their first mitosis. Using an autoradiographic technique, lymphocytes were pulse-labelled with [3H]thymidine at 48 h of culture, allowed to proceed through mitosis, identified by autoradiography between 72 and 84 h and micronuclei were scored in them. It was not possible to select a concentration of radiolabel which did not itself produce micronuclei and consequently the method was of no value for measuring pre-existing chromosomal damage present in vivo. However, it was capable of quantitating micronuclei produced by irradiation of lymphocytes in vitro. In the second method, cytokinesis was blocked using cytochalasin B. Micronuclei were scored in cytokinesis-blocked cells. These were easily recognisable owing to their binucleate appearance and a large number could be accumulated by adding 3.0 micrograms/ml cytochalasin B at 44 h and scoring at 72 h. Cytochalasin B did not itself produce micronuclei. The cytokinesis-block method was simple to perform; the 'in vivo' micronucleus frequency in normal individuals was 4.4 +/- 2.6 micronuclei/500 cytokinesis-blocked cells; and for lymphocytes irradiated in vitro there was a linear relationship between dose of radiation and number of induced micronuclei. The cytokinesis-block method appears to be the procedure of choice for quantitating micronuclei in lymphocytes.     

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.     

Cytogenetic effects of diethylstilbestrol-diphosphate (DES-dp) on mouse bone marrow monitored by the micronucleus test.

6 dosages of diethylstilbestrol-diphosphate (DES-dp), ranging from 0.01 to 500 mg per kg of body weight were compared to saline and phosphate buffered saline (negative controls) and two dosages of cyclophosphamide (positive control) in the micronucleus test with 115 ICR mice. DES-dp failed to generate a significant increase in micronucleated polychromatic erythrocytes over negative controls. Cyclophosphamide produced a dose-related increase in micronuclei similar to previously published reports. Iit was therefore determined that the micronucleus test did not detect the types of chromosomal changes known to be generated by DES-dp and DES.     

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     

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.     

Micronucleus formation in cultured human keratinocytes following exposure to mitomycin C and cyclophosphamide

A method is described to investigate the induction of micronuclei in cultured human keratinocytes after short-term exposure to known clastogenic agents. The cytokinesis-block method was applied to facilitate the scoring of micronucleated cells. Mitomycin C, a direct-acting compound, caused a 5-20-fold increase in micronuclei over the controls at the highest concentration tested (1 microgram/ml). Cyclophosphamide, an agent requiring metabolic activation, did not induce the formation of micronuclei in cultured keratinocytes. However, after pretreatment of the keratinocyte cultures with Aroclor 1254 for 72 h, exposure to cyclophosphamide resulted in a 3-fold increase in micronucleus frequency over the controls. No cytogenetic effect of Aroclor 1254 was observed in control experiments.     

Effects of chemically- and environmentally-induced hypothermia on micronucleus induction in rats

We investigated micronucleus induction in rats treated with chlorpromazine and reserpine, drugs that induce hypothermia. We administered chlorpromazine (31.3--250mg/kg) or reserpine (500--2000 mg/kg) intraperitoneally and measured temperature rectally. Chlorpromazine at 62.5-250mg/kg and reserpine at all doses significantly decreased rectal temperature, although the hypothermic response was weaker than previously reported in mice. Only chlorpromazine at 250mg/kg decreased rectal temperature transiently to <33 degrees C for 20h and induced a statistically significant increase in micronucleated polychromatic erythrocyte frequency. When rats treated with reserpine at 500mg/kg were exposed to an environmental temperature of 16 degrees C for 6, 12, or 24h to keep their body temperature under 33 degrees C, only the 24h treatment group significantly induced micronuclei. In addition, relatively large micronuclei (diameter of micronucleus> or = 1/4 diameter of cytoplasm) accounted for 33.0% of the induced micronuclei, suggesting that hypothermia affected the mitotic apparatus. The hypothermic response to chlorpromazine and reserpine was weaker in rats than in mice, and it was correspondingly more difficult to induce micronuclei in rats with those drugs.