Induction of micronuclei in polychromatic erythrocytes from mouse bone marrow following intraperitoneal administration of halogenated benzenes

The mutagenicity of halogenated benzenes, including three isomers of tri- and tetrachlorobenzenes (TCB, TeCB) was studied on male Swiss CD1 mice by MN test. The data presented show that all the halogenated benzenes tested were found to be clastogenic apart from 1,2,3,5-TeCB. No significant differences were observed in the clastogenic activities of TCB-isomers and TeCB-isomers.     

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.     

Kinetochore identification in micronuclei in mouse bone-marrow erythrocytes: an assay for the detection of aneuploidy-inducing agents

An in vivo micronucleus assay using mouse bone marrow for identifying the ability of chemicals to induce aneuploidy and/or chromosome breaks is described. Micronucleus formation in bone-marrow erythrocytes of mice is commonly used as an index for evaluating the clastogenicity of environmental agents. However, micronuclei may also originate from intact lagging chromosomes resulting from the effect of aneuploidy-inducing agents. We have used immunofluorescent staining using anti-kinetochore antibodies to classify micronuclei for the presence or absence of kinetochores. Micronuclei positive for kinetochores are assumed to contain intact chromosomes and result from induced aneuploidy; while those negative for kinetochores contain acentric chromosomal fragments and originate from clastogenic events. The assay was evaluated using X-irradiation (a known clastogen) and vincristine sulfate (an aneuploidy-inducing agent). A dose-related response for the induction of micronuclei was observed for both agents. Micronuclei induced by X-irradiation were negative for kinetochores while the majority of the micronuclei resulting from vincristine treatment contained kinetochores. Thus, the micronucleus assay in combination with immunofluorescent staining for kinetochores may provide a useful method to simultaneously assess the ability of chemicals to induce aneuploidy and/or chromosome breaks.     

Formation of micronucleated erythrocytes in mouse bone-marrow under conditions of hypothermia is not associated with stimulation of erythropoiesis

Conditions of marked and long-lasting hypothermia have been shown to increase the formation of micronucleated polychromatic erythrocyte (MNPCE) in mouse bone-marrow. Stimulation of erythropoiesis as a consequence of anoxic conditions associated with decreased body temperature has been suggested as a possible mechanism for hypothermia-induced micronucleus formation. We examined whether chemically induced hypothermic conditions that produced increased MNPCE formation were associated with stimulation of erythropoiesis by measuring erythropoietin (EPO) concentrations in blood. Marked and long-lasting hypothermia was induced in male mice by oral administration of the antipsychotic compounds E-5842 (200 mg/kg) or chlorpromazine (100 mg/kg). Maximum decreases from the basal temperature, achieved 8 h after treatment, were 14.8 and 12.8 degrees C, respectively. A statistically significant increase in bone-marrow MNPCE frequency was observed 48 h after administration of E-5842 (p<0.01) or chlorpromazine (p<0.05). Mice made anaemic by retro-orbital bleeding (0.5 ml), which acted as positive control for stimulation of erythropoiesis, showed no relevant variation in mean rectal temperature and a slight non-statistically significant increase in MNPCE frequency after 48 h. Blood samples for determination of EPO levels were obtained 4 (bleed-control animals only), 8, 16 and 24 h after treatment. In spite of the induced hypothermia, no significant variation in EPO blood levels was observed after administration of E-5842 or chlorpromazine. Bleed-induced anaemic mice showed a clear increase in EPO blood levels at all sampled time points, differences from baseline values being statistically significant (p<0.001) at the 8-h samplings and beyond. These results indicate that induction of MNPCE secondary to chemically induced hypothermia is not mediated by stimulation of erythropoiesis.     

The effect of diphenylhydantoin on the frequency of micronuclei in bone-marrow polychromatic erythrocytes of mice

Using the micronucleus test to evaluate the mutagenic effect of 5,5-diphenylhydantoin (DPH) on bone marrow polychromatic erythrocytes, male Balb-C mice were treated with the drug in single and multiple injection tests. A significant increase in the frequency of micronucleated polychromatic erythrocytes (MPE), P less than 0.05, was found when the mice received a single injection of DPH at doses of 0.5 and 1.0 mg/kg, and this frequency did not increase at higher doses. When mice were treated 3 times, at 24-h intervals, with 1.0 mg/kg of DPH, a significant increase in MPE was also observed (P less than 0.05) but this was lower than when they received a single injection of the same dose. A cytotoxic effect of NaOH, 0.1 N, which was used as solvent, was also observed either when alone or when DPH (1.0 mg/kg) was injected 3 times. This effect was comparable to the one produced by mitomycin C (MMC) at a dose of 0.5 mg/kg.     

The effect of hycanthone and maleic hydrazide on the frequency of micronuclei in the bone-marrow erythrocytes of mice.

Male Swiss mice were assigned to 6 groups of either 3 or 4 animals each. 3 groups were given hycanthone methanesulfonate intraperitoneally, at 40, 80 or 120 mg/kg, respectively; the dose was repeated after an interval of about 24 h. At the same time 2 groups received maleic hydrazide at 100 or 200 mg/kg, and the remaining group was given dimethyl sulfoxide which was used as a solvent for both drugs. 6 h after the second injection, the mice were killed and bonemarrow preparations were made. Hycanthone induced a significant increase in the frequency of micronuclei in the polychromatic erythrocytes and suppressed the P/N ratio significantly. However, there was no dose-response relationship. Maleic hydrazide, on the other hand, failed to influence the incidence of micronuclei or the ratio of poly- to normo-chromatic erythrocytes.     

Comparison of the effects of arsenic and cadmium on benzo(a)pyrene-induced micronuclei in mouse bone-marrow

This study was undertaken to investigate the genotoxic interactions between the common environmental pollutants: arsenic (As), cadmium (Cd) and benzo(a)pyrene (BaP), which are known to be human carcinogens. C57BL/6J/Han mice were pre-treated with 100mg cadmium chloride (Cd(2+))/L or 50mg sodium arsenite (As(3+))/L in drinking water for 7 days and then given a single dose of 200mg BaP/kg bw by intra-peritoneal injection. A third group of mice did not receive the pre-treatment and was given BaP alone. Mice were sacrificed before or at 12, 24, 48 or 72h after BaP administration. Chromosome damage in bone-marrow cells was assessed by use of the micronucleus test. The study revealed that BaP induced a statistically significant increase in micronucleus (MN) frequency at 48h after administration. In animals exposed to Cd in drinking water no enhancement of genotoxicity was observed compared with the control group that was given tap water only. In Cd/BaP co-exposed animals, the MN frequency at respective time points did not differ from that for the animals exposed solely to BaP. A statistically higher MN frequency was found in bone marrow of animals exposed to As compared with controls that received tap water (0.92+/-0.29% versus 0.38+/-0.13%, respectively). This effect was even more pronounced after combined exposure to As and BaP. In the co-exposed animals, significantly elevated levels of MN were detected in samples examined at 12, 24 and 48h after BaP administration, compared with animals receiving BaP alone (1.14+/-0.31%, 1.26+/-0.3% and 2.02+/-0.45% versus 0.44+/-0.13%, 0.44+/-0.11% and 1.04+/-0.44%, respectively). These findings imply strong interactions between As and BaP, but not between Cd and BaP, in inducing DNA damage in polychromatic erythrocytes in mouse bone-marrow.     

Changes in mouse brain diazepam receptor binding after phenobarbital administration

Specific ³H-diazepam binding was measured $\text{in vitro}$ in adult mouse (strain, Crl=CD-1) brain after four days of an inductive dose of phenobarbital pretreatment (i.p.). Sexual dimorphism was observed in ³H-diazepam brain binding, female mice had significantly higher benzodiazepine binding than males without any differences in apparent affinity constants (K_D). Phenobarbital pretreatment caused a significant decrease in the maximal number of binding sites (Bmax) as well as in dissociation rate constants in both sexes.     

Effect of chlorophyllin on chromium trioxide-induced micronuclei in polychromatic erythrocytes in mouse peripheral blood

The effect of chlorophyllin on micronucleated polychromatic erythrocytes (MN-PCE) induction by chromium trioxide (CrO(3)) exposure in peripheral blood of mice was studied. Animals were treated with a single intraperitoneal dose of chlorophyllin (CHL) (20mg/kg), CrO(3) (20mg/kg), and CHL (20mg/kg) 4h before (CHL-CrO(3)) or 4h before and 20h after chromium treatments (20mg/kg; CHL-CrO(3)-CHL). Peripheral blood samples were drawn from the caudal vein at 0, 12 and 48h, and analyzed by the acridine orange (AO) technique. The results obtained in present study shown that CHL injection did not modify the number of MN-PCE. CrO(3) treatment resulted in a significantly increases 12 and 48h after the injection, reaching a four-fold increase 48h after CrO(3) administration. Whereas treatment with 20mg/kg of CHL prior to chromium, decreased the MN frequency induced by chromium in the 12h samples. When the samples were analyzed 48h after CrO(3) injection, no significant differences between CHL-CrO(3) and CHL-CrO(3)-CHL in comparison with CrO(3) treatment, were observed. These results indicate that increase of MN-PCE by CrO(3) is CHL-blocked in both protocols used (CHL-CrO(3) and CHL-CrO(3)-CHL) at 12h after treatment, but it was unable to modify the frequency of MN-PCE measured 48h after CrO(3) injection. The absence of a protective effect by CHL in the MN-PCE induction by CrO(3) at 48h, show that CHL has action only on one of the times of MN induction and suggests the possible action of CrO(3) by two different mechanisms, and not by CHL time-limited in vivo.     

Failure of antimony trioxide to induce micronuclei or chromosomal aberrations in rat bone-marrow after sub-chronic oral dosing

Antimony trioxide (Sb2O3, CAS 1309-64-4) is widely used as a flame retardant synergist in a number of household products, as a fining agent in glass manufacture, and as a catalyst in the manufacture of various types of polyester plastics. It does not induce point mutations in bacteria or mammalian cells, but is able to induce chromosomal aberrations (CA) in cultured cells in vitro. Although no CA or micronuclei (MN) have been induced after acute oral dosing of mice, repeated oral dosing for 14 or 21 days resulted in increased CA in one report, but did not result in increased MN in another. In order to further investigate its in vivo genotoxicity, Sb2O3 was dosed orally to groups of rats for 21 days at 250, 500 and 1000 mg/kg day. There were no clinical signs of toxicity in the Sb2O3-exposed animals except for some reductions in body-weight gain in the top dose group. Toxicokinetic measurements in a separate study confirmed bone-marrow exposure, and at higher levels than would have been achieved by single oral dosing. Large numbers of cells were scored for CA (600 metaphases/sex group) and MN (12,000 PCE/sex group) but frequencies of CA or MN in Sb2O3-treated rats were very similar to controls, and not biologically or statistically different, at all doses. These results provide further indication that Sb2O3 is not genotoxic to the bone marrow of rodents after 21 days of oral administration at high doses close to the maximum tolerated dose.     

Induction of micronuclei in mouse and rat by glycidamide,genotoxic metabolite of acrylamide

Male CBA mice and male Sprague-Dawley rats were treated by i.p. injection of glycidamide (GA), the presumed genotoxic metabolite of acrylamide (AA). GA was obtained through a new way of synthesis. As an endpoint of chromosome damage, micronucleus (MN) induction in erythrocytes was measured. Hemoglobin (Hb) adducts were used as a measure of in vivo dose of GA. GA induced linear dose-dependent increases in adduct levels in both species. Rats exhibit, compared with mice, 30% higher Hb adduct levels per unit of administered amount of GA. The incremental MN frequencies per administered dose of GA in mice showed a linear-quadratic dose-dependent curve. In the rat no positive dose-response relationship was obtained, probably due to toxic effects to the bone marrow. The main result of this study is the finding that after treatment with synthetic GA the MN frequency per unit of the in vivo dose of GA in the mouse is very similar to that obtained in a previous study, where animals were treated with AA and GA as a metabolite. This equality in potency of GA, whether its in vivo dose is established by injection of synthetic GA or through metabolism of AA, supports the view that GA is the predominant genotoxic factor in AA exposure.     

Induction of micronuclei by X-radiation in human, mouse and rat peripheral blood lymphocytes

We compared the radiosensitivity of human, rat and mouse peripheral blood lymphocytes (PBLs) by analyzing micronuclei (MN) in cytochalasin B-induced binucleated (BN) cells. For each species and dose 4-ml aliquots of whole blood were X-irradiated to obtain doses of 38, 75, 150 or 300 cGy. Controls were sham-irradiated. After exposure to X-rays, mononuclear leukocytes were isolated using density gradients and cultured in RPMI 1640 medium containing phytohemagglutinin to stimulate mitogenesis. At 21 h cytochalasin B was added to produce BN PBLs, and all cultures were harvested at 52 h post-initiation using a cytocentrifuge. Significant dose-dependent increases in the percentage of micronucleated cells and the number of MN per BN cell were observed in all three species. The linear-quadratic regression curves for the total percentage of micronucleated cells for the three species were similar; however, the curve for the mouse PBLs had a larger quadratic component than either of the curves for the rat or human PBLs. Although the correlation between the percentage of cells with MN and those with chromosome aberrations was high (r2 greater than 0.95), the mouse and rat PBLs were over twice as efficient as human PBLs in forming MN from presumed acentric fragments. These data indicate that the induction of MN in BN cells following ionizing radiation is similar in human, rat and mouse PBLs, but care must be taken in using the MN results to predict frequencies of cells with chromosomal aberrations.     

Induction of micronuclei in plant cells after exposure to accelerated ion irradiation

The root apex cells ofPisum sativum were irradiated in an U-240 isochronous cyclotron at the Institute for Nuclear Research of the Ukrainian National Academy of Sciences, Kyiv. Several types of micronuclei, differing in structural peculiarities, were observed in the 84 h following exposure to beams of accelerated¹H,⁴He, and¹⁴N ions with linear energy transfer (LET) of 0.95, 9.34, and 221 keV/µym, respectively. The maximum micronucleus induction was observed after irradiation with helium ions. Results obtained show that the micronucleus assay is a responsive test for investigations of cytogenetic damage produced by high LET beams in dividing cell systems in vivo.     

Relationship between the number of erythrocytes with micronuclei and immunologic status in humans

26 persons, having high level of erythrocytes with micronuclei were distinguished among clinically healthy people. While examining their health changes were registered in the systolic and pulse pressures, number of T-suppressors and T-helpers, natural killers, level of immunoglobulins and immune complexes, phagocytosis activity and peroxidases reaction of the leukocytes. Increase in the level of the cytogenetic aberrative lymphocytes was observed in all the cases. DNA-reparative synthesis was reduced only in 5 persons.