Genotoxic action of fungicide Conan 5FL (hexaconazole) on mammalian cells in vivo and in vitro

The genotoxic effects of fungicide Conan 5FL (containing 50 g/L hexaconazole) in mouse bone-marrow cells and human lymphocytes have been evaluated. Three different concentrations of Conan 5FL (17.50, 35.00 and 70.00 microg/mL for human lymphocytes and 17.50, 35.00 and 70.00 mg/kg for mouse bone marrow cells) were studied. Conan 5FL induced significant increases (except 17.50 mg/kg for mouse bone marrow) in the frequency of chromosomal aberrations (CAs) in both test systems. This fungicide caused structural and numerical abnormalities in both mammalian cells. These are sister chromatid union, chromatid and chromosome breaks, fragments, dicentric and ring chromosomes, and polyploidy. Significant increase was found in induction and in minimum-maximum numbers of sister chromatid exchanges (SCEs) at all treatments compared with the negative control. Conan 5FL did not affect the replication index (RI) in human lymphocyte cultures, however, it significantly decreased the mitotic index (MI) in all treatment concentrations in both test systems. Using of Conan 5FL should be reconsidered due to its possible cytotoxic, clastogenic and mutagenic effects.     

In vivo induction of sister chromatid exchanges (SCEs) and chromosomal aberrations (CAs) by lynestrenol

Genotoxicity study of synthetic progestin lynestrenol, was carried out on mouse bone marrow cells using sister chromatid exchanges (SCEs) and chromosomal aberrations (CAs) as parameters. Lynestrenol was studied at three different doses (6.87, 13.75 and 27.50 mg/kg body wt.). SCE and CA increased significantly as compared to normal control when treated with lynestrenol at 13.75 and 27.50 mg/kg body wt. The present results suggest that lynestrenol has both a genotoxic and cytotoxic effects in mouse bone marrow cells.     

Clastogenicity of the fungicide afugan in cultured human lymphocytes

The genotoxic effects of the fungicide afugan were analysed by measuring chromosomal aberrations (CAs), sister chromatid exchange (SCE) and micronuclei (MN) in cultured human peripheral lymphocytes. Concentrations of 2.5, 5, 10 and 20 microg/ml of afugan were used during 24 and 48 h. Afugan significantly increased the frequency of CAs at 5, 10 and 20 microg/ml concentrations during a 48 h treatment period. A significant increase was observed for induction of SCE and MN at all treatments compared with the negative control. A significant dose-response correlation was found in all tests. Afugan did not affect the replicative index (RI), however it significantly decreased the mitotic index (MI) at all treatment concentrations except 2.5 microg/ml, and at both treatment times. The present results indicate that afugan is clastogenic and cytotoxic to cultured human lymphocytes.     

A comparison of sister chromatid exchange frequencies in peripheral lymphocytes and bone marrow cells of Fischer 344 and Sprague-Dawley rats

We have investigated spontaneous sister chromatid exchange (SCE) frequencies in peripheral lymphocytes and bone marrow cells explanted from two strains of the laboratory rat, Fischer 344 and Sprague-Dawley. A small, but significant difference was noted for both cell types, with the Fischer 344 rat being consistently higher. Other cell parameters, such as the mitotic index and the replicative index, were similar in the two strains. SCE levels in cultured peripheral lymphocytes after intraperitoneal administration of the alkylating drug cyclophosphamide (10 mg/kg) were similar for the two strains. Fischer 344 rats are known to have approximately a 10-fold higher incidence of spontaneous leukemia than do Sprague-Dawley rats. Since SCE frequency is a sensitive measure of DNA damage, our observations suggest that high leukemia incidence in the Fischer 344 rat may be related to a higher level of spontaneous DNA damage.     

Genotoxicity testing of fluconazole in vivo and in vitro

The genotoxic effects of the antifungal drug fluconazole (trade name triflucan) were assessed in the chromosome aberration (CA) test in mouse bone-marrow cells in vivo and in the chromosome aberration, sister chromatid exchange (SCE) and micronucleus (MN) tests in human lymphocytes. Fluconazole was used at concentrations of 12.5, 25.0 and 50.0 mg/kg for the in vivo assay and 12.5, 25.0 and 50.0 microg/ml were used for the in vitro assay. In both test systems, a negative and a positive control (MMC) were also included. Six types of structural aberration were observed: chromatid and chromosome breaks, sister chromatid union, chromatid exchange, fragments and dicentric chromosomes. Polyploidy was observed in both the in vivo and in vitro systems. In the in vivo test, fluconazole did not significantly increase the frequency of CA. In the in vitro assays, CA, SCE and MN frequencies were significantly increased in a dose-dependent manner compared with the negative control. The mitotic, replication and cytokinesis-block proliferation indices (CBPI) were not affected by treatments with fluconazole. According to these results, fluconazole is clastogenic and aneugenic in human lymphocytes, but these effects could not be observed in mice. Further studies should be conducted in other test systems to evaluate the full genotoxic potential of fluconazole.     

Reduced frequencies of Mitomycin-C induced sister chromatid exchanges in AKR Mice

Summary The frequencies of base-line and Mitomycin-C (MMC) induced sister chromatid exchanges (SCE) were surveyed in four inbred strains of mice. In contrast to the C57B1/6J, CBA/J, and A/J strains where frequencies of SCE increased linearly with increasing dose of MMC, levels of SCE were significantly lower in AKR/J mice at high MMC concentrations. At a dose of 5 mg/kg MMC, chromosomal aberrations were more frequent in bone marrow cells of AKR/J mice than in C57B1/6J mice. These observations suggest an altered response to DNA damage in the AKR mouse strain.     

In vivo sister chromatid exchange analysis of a mouse plasmacytoma cell line NP-38

In vivo sister chromatid exchange (SCE) frequencies have been compared between the mouse plasmacytoma NP-38 and normal bone marrow cells of the host BALB/c mouse. NP-38 cells, transplanted subcutaneously showed a two-fold increase in SCEs (4.35-5.76/cell) compared with the bone marrow cells of the host (1.65-2.14/cell). Such an increase in SCE rates was also observed in NP-38 cells metastasized in spleen, bone marrow, liver, or mesentery, upon inoculation of NP-38 cells by intravenous injection. Even in such tumor-bearing mice, the SCE rates of the bone marrow cells were equivalent to the SCE level found in uninfected mice. These results indicate that the high SCE incidence in NP-38 cells is an inherent characteristic of this tumor cell line.     

Bromodeoxyuridine tablet methodology for in vivo studies of DNA synthesis.

5-Bromodeoxyuridine (BrdU) tablets with different physical characteristics are useful in a wide variety of studies requiring detection of DNA replication in vivo. These tablets can effect a high substitution of BrdU in DNA, thereby permitting sister chromatid differentiation in chromosomes stained with 33258 Hoechst alone or in conjunction with Giemsa. Baseline and cyclophosphamide-induced in vivo sister chromatid exchange frequencies in mouse spleen, marrow, and thymus were measured and found to be significantly greater than those in spermatogonia. Sister chromatid exchange analysis was also extended to mouse liver and to Chinese hamster and Armenian hamster marrow cells. Sister chromatid differentiation was observed in Armenian hamster meiotic tissue, and evidence for interhomolog chromatid exchange obtained.     

Genotoxicity assessment of new synthesized acridine derivative--3,6-diamino-10-methyl-9,10-dihydroacridine.

A new synthesized acridine derivative, 3,6-diamino-10-methyl-9, 10-dihydroacridine (AcrH), was tested for in vitro reverse mutations with Salmonella TA strains, chromosome aberrations and sister chromatid exchanges (SCE) in human lymphocytes, and for in vivo chromosome aberrations in bone marrow of mice. Using the classic plate incorporation method, mutagenicity of AcrH in bacterial cells (TA97a, TA98, TA100 and TA102) was observed in the experiments performed with, and without, rat liver S9 metabolic activation. The reverse mutation assay showed no difference in mutagenic activity between AcrH and acriflavine (Acr(+)) in the test with TA97. The results of in vitro chromosome aberrations assay revealed potential clastogenicity. The test using macroculture of human lymphocytes induced mainly chromatid gaps. The experiments with human lymphocytes revealed SCE-inducing effect of AcrH and Acr(+). In an in vivo study, AcrH given intraperitoneally to Balb/c mice did not cause any significant increase in the percentage of cells with aberrations compared to the negative control.     

Sanguinarine: an evaluation of in vivo cytogenetic activity

Sanguinarine (SG), a benzophenanthridine alkaloid, has been shown to possess anti-microbial, anti-inflammatory and antioxidant properties. In the UK and USA its salts has been in use in mouthwashes and toothpastes to inhibit dental plaque and improve gingival health. In India and Nepal consumption of mustard oil contaminated with argemone seeds containing sanguinarine, was associated with "dropsy" syndrome. In the present study, SG was evaluated in vivo in mouse bone marrow cells for its ability to induce clastogenicity and DNA damage in terms of increased sister chromatid exchange (SCE) frequencies. Doses of 5, 10, and 15 mg/kg body weight of SG given intra peritoneally induced a positive dose-dependent significant clastogenicity and SCE frequency increases (trend test alpha < or = 0.05). The minimum effective concentration to induce clastogenic and DNA damage was 10mg of SG/kg body weight. In addition to examining SCEs, the BrdUrd-differential technique was utilized to assess the effect of SG on cell replication. The analysis revealed that SG treatment did not significantly affect the distribution of cells among the different phases of the cell cycle. The proliferation rate index and average generation time data were statistically non-significant. This indicated that the alkaloid was not cytotoxic to the bone marrow cells at the doses tested. Based on the results of the present findings, the use of this alkaloid should be restricted.     

Correlation of the sister chromatid exchange level with chromosome aberrations induced by chemical mutagens in vivo

The data on the dose dependencies of the induction of sister chromatid exchanges (SCE) and chromosomal aberrations during exposure of mouse bone marrow cells in vivo to 5 alkylating substances are provided. The efficacy of SCE induction was found to be higher than that of chromosomal aberrations. It was established that SCE induced by chemical mutagens in vivo and in vitro are more sensitive and stable tests than chromosomal aberrations.     

Comparison of the levels of chromosome aberration and sister chromatid exchange induced by chemical mutagens in vitro

Dose dependencies of the induction of sister chromatid exchanges (SCEs) and chromosome aberrations were studied under in vivo exposure of mouse bone marrow cells to 5 alkylating agents. The efficacy of the induction of SCEs for all the substances was 20 to 60 times higher than that of the induction of chromosome aberrations. It was demonstrated that SCEs induced by chemical mutagens in vivo and in vitro are more sensitive tests than chromosome aberrations.     

Sister chromatid exchange induced by anti-herpes drugs.

The rate of sister chromatid exchange induced by several anti-herpes agents was measured to assess their potential mutagenicity. The agents--5-iodo-deoxyuridine (IDU), 5-trifluoromethyl-deoxyuridine (TFT), and [E]-5-(2-bromovinyl)-deoxyuridine (BVDU)--were incubated at various concentrations with human lymphocytes and fibroblasts, and that rate of sister chromatid exchanges was measured. In lymphocytes and fibroblasts BVDU and IDU did not induce exchange except at concentrations of 50 mg/l, while TFT increased the rate of exchange at a concentration of 0.5 mg/l. The rate of sister chromatid exchange is a sensitive index of chromosomal damage, and these findings provide information on the safety of some of the antiherpes agents tested. TFT increased the rate of exchange at a concentration that coincides with its minimal antiviral concentration, but BVDU did not induce exchange at therapeutic concentrations.     

Chromosomal changes induced in mouse bone marrow cells following six weeks inhalation of cyclohexanol

The effects of low doses of cyclohexanol exposure were studied in mouse bone marrow cells including chromosome aberrations (CA), micronucleus (MN) and sister chromatid exchanges (SCE) as biomarkers. Capillaries with a tested agent that was evaporated continuously were placed in an experimental chamber for six weeks. No clastogenic and/or aneugenic effect of CA and MN induction was observed. A significant elevation of induced damage was achieved in the SCE study (p < 0.001) that has confirmed the early exposure of cyclohexanol to mice.     

Genotoxic effect of raw betel-nut extract in relation to endogenous glutathione levels and its mechanism of action in mammalian cells

The mutagenic and carcinogenic potency of betel-nut components is well established. This study was undertaken to determine the genotoxic potency of an aqueous extract of raw betel nut (AEBN) in relation to the endogenous glutathione (GSH) level in mouse bone marrow cells (BMC) and human peripheral blood lymphocytes (PBLs), and to find out whether arecoline (ARC), an alkaloid of betel nut, could generate reactive oxygen species (ROS) in these cells. It was observed that AEBN has genotoxic properties, which is further enhanced by depletion of endogenous GSH levels. However, the degree of enhancement varies with the type of parameter and cell system studied. The present data indicate that the generation of ROS by ARC could partially contribute to the induction of chromosomal aberrations (CAs), since the frequency of ARC-induced CAs was reduced either by post-treatment with superoxide dismutase (SOD) or in anoxic conditions. However, the induction of sister chromatid exchanges (SCEs) probably involves p53-dependent changes in cell proliferation and allowing some repair of DNA damage. The extent of damage for each parameter was higher when the mice were exposed to AEBN for 30 days than 5 days. Longer exposure showed higher level of p53 expression in mouse BMC, which could block the damaged cells from proliferation and allow the cells to repair the DNA damage.     

Effects of styrene oxide on chromosome aberrations, sister chromatid exchange and hepatic drug biotransformation in chinese hamsters in vivo

In vivo inhalation exposure to styrene oxide (25, 50, 75 and 100 ppm) for 2, 4 or 20 days (25 ppm only) had no effects on chromosomal aberration rates or sister chromatid exchange (SCE) frequencies (BrdU/labelling performed in vitro) in the bone marrow cells of Chinese hamsters. The only positive response in aberration frequency was obtained when styrene oxide was injected in lethal concentration (500 mg/kg body weight, i.p.) into the animal. One animal out of six showed slightly elevated SCE values after this high dose. The response of the hepatic drug metabolizing enzymes to styrene oxide exposure was found to be rather weak, which may be due to rather high activity of epoxide hydratase in Chinese hamsters as compared to e.g. mouse.     

Effect of chlorophyllin on gamma ray induced micronuclei in polychromatic erythrocytes of murine peripheral blood determined by the ABC strategy

The effect of chlorophyllin on micronucleated polychromatic erythrocyte (MN-PCE) induction by gamma ray exposure in peripheral blood of mice was studied. The area beneath the curve (ABC) of MN-PCE frequency versus time was used as an index of total MN-PCE induction. The dose of 200 mg chlorophyllin per kg of body weight caused a slight, but not significant, reduction of the MN-PCE caused by 1.0 Gy exposure. This result indicates that chlorophyllin did not protect the cells against MN induction. In previous studies it was observed that the same chlorophyllin dose was able to protect 100% against sister chromatid exchange (SCE) induction by 1.0 gamma rays in both murine spermatogonia and bone marrow cells. These contradictory results indicate that chlorophyllin did not protect cells by scavenging free radicals, but by other mechanism, i.e. stimulating repair of lesions involved in SCE induction.     

Cytogenetic effects of malathion insecticide on somatic and germ cells of mice

Male mice dermally exposed to single or multiple treatment (5 days/2 weeks) showed that the ability of malathion to induce chromosome aberrations in somatic (bone marrow) and germ cells (primary spermatocytes) was related to the type of treatment and dose used. Statistically significant increases of chromosome aberrations in bone marrow cells occurred after single treatment (500 and 2000 mg/kg body wt) when chromatid gaps were included and after multiple treatment (250 and 500 mg/kg) when they were excluded. No dose-response relationships were observed for either treatment. In germ cells, malathion induced a significant increase of univalents in both types of treatment but structural chromosome aberrations were induced only by multiple treatment. Malathion induced a significant decrease of the mitotic indices in the bone marrow.     

In vivo BrdU-33258 Hoechst analysis of DNA replication kinetics and sister chromatid exchange formation in mouse somatic and meiotic cells

BrdU (5-bromodeoxyuridine)-33258 Hoechst methods have been adapted for in vivo analyses of replication kinetics, sister chromatid differentiation and sister chromatid exchange (SCE) formation in mice. Sufficient in vivo BrdU substitution for cytological detection was effected with multiple intraperitoneal injections of the analogue. The combination of centromere staining asymmetry and sister chromatid differentiation at metaphase permits unambiguous determination of the number of replications in BrdU and dT (deoxythymidine) undergone by individual cells. Late-replicating regions in marrow and spermatogonial chromosomes are highlighted by bright fluorescence after sequential incorporation of BrdU followed by dT during a single DNA synthesis period. SCEs are analyzed in marrow and spermatogonial metaphases after successive complete cycles of BrdU and dT incorporation. Significant induction of SCE was observed with both mitomycin C and cyclophosphamide; the latter drug requires host-mediated activation to be effective. In meiotic metaphase cells harvested two weeks after BrdU incorporation, satellite DNA asymmetry, sister chromatid differentiation and SCE could be detected in a few chromosomes, most frequently the X and the Y.     

Increased sister chromatid exchange in bone marrow and blood cells from Bloom's syndrome.

Bone-marrow cells from a patient with Bloom's syndrome cultured for 48 h in the presence of BudR exhibited a striking increase in the number of sister chromatid exchanges (SCEs) in comparison to that in the marrow cells of a patient with treated polycythemia vera (PV). Thus, it appears that an increased incidence of SCE in Bloom's syndrome occurs in various differentiated types of cells, not just blood lymphocytes, and constitutes the syndrome's most characteristic cytogenetic feature. In contrast, the incidence of SCE was not increased in marrow cells and lymphocytes of the particular PV patient studied here, whose cells did exhibit increased numbers of chromatid and chromosome gaps and breaks, presumably as result of the patient's earlier treatment. An increased frequency of SCE was demonstrated in Bloom's syndrome lymphocytes using both a technique based on BudR incorporation and one based on labeling with tritated deoxycytidine. This observation constitutes evidence against the increase of SCE being due to an unusual reaction to BudR. By conventional cytogenetic techniques, chromosome instability, including chromatid and chromosome breaks, but no homologous chromatid interchanges were also recognized in Bloom's syndrome bone-marrow cells incubated in vitro (without BudR) for either 1.k or 16 h. This observation points to the existence of chromosome instability in vivo.