Cytogenetic studies on the effect of feeding mice with stored wheat grains treated with malathion

The cytogenetic effect of malathion residues in wheat grains stored for different periods of time (4, 12, 24 weeks) was evaluated in Swiss mice. The studies included: (1) chromosomal aberrations analysis in bone-marrow and spermatocyte cells; (2) chromosomal aberrations and sister chromatid exchange (SCE) analysis in spleen cell culture from mice fed with stored wheat grains. The tested doses were 8.36 (applied dose), 25.08 and 41.80 mg malathion kg⁻¹ wheat grains. The results demonstrated that the cytogenetic effect induced in different mouse tissues by malathion residues was dose-dependent and increased with increasing of both feeding and storage periods.Feeding mice with wheat grains stored for 4 weeks had a non-significant effect with respect to the induction of chromosomal aberrations or SCEs. Significant chromosome damage and increase of SCEs were observed in mice fed with wheat grains stored for 12 weeks. The maximum effect was recorded in mice fed for 12 weeks with the grains treated with the highest tested dose and stored for 24 weeks. However, mitomycin C i.p.-injected in mice at 1 mg kg⁻¹ body weight (b.w.) (positive control) induced a higher effect. The percentage of chromosome aberrations reached 13.60±0.98, 13.60±0.77 and 11.73±0.98 (P<0.01) in bone-marrow, cultured spleen cells and spermatocytes, respectively. The significant increase of abnormalities in spermatocytes was seen for univalent formation only, predominantly of the sex chromosomes. The frequency of SCEs was 10.76±0.62 per cell (P<0.01) in cultured spleen cells compared with 5.46±0.45 per cell for control and 14.66±0.54 per cell for the positive control.The obtained results indicate that malathion residues in stored wheat grains have potential genotoxic effect in mice under the conditions tested.     

Induction and persistence of micronuclei, sister-chromatid exchanges and chromosomal aberrations in splenocytes and bone-marrow cells of rats exposed to ethylene oxide

Studies on the induction and persistence of ethylene oxide (EO) induced chromosomal alterations in rat bone-marrow cells and splenocytes following in vivo exposure were carried out. Rats were exposed to ethylene oxide either chronically by inhalation (50-200ppm, 4 weeks, 5 days/week, 6h/day) or acutely by intraperitoneal injection (i.p.) at dose levels of 50-100ppm.Spontaneous- and induced-frequencies of micronuclei (MN), sister-chromatid exchanges (SCEs) and chromosomal aberrations were determined in rat bone-marrow cells, and in splenocytes following in vitro mitogen stimulation. Unstable chromosomal aberrations were studied in whole genome using standard Giemsa staining technique and fluorescence in situ hybridisation using probe for chromosome #2 was employed to detect chromosome translocations.Following chronic exposure, the cytogenetic analyses were carried out at days 5 and 21 in rat splenocytes, to study the induction and persistence of sister-chromatid exchanges. Following chronic exposure, ethylene oxide was effective in inducing SCEs, and markedly cells with high frequency SCEs were observed and they in-part persisted until day 21 post-exposure. However, no significant effect was observed in rat splenocytes for induction of MN and chromosomal aberrations. Following acute exposure, both SCEs and MN were increased significantly in rat bone-marrow cells as well as splenocytes.In conclusion, this study indicates that ethylene oxide at the concentrations employed by intraperitoneal injection or inhalation in adult rats is mutagenic and can induce both SCEs and MN.     

Studies on the genotoxic effect of beryllium chloride and the possible protective role of selenium/vitamins A, C and E

The genotoxic potential of beryllium chloride (BeCl2) was evaluated in vivo in mice using different endpoints. Chromosomal aberrations in bone marrow cells and in spermatocytes as well as sperm abnormalities were determined in the tested mice. The protective role of an orally administered drug consisting of selenium and vitamins A, C and E (selenium-ACE) was also studied. For analysis of chromosomal aberrations, both single and repeated oral treatments for a period of 3 weeks were performed. The doses used were 93.75, 187.50, 375, and 750 mg BeCl2/kg bw, which corresponds to 1/16, 1/8, 1/4, and 1/2 of the experimental LD50. BeCl2 induced a statistically significant increase in the percentage of chromosomal aberrations in both somatic and germ cells, with a dose- and time-response. The percentage of induced chromosomal aberrations was significantly reduced in all BeCl2-treated groups after oral administration of selenium-ACE. Beryllium chloride also induced a significant increase in the percentage of abnormal sperm. This percentage reached values of 9.62 +/- 0.32 and 5.56 +/- 0.31 in mice treated with the highest test dose of BeCl2 and with BeCl2+selenium-ACE, respectively, compared with 1.96 +/- 0.14 for the control. In conclusion, the results demonstrate the genotoxic effect of beryllium chloride and confirm the protective role of selenium-ACE against the genotoxicity of beryllium chloride.     

Constitutive heterochromatin polymorphism and chromosome damage in viral hepatitis

The frequencies of chromosomal aberrations and sister-chromatid exchanges (SCEs) were scored in relation to constitutive heterochromatin in 100 patients with viral hepatitis B, 100 patients with viral hepatitis A and 100 age- and sex-matched normal controls. 23.4%, 15% and 4% of the cells showed chromosomal aberrations in patients with hepatitis B, hepatitis A and normal controls respectively. Non-random involvement of chromosomal aberrations were also noted in chromosome 1 of patients with hepatitis B and A as compared to normal controls. The frequencies of SCEs (mean +/- S.D.) were found to be 10.40 +/- 2.83 in hepatitis B and 8.70 +/- 2.34 in hepatitis A. These values were significantly higher than the SCE frequency (mean +/- S.D.) of 5.88 +/- 2.25 observed in normal controls (P less than 0.001). The intra-chromosomal distribution of SCEs revealed a relatively increased incidence of SCEs in chromosome 1 of patients with hepatitis B and A as compared to normal controls. Analysis of constitutive heterochromatin polymorphism showed chromosome 1 qh+ to be the most frequent variant in patients with hepatitis B and A as compared to normal controls. The increased involvement of C-band variant 1 qh+ in patients with hepatitis B and A as compared to normal controls may indicate that extra heterochromatin offers additional sites for viral integration.     

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.     

Cytological characterization of repair-deficient CHO cell line 43-3B. I. Induction of chromosomal aberrations and sister-chromatid exchanges by UV and its modulation with 3-aminobenzamide

The induction of chromosomal aberrations and sister-chromatid exchanges (SCEs) by short-wave ultraviolet (UV) and X-irradiation was studied in Chinese hamster ovary (CHO) wild-type (WT) cells and one of its UV-hypersensitive mutants, 43-3B. The results indicate that CHO 43-3B show high levels of spontaneously occurring chromosomal aberrations and SCEs; these levels are, respectively, approximately 4 and 1.7 times those found in WT CHO. Treatment with UV produced a considerable delay in the cell-cycle progression of the mutant cells compared to the WT cells. Doses of UV that had no effect on WT cells, significantly induced chromosomal alterations in the mutant in a dose-dependent manner. An approximately 5-fold increase in the induced frequencies of SCEs was obtained in 43-3B cells after UV treatment. No synergistic effect was observed with UV irradiation and the inhibitor of poly(ADP-ribose) synthetase, 3-aminobenzamide (3AB), in either cell type. The frequency of SCEs in the mutant cell lines was lower than would be expected if the effects of UV and the inhibitor were additive. X-Ray alone in G1 and in combination with 3AB in G2 did not induce increased frequencies of chromosomal aberrations in mutant cells in comparison to the WT cells.     

Evaluation of the mutagenic potential of the hair dye N-methyl-amino-2-nitro-4-N',N'-bis-(2-hydroxyethyl)-aminobenzene in a battery consisting of Drosophila and mammalian cytogenetic assays

The compound N-methyl-amino-2-nitro-4-N', N'-bis(2-hydroxyethyl)-aminobenzene was tested for mutagenic activity in the sex-linked recessive lethal test with Drosophila melanogaster, the induction of chromosomal aberrations and sister-chromatid exchanges (SCEs) with Chinese hamster ovary (CHO) cells in vitro, and the micronucleus test with mouse bone-marrow cells in vivo. Consistently negative results were obtained with the 3 tests. The SCE tests gave positive results with prolonged treatments. It is concluded that reliable decisions about mutagenic activity cannot be based on the induction, in vitro, of SCEs alone.     

Studies on the influence of photoreactivation on the frequencies of UV-induced chromosomal aberrations, sister-chromatid exchanges and pyrimidine dimers in chicken embryonic fibroblasts

Chicken embryonic fibroblasts, which possess photoreactivating enzyme were used to study the influence of photoreactivating light on the induction of pyrimidine dimers, sister-chromatid exchanges (SCEs) and chromosomal aberrations by 254 nm UV. While photoreactivation (PR) efficiently removed most of the induced dimers (75-95%), the frequencies of SCEs and chromosomal aberrations were reduced only by about 30-65%, in parallel experiments. Since pyrimidine dimers are the only photoreactivable photolesions known, the reduction in the frequencies of SCEs and chromosomal aberrations on PR has been interpreted as due to disappearance of pyrimidine dimers, implying that these lesions are the primary events responsible for the induction of the biological end points studied. The possible reasons for the lack of quantitative relationship between the frequencies of dimers and the frequencies of SCEs and chromosomal aberrations are discussed.     

Genotoxic effects of malathion: an organophosphorus insecticide, using three mammalian bioassays in vivo

The genotoxic effects of malathion was evaluated using chromosome aberration, sister chromatid exchange (SCE) and sperm abnormality assays in mice. All the three acute doses (2.5, 5 and 10mg/kg) of malathion tested in the present study, induced significant dose-dependent increase in the frequency of chromosome aberrations and sperm abnormalities, but did not affect the total sperm count. The highest acute dose induced a >12-fold increase in the frequency of chromosome aberrations, two-fold increase in the frequency of SCEs and four-fold increase in the frequency of sperms with abnormal head morphology following intraperitoneal (i.p.) exposure. Further, a significant increase in the frequency of SCEs was observed, but the increase was not dose-dependent. At higher doses, malathion induced a moderate delay in cell cycle as evident from the increase in average generation time (AGT). The present findings suggest that technical grade malathion is a potent genotoxic agent and may be regarded as a potential germ cell mutagen also.     

Differential sensitivity of peripheral blood lymphocytes of untreated leprosy patients to mitomycin C

The effects of a bifunctional alkylating agent mitomycin C (MMC), an effective inducer of chromosome aberrations and sister-chromatid exchanges (SCEs), have been studied in untreated leprosy patients. This was done to study the mutagen sensitivity of the leprosy patients. The frequency of chromosomal aberrations induced by MMC (conc. 0.01 microgram/ml) was 2.5% in controls, 3.6% in paucibacillary (PB), and 6.8% in multibacillary (MB) patients. The difference in the frequency of MMC-induced chromosome aberrations between the 3 groups studied was highly significant (p less than 0.01). Cultures grown with MMC showed the frequency of SCEs/cell to be 12.70 +/- 1.19 in controls, 19.97 +/- 3.51 in PB, and 29.66 +/- 5.92 in MB patients. The differences in the frequency of MMC-induced SCEs between the 3 groups were found to be highly significant (p less than 0.01). The enhanced frequencies of spontaneous and MMC-induced chromosome aberrations and SCEs observed in PB and MB patients indicate a clear differential mutagen sensitivity between PB and MB patients who are known to have different immunological status and thereby differ in the severity of the disease.     

Dose-responsive increase in sister-chromatid exchanges in bone-marrow cells of mice exposed nose-only to whole cigarette smoke

The effect of whole cigarette smoke exposure on bone-marrow sister-chromatid exchanges (SCEs) was studied in B6C3F1 mice. Animals were exposed nose-only to 10% (v/v) cigarette smoke 5 days/week for 2 weeks. Four dose levels of cigarette smoke (1, 4, 9 and 18 exposures/day) were studied using 2 cigarette types, Kentucky reference 3A1 (3A1) and American Blend (AB). A single exposure represented approximately 1 cigarette. A dose-dependent increase in SCEs was observed for both the 3A1 and AB cigarettes at dose levels which had no effect on bone-marrow cell-replication kinetics. These findings represent the first demonstration of a dose-responsive increase in cigarette smoke-induced SCEs in a rodent model system.     

Cytogenetical characterization of Chinese hamster ovary X-ray-sensitive mutant cells xrs 5 and xrs 6. III. Induction of cell killing, chromosomal aberrations and sister-chromatid exchanges by bleomycin, mono- and bi-functional alkylating agents

Two X-ray-sensitive mutants of CHO-K1 cells, xrs 5 and xrs 6, were characterised with regard to their responses to genotoxic chemicals, namely bleomycin, MMS, EMS, MMC and DEB for induction of cell killing, chromosomal aberrations and SCEs at different stages of the cell cycle. In addition, induction of mutations at the HPRT and Na+/K+ ATPase (Oua) loci was evaluated after treatment with X-rays and MMS. Xrs 5 and xrs 6 cells were more sensitive than wild-type CHO-K1 to the cell killing effect of bleomycin (3 and 13 times respectively) and for induction of chromosomal aberrations (3 and 4.5 times). In these mutants a higher sensitivity for induction of chromosomal aberrations to MMS, EMS, MMC and DEB was observed (1.5-3.5 times). The mutants also showed increased sensitivity for cell killing effects of mono- and bi-functional alkylating agents (1.7-2.5 times). The high cell killing effect of X-rays in these mutants was accompanied by a slight increase in the frequency of HPRT mutation. The xrs mutants were also more sensitive to MMS for the increased frequency of TGr and Ouar mutants when compared to wild-type CHO-K1 cells. Though bleomycin is known to be a poor inducer of SCEs, an increase in the frequency of SCEs in xrs 6 cells (doubling at 1.2 micrograms/ml) was found in comparison to no significant increase in xrs 5 or CHO-K1 cells. The induced frequency of SCEs in all cell types increased in a similar way after the treatment with mono- or bi-functional alkylating agents. MMS treatment of G2-phase cells yielded a higher frequency of chromatid breaks in the mutants in a dose-dependent manner compared to no effect in wild-type CHO-K1 cells. Treatment of synchronised mutant cells at G1 stage with bleomycin resulted in both chromosome- and chromatid-type aberrations (similar to the response to X-ray treatment) in contrast to the induction of only chromosome-type aberrations in wild-type CHO-K1 cells. The frequency of chromosomal aberrations chromosome and chromatid types) also increased with MMC treatment in G1 cells of xrs mutants. DEB treatment of G1 cells induced mainly chromatid-type aberrations in all cell types. The possible reasons for the increased sensitivity of xrs mutants to the chemical mutagens studied are discussed and the results are compared to cells derived from radiosensitive ataxia telangiectasia patients.     

Sister chromatid exchange and chromosome aberrations induced by curcumin and tartrazine on mammalian cells in vivo

Sister chromatid exchanges (SCEs) and chromosomal aberrations induced by curcumin (a natural dye) and tartrazine (a synthetic dye) were studied on bone marrow cells of mice and rats following acute and chronic exposure via the diet. Except for two low concentrations in the curcumin and one low concentration in the tartrazine treated series a significant increase in SCEs was observed in all the concentrations of the two dyes tested. Except for two high concentrations during the 9 months treatment no significant increase in chromosomal aberrations was observed in the curcumin treated series, whereas tartrazine showed a significant increase in chromosomal aberrations in some of the higher concentrations in all the series tested. The results indicate that tartrazine is more clastogenic than curcumin.     

Comparison of frequencies of radiation-induced stable chromosomal aberrations in somatic and germ tissues of the mouse

Radiation-induced stable chromosome aberrations have been studied in the testes and bone-marrow of the mouse (Mus musculus). 60 days after whole-body irradiation with a dose of 400 rad X-rays, the frequency of visible chromosome aberrations in bone-marrow cells was 19.8%. The frequency of chromosome aberrations in spermatogonia of the same mice, scored as multivalents in spermatocytes, was considerably lower — only 4.7%. The possible mechanisms underlying this marked difference in translocation yield are discussed.     

Cytogenetic effects of pesticides. IV. Cytogenetic effects of the insecticides Gardona and Dursban.

The cytogenetic effects of the insecticides Gardona and Dursban were investigated. The toxicity and ability of both insecticides to induce chromosome aberrations and sister-chromatid exchange in vitro was tested in a primary culture of mouse spleen cells, in order to assess the potential mutagenicity of both insecticides. The concentrations 10(-7)-10(-3) M were used for testing the toxic effects of the insecticides. Both Gardona and Dursban were toxic to spleen cell cultures and the percentage of viable cells decreased as the concentration of the insecticide was increased. It reached 76.8% and 77.8% of control after treatment with the highest concentration tested (10(-3) M) of Gardona and Dursban respectively. Gardona at 0.25, 0.50, 1.0 and 2.0 micrograms/ml, and Dursban at 0.50, 1.0, 2.0 and 4.0 micrograms/ml were tested for the induction of chromosome aberrations and sister-chromatid exchanges. All of the tested concentrations of both insecticides induced a high percentage of metaphases with chromosomal aberrations in cultured mouse spleen cells after 4-h treatment. The frequency of SCEs/cell increased with increasing concentration of the insecticides. It reached 11.92 +/- 0.14/cell and 13.40 +/- 0.20/cell after treatment with Gardona (2 micrograms/ml) and Dursban (4 micrograms/ml), respectively, compared with 8.2 +/- 0.19/cell and 7.6 +/- 0.15/cell in the solvent control. The presented results indicate that both Gardona and Dursban in the tested concentrations are mutagenic in mouse spleen cell cultures.     

Evaluation of the mutagenic potential of the hair dye N-methyl-amino-2-nitro-4-N′,N′-bis-(2-hydroxyethyl)-aminobenzene in a battery consisting of Drosophila and mammalian cytogenetic assays

The compound N-methyl-amino-2-nitro-4-N′,N′-bis-(2-hydroxyethyl)-aminobenzene was tested for mutagenic activity in the sex-linked recessive lethal test with Drosophila melanogaster, the induction of chromosomal aberrations and sister-chromatid exchanges (SCEs) with Chinese hamster ovary (CHO) cells in vitro, and the micronucleus test with mouse bone-marrow cells in vivo. Consistently negative results were obtained with the 3 tests. The SCE tests gave positive results with prolonged treatments. It is concluded that reliable decisions about mutagenic activity cannot be based on the induction, in vitro, of SCEs alone.     

Genotoxic effect of 2,4-dichlorophenoxy acetic acid and its metabolite 2,4-dichlorophenol in mouse.

The cytogenetic effect of 2,4-dichlorophenoxy acetic acid (2,4-D) and its metabolite 2,4-dichlorophenol (2,4-DCP) was studied in bone-marrow, germ cells and sperm head abnormalities in the treated mice. Swiss mice were treated orally by gavage with 2,4-D at 1.7, 3.3 and 33 mg kg(-1)BW (1/200, 1/100 and 1/10 of LD(50)). 2,4-DCP was intraperitoneally (i.p.) injected at 36, 72 and 180 mg kg(-1)BW (1/10, 1/5, 1/2 of LD(50)). A significant increase in the percentage of chromosome aberrations in bone-marrow and spermatocyte cells was observed after oral administration of 2,4-D at 3.3 mg kg(-1)BW for three and five consecutive days. This percentage increased and reached 10.8+/-0.87 (P<0.01) in bone-marrow and 9.8+/-0.45 (P<0.01) in spermatocyte cells after oral administration of 2,4-D at 33 mg kg(-1)BW for 24 h. This percentage was, however, lower than that induced in bone-marrow and spermatocyte cells by mitomycin C (positive control). 2,4-D induced a dose-dependent increase in the percentage of sperm head abnormalities. The genotoxic effect of 2,4-DCP is weaker than that of 2,4-D, as indicated by the lower percentage of the induced chromosome aberrations (in bone-marrow and spermatocyte cells) and sperm head abnormalities. Only the highest tested concentration of 2,4-DCP (180 mg kg(-1)BW, 1/2 LD(50)) induced a significant percentage of chromosome aberrations and sperm head abnormalities after i.p. injection. The obtained results indicate that 2,4-D is genotoxic in mice in vivo under the conditions tested. Hence, more care should be given to the application of 2,4-D on edible crops since repeated uses may underlie a health hazard.     

Comparative effectiveness in the induction of sister chromatid exchanges and chromosome aberrations

The dose curves for 5 chemicals were studied to compare the efficiency of induction of SCEs and chromosomal aberrations by "polycentric" mutagens. The number of SCEs was found to increase linearly with the dose while that of chromosomal aberrations--nonlinearly. The efficiency of SCEs induction by these mutagens was found to be 25-50 times as high as in the induction of chromosomal aberrations. Division of alkylating mutagens into "monocentric" and "polycentric" is shown to be useful. It reflects their different efficiency in damaging one or simultaneously two DNA strands. The correlation between SCEs and formation of aberrations of the chromatid type is stated.     

Influence of incorporated bromodeoxyuridine on the induction of chromosomal alterations by ionizing radiation and long-wave UV in CHO cells

Incorporation of BrdUrd into nuclear DNA sensitizes CHO cells (1) to the induction of chromosomal aberrations by X-rays and 0.5 MeV neutrons and (2) to induction of chromosomal aberrations and SCEs by lw-UV. We have attempted to establish a correlation between induced chromosomal alterations and induced single- or double-strand breaks in DNA. The data show that while DSBs correlate very well with X-ray-induced aberrations, no clear correlation could be established between lw-UV induced SSBs (including alkali-labile sites) and chromosomal alterations.

In addition the effect of 3-aminobenzamide (3AB) on the induction of chromosomal aberrations and SCEs induced by lw-UV has been determined. It is shown that 3AB is without any effect when lw-UV-irradiated cells are posttreated with this inhibitor.

The significance of these results is discussed.     

Clastogenic action of ellipticine over the cell cycle of human lymphocytes and influence of posttreatments with caffeine and ara-C at G2

The clastogenic potential of the intercalating compound ellipticine, an antitumor alkaloid, has been demonstrated in mammalian cells. To characterize the mechanism of action of this drug over the cell cycle, human lymphocyte cultures from 2 healthy donors were treated with 3 micrograms/ml ellipticine in 30-min pulses during different phases of the cell cycle and analyzed for chromosomal aberrations and sister-chromatid exchanges. The G2 phase was most sensitive in terms of induction of aberrations, followed by S and G1. Chromatid-type aberrations were the most common type of chromosomal damage. Induction of SCEs was significantly high only after treatment at G1, when the frequencies of SCEs doubled. The post-treatment effect of lymphocytes with inhibitors of DNA repair, 10(-3) M caffeine and 5 x 10(-6) M 1-beta-D-arabinofuranosylcytosine, was also tested by adding 3 micrograms/ml ellipticine at G2 in 30-min pulses and immediately followed by caffeine and/or ara-C during the last 3 h before harvesting. Three experiments performed on blood from 3 donors showed a moderate potentiation effect on the frequency of chromatid-type aberrations (about 2-3 times) by both inhibitors. Likewise, a 3-fold increase was observed in the frequencies of chromosomal aberrations when caffeine and ara-C were combined. The present data demonstrate that posttreatment with caffeine and ara-C at G2 can modify the response of human lymphocytes treated with ellipticine by increasing the clastogenic action of this compound or by changing the cell-cycle progression.