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.     

Chromosomal abberations and sister-chromatid exhanges (SCEs) in patients undergoing long-term Imuran therapy

13 patients treated daily for an extended time with Imuran and prednisone and 4 patients treated in the samy way with Imuran only were cytogenetically analysed for the induction of structural chromosomal aberrations and SCEs. There was an increase in the number of aberrations and SCEs in nearly all patients analysed. However, we did not find any dose-dependent cumulative effect on chromosomal damgae, with the exception of 1 patient tested in a small group of 4 patients involved in a prospective cytogenetic study, who showed a significant time-dependent increase in the number of aberrations.     

Chromosomal aberrations induced by secondary and tertiary amine-containing dyes and in combination with nitrite in vivo in mice

Chromosomal aberrations induced by indigo carmine (secondary amine-containing dye), fast green FCF (tertiary amine-containing dye) and sodium nitrite, singly and in combination, were studied in mice after prolonged feeding in the diet. A significant increase in the chromosomal aberrations was observed for all the dyes and in the nitrite treated series when compared with distilled water controls. However, a combination of half the concentration of dye and nitrite gave additive effects.     

Cytogenetic monitoring of petrochemical workers

The frequencies of chromosomal aberrations and sister-chromatid exchanges (SCEs) in the peripheral blood lymphocytes of 360 persons, 180 workers in a petrochemical corporation and 180 appropriate controls, were studied. A significant increase in chromosomal aberrations and SCEs, compared to the control group, was observed in two sewage-treatment workshop workers; however, there were no significant differences between petrochemical workers from four workshops and a control group.     

The effects of beta-radiation on sister-chromatid exchanges in cultured human lymphocytes.

The incidence of Sister-Chromatid Exchanges (SCEs) due to beta-radiation was investigated in cultured human lymphocytes using the BrdU/Giemsa technique. Cultures treated continuously with 0.001 and 0.01 microCi of [3H]uridine showed no increase in either chromosome abnormalities or SCEs. Continuous treatment with 0.1 microCi resulted in a significant increase in chromosome aberrations but no increase in SCEs, while treatment with 0.2 microCi gave both an increase in chromosome aberrations and SCEs. Cultures given a 4-h pulse with 1.0 microCi showed a significant increase in both SCEs and chromosome aberrations. The results indicate that low levels of beta-radiation do not cause an increase in SCEs in human lymphocytes, and, that a number, if not all the exchanges observed at low levels of beta-radiation with autoradiography, may be spontaneous events.     

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.     

Effects of CCNU therapy on human chromosomes

Peripheral blood lymphocytes of 9 patients under CCNU therapy were examined for frequency of sister-chromatid exchanges (SCEs) and chromosomal aberrations (CAs). 7 out of 9 patients were treated with only CCNU, whereas the remaining 2 were treated with other chemotherapeutic agents in combination with CCNU. Compared to normal individuals, a significantly increased frequency of SCE was observed in the patients before starting anticancer therapy (P less than 0.001). Increased incidences of structural changes in chromosomes were observed in cells from all the treated patients. The most frequent aberrations were of chromatid type. After administration of a single dose of CCNU, an increase in SCE frequencies was observed which remained elevated even after 6 weeks. It was concluded that increases in SCEs and CAs in lymphocytes were caused by CCNU treatment. Further studies are needed to elucidate whether any CAs observed in the present study could participate in the induction of second neoplasm.     

Chromosomal aberrations and sister-chromatid exchanges in workers exposed to styrene

Few studies exist about chromosomal damage in workers occupationally exposed to styrene. In the present study, chromosomal aberrations and SCEs were analyzed from cultures of peripheral lymphocytes of workers employed in 6 different reinforced-plastics industries with styrene air exposure levels ranging from 30 to 400 mg/mc. A control group was selected on the base of sex, age and smoking habit. We examined 50-h cultures (for chromosomal-aberrations) and 72-h cultures (for SCEs) for each individual. All workers exposed to styrene, as compared with controls, showed significantly increased frequencies of chromosomal aberrations, while SCEs were significantly increased at 4 of the 6 plants. High SCE values appeared with styrene air concentrations higher than 200 mg/mc. Apart from the possible presence and role of other interfering chemicals in the various plants, chromosomal aberrations seem to be more sensitive than SCEs for the detection of chromosomal damage caused by exposure to low doses of styrene.     

The mutagenic effect of benzene, toluene and xylene studied by the SCE technique.

In experiments in vitro, neither benzene, toluene nor xylene changed the number of sister-chromatid exchanges (SCEs) or the number of chromosomal aberrations in human lymphocytes. Toluene and xylene caused a significant cell growth inhibition which was not observed with benzene in the same concentrations.     

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.     

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.     

In vivo cytogenetic effects of cooked food mutagens

Using a variety of in vivo cytogenetic endpoints, we have investigated the effects of several compounds formed during the cooking of meat. C57Bl/6 mice were used to test for an increase in the frequency of sister-chromatid exchanges (SCEs), chromosomal aberrations, and micronucleated erythrocytes by 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx). 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (DiMeIQx), and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). MeIQx and DiMeIQx did not induce SCEs in mouse bone marrow cells. PhIP induced sister-chromatid exchanges, but not chromosomal aberrations in bone marrow. In peripheral blood lymphocytes, PhIP did induce aberrations at 100 mg/kg, the highest dose tested. PhIP induced a low but significantly increased frequency of micronuclei in normochromatic but not polychromatic erythrocytes in bone marrow and peripheral blood. However, dose responses were not observed. With the exception of the SCEs induced by PhIP, these results contrast with observations made in vitro, where these compounds were found to have significant genotoxicity in mammalian cells and a very high mutation frequency in prokaryotic systems.     

Relative ranking of culture media based on proliferation kinetics and spontaneous chromosomal aberrations in PHA-responsive human peripheral blood lymphocytes.

Proliferation kinetics and spontaneous yield of chromosomal aberrations phytohemagglutinin (PHA)-responsive peripheral blood lymphocytes were studied from blood samples collected from 45 individuals in 4 different synthetic media. Except for a significant difference for Eagle's MEM and RPMI 1640, the other media did not show difference for the yield of chromatid or chromosome type of aberrations. Differences were however noticed in the proliferation kinetics (mitotic and proliferative rate indices) of cells among the media used. The study indicated that (i) the intrinsic properties of media which influence proliferation rate and yield of chromosomal aberrations are independent of each other as higher proportion of first division cells do not correspond with higher frequency of chromosomal aberrations, (ii) the amount of free-radical scavengers present in the medium, apart from the genetic make-up of the individuals, may contribute to the spontaneous yield of chromosomal aberrations and (iii) RPMI 1640 medium, which showed higher transformation and faster cycling rate for the lymphocytes, may be considered as medium of choice for analysing two main cytogenetic end-points, chromosomal aberrations and sister chromatid exchanges (SCEs).     

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.     

Chromosomal analysis in vinyl chloride exposed workers: Comparison of the standard technique with the sister-chromatid exchange technique

A group of 21 workers occupationally exposed to vinyl chloride and 6 controls were examined for the presence of chromosomal aberrations or sisterchromatid exchanges in their peripheral lymphocytes. These people comprised a second sampling from a group of exposed workers and controls first examined 18 months earlier. The vinyl chloride exposed workers showed levels of chromosomal aberrations elevated above those of the controls, but there was only a slight increase in sister-chromatid exchanges (per cell or per chromosome) and this increase was not statistically significant. Sister-chromatid exchanges (SCEs) were also examined from in vitro cultures of lymphocytes exposed in G₀/early G₁ and late G₁/early S phase to vinyl chloride, both with and without metabolic activation. There was no increase in SCEs in vitro without metabolic activation but there was a marked increase with metabolic activation and this increase was shown to be independent of cell-cylce phase. It thus was apparent that the small increases of SCEs in workers were not due to the inability of vinyl chloride to induce SCEs in human lymphocytes but were probably because of low exposures and SCE levels could have returned to normal relatively quickly after exposure. The present study suggested that the analysis of longer-living conventional chromosomal aberrations appeared to be a more sensitive monitor of exposure to vinyl chloride in exposed workers than the estimation of SCEs; however, it should be noted that in a 3rd sampling taken 24 months later the exposed workers had chromosomal aberration levels similar to the controls.     

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.     

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 of chromosomal aberrations, sister-chromatid exchanges and cell-cycle delay in cow peripheral blood lymphocytes treatment with two N-nitroso compounds in vitro

We investigated the effect of NDMA and DNSGU on the induction of chromosomal aberrations and sister-chromatid exchanges (SCEs), as well as the influence of the former compound on cell-cycle kinetics in cultured cow peripheral lymphocytes. A clastogenic effect was observed in treated cell cultures at 6 or 12 × 10⁻⁵ M concentrations of NDMA and DNSGU, respectively, but no increase of chromosomal breaks was seen at the lowest dose. NDMA at 6 × 10⁻⁴ M was toxic to cow lymphocytes. NDMA and DNSGU induced statistical increases of SCEs at the test doses (6 or 12 × 10⁻⁶ and 6 or 12 × 10⁻⁵ M, respectively). In addition, treatment with NDMA at a dose of 6 × 10⁻⁵ M revealed significant heterogeneity of the first, second and third metaphases between treated and untreated groups. A reduction of the proliferation index and proliferation delay per cycle was shown too.     

The effect of continuous gamma irradiation on formation of sister chromatid exchanges (SCEs) and chromosomal aberrations in meristematic cells ofVicia faba

Germinated seeds ofVicia faba were continuously irradiated at low dose rate of gamma rays (0.05 Gy h-¹) up to a total accumulated dose of 2 Gy. The FPG (fluorescence plus Giemsa) technique of differential chromatid staining was used to monitor the frequency of sister chromatid exchanges (SCEs) in irradiated root tip meristem cells. The results of the experiments have demonstrated that SCE frequency is raised by continuous gamma irradiation only in plant cells containing BrdU in the chromosomal DNA. No effect concerning SCE formation was recorded at continuous irradiation of meristematic cells of Vicia faba with native, i. e. BrdU-nonsubstituted, DNA. In contrast to SCEs, a significant increase was found in the yield of chromosomal aberrations in all variants of irradiation.     

Bimodal distribution of sensitivity to SCE induction by diepoxybutane in human lymphocytes. I. Correlation with chromosomal aberrations

We carried out a cross-sectional analysis of sister-chromatid exchanges (SCEs) and chromosomal aberrations induced by diepoxybutane (DEB) in lymphocyte cultures from 58 normal blood donors. DEB-induced SCE frequencies were measured in all subjects and chromosomal aberrations in 18. Analysis of variance was used to assess the contributions of exposure to organic solvents, age, smoking history, alcohol and coffee consumption, and red and white blood cell counts to variations in DEB-induced SCEs. In 10 individuals, the epoxide-detoxifying enzyme, glutathione (GSH)-S-transferase mu, was also measured. We observed a bimodal distribution of DEB-induced SCEs in the study population. Approx. 24% of the individuals were twice as sensitive to the induction of SCEs by DEB as the remaining 76%. Lymphocytes from persons sensitive to SCE induction by DEB contained a 4.4-fold increase in the number of DEB-induced chromatid deletions and exchanges. Within sensitive and resistant groups, significant interindividual variations in DEB-induced SCE frequencies were noted. Cigarette smoking was weakly associated with lower SCE frequencies within each group. Genetic deficiency in GSH-S-transferase mu was not correlated with increased sensitivity to SCE induction by DEB. Sensitivity to induction of SCEs by DEB can be rapidly determined and may be a marker of sensitivity to the induction of genotoxicity by certain classes of mutagens.