Clastogenic effects of bleomycin, cyclophosphamide, and ethyl methanesulfonate on resting and proliferating human B- and T-lymphocytes

The effects of bleomycin (BM), cyclophosphamide (CP), and ethyl methanesulfonate (EMS) on the frequencies of chromosomal aberrations were tested in mitogen-stimulated highly purified human B- and T-lymphocytes. In unstimulated G0/G1 B- and T-lymphocytes the clastogen induction of chromosome fragments was investigated in prematurely condensed chromosomes (PCC) induced by cell fusion with xenogenic mitotic cells. BM, CP (with metabolic activation), and EMS induced a significant increase in chromosome aberrations in proliferating human B- and T-lymphocytes. There were no significant differences in the BM-induced aberration rates between the cell populations. CP and EMS induced more aberrations in T- than in B-lymphocytes. In the PCC tests, BM-exposed G0/G1 lymphocytes showed dose-dependent high yields of chromosome fragments. No significant differences between B- and T-lymphocytes were observed. CP and EMS induced no clear increase in fragments in either cell population.     

Sister-chromatid exchange analysis in a rural population of Mexico exposed to pesticides.

Cytogenetic damage was evaluated by means of the analysis of sister-chromatid exchange (SCE) in a rural population of Tlaxcala, Mexico, in occupational contact with pesticides. We studied 170 men, 94 exposed and 76 not exposed. It was shown that SCE followed a normal distribution and Student's t test did not present differences between the two groups (P = 0.4). The frequency of SCE was not correlated with the duration of exposure of the rural workers (r = -0.06), the multiple covariance analysis applied to the data of duration of exposure, tobacco intake and alcohol ingestion demonstrated a lack of statistical significance. In the exposed people we observed no symptoms provoked by these compounds.     

Sister-chromatid exchange in 4 human races

The frequencies of sister-chromatid exchanges (SCE) were investigated in lymphocytes in 32 normal adult individuals of both sexes with no interracial familial backgrounds from Caucasian, American black, oriental and native American races. There was no significant difference in the average frequency of SCEs in the 4 races.     

Proliferation kinetics of human B- and T-lymphocytes

Human peripheral blood B- and T-lymphocytes, highly purified by immunological methods, were supplemented with gamma-irradiated unseparated autologous mononuclear cells to restore helper functions and stimulated with pokeweed mitogen and phytohaemagglutinin, respectively. Proliferation kinetics of the cell populations were investigated using 5-bromodeoxyuridine (BrdU) labelling of the cell cultures and chromosome preparation at different times after stimulation. The percentages of metaphase cells having replicated for one, two or three generations in the presence of BrdU were determined following sister chromatid differential staining. In all donors, the changes in these percentages were faster in B- than in T-lymphocytes, indicating a longer cell cycle time in the latter population.     

Proliferation kinetics of human B- and T-lymphocytes

Abstract Human peripheral blood B- and T-lymphocytes, highly purified by immunological methods, were supplemented with γ-irradiated unseparated autologous mononuclear cells to restore helper functions and stimulated with pokeweed mitogen and phytohaemagglutinin, respectively. Proliferation kinetics of the cell populations were investigated using 5-bromodeoxyuridine (BrdU) labelling of the cell cultures and chromosome preparation at different times after stimulation. The percentages of metaphase cells having replicated for one, two or three generations in the presence of BrdU were determined following sister chromatid differential staining. In all donors, the changes in these percentages were faster in B- than in T-lymphocytes, indicating a longer cell cycle time in the latter population.     

Sister-chromatid exchange and cell kinetics in CHO-K1 cells, human fibroblasts and lymphoblastoid cells exposed in vitro to asbestos and glass fibre

Possible mutagenic activity of the asbestos dusts crocidolite and chrysotile, and fine and coarse glass, was assessed in CHO-K1 cells, human fibroblasts and human lymphoblastoid cells using the sister-chromatid exchange assay and by examining the effects on cell kinetics. Asbestos caused no dose-related increase in sister-chromatid exchange levels in any of the cell types. However, mitotic delay was induced in CHO-K1 cells and human fibroblasts. The order of magnitude of induced delay in CHO-K1 cells was chrysotile greater than fine glass greater than crocidolite greater than coarse glass. Mitotic inhibition was more pronounced in these cells if they were still in suspension when initially exposed to the dusts compared with 1 h after plating.     

Sister-chromatid exchanges in human lymphocytes exposed in vitro to therapeutic ultrasound

Human lymphocytes were exposed in vitro to therapeutic levels of ultrasound (1 W/cm2, CW, 0.87 MHz, durations of 80 and 160 sec). There were no significant differences in sister-chromatid exchange frequencies between controls and ultrasound-exposed cells. Exposure of lymphocytes to the positive control (mitomycin C) resulted in a significant increase in sister-chromatid exchanges. The data do not verify a report by Stella et al. (Mutation Res., 138 (1984) 75-85) that such exposures result in increased frequencies of SCEs.     

Sister-chromatid exchange (SCE) among individuals chronically exposed to arsenic in drinking water

A study was carried out on human subjects of various ages and backgrounds who had been drinking water containing more than 0.13 mg/l (0.13 ppm) arsenic for a period of at least 20 years. The main aim was not only to correlate the frequency of sister-chromatid exchanges in the lymphocytes with the amount of arsenic in water and urine but also to correlate the frequency of SCE with sex and age. In addition, family background regarding skin alterations or other arsenic-related symptoms was explored, so that individual health conditions could be assessed. External factors such as exposure to other chemical or contaminating agents (pesticides, battery manufacturing plants, foundries) were also taken into consideration. The data on sister-chromatid exchanges (282 exposed and 155 control individuals) showed that arsenic at concentrations used by our population (0.13 mg/l) induced a significantly elevated response. Other health effects of arsenic at these concentrations were found, e.g., hyperkeratosis, melanosis, actinic keratosis, basal cell carcinoma.     

Sister-chromatid exchanges in human lymphocytes exposed during Go to four classes of DNA-damaging chemicals.

Human lymphocytes were treated prior to mitogenic stimulation with varying concentrations of 6 cytostatic drugs representing 4 classes of DNA-damaging chemicals. Afterwards the cells were washed to remove residual chemical and cultured in the presence of bromodeoxyuridine for analysis of sister-chromatid exchanges (SCEs). A dose-related increase in SCEs was observed in cells exposed during Go to the alkylating chemicals mitomycin C, chlorambucil, and thiotepa, while significant increases in SCEs were not noted in cultures exposed to methotrexate, cytarabine, or bleomycin. These findings suggest that not all classes of clatogenic chemicals which induce SCEs in proliferative cells substituted with BUdR are capable of inducing long-lived lesions in the DNA of Go lymphocytes that can lead to SCE formation.     

Sister-chromatid exchange (SCE) analysis in mothers exposed to DNA-damaging agents and their newborn infants

The incidence of SCE in the lymphocytes of mothers and their newborn infants was determined. A detailed antenatal history of parental habits such as smoking, alcohol consumption and possible exposure to DNA-damaging agents was documented. The results showed that the SCE rate in the newborn is significantly less than that of their mothers. Mothers who consumed alcohol, but not cigarette smokers, had a significantly increased SCE rate compared to control mothers. However, these maternal habits did not affect the SCE rate of their infants. Neonates with neural tube defects showed a significantly increased SCE rate compared to normal babies.     

Sister-chromatid exchange in highly purified human CD4+ and CD8+ lymphocytes.

Sister-chromatid exchange (SCE) frequencies were determined in human peripheral blood CD₄⁺ and CD₈⁺ T lymphocyte subpopulations which were rapidly and highly purified from pooled T lymphocytes by immunological methods. The purified lymphocytes were stimulated with phytohemagglutinin (PHA) for 4 days. CD₄⁺ lymphocytes showed significantly higher SCE frequencies than autologous CD₈⁺ lymphocytes when measured simultaneously after identical bromodeoxyuridine (BrdU) incubation times. Differences in SCE frequencies between CD₄⁺ and CD₈⁺ lymphocytes were also detected when mitomycin C (MMC) was added to the cultures. Higher SCE frequencies in CD₄⁺ lymphocytes were associated with lower proliferating rate indices (PRI) as compared to autologous CD₈⁺ lymphocytes. Abnormalities in CD₄⁺ T lymphocyte function and number in peripheral blood have been observed in several diseases characterized by immunological disorders. Thus, our data may suggest a link between some immunological disturbances and abnormal SCE frequencies in T lymphocyte subsets.     

Sister-chromatid exchanges and chromosomal aberrations in a population exposed to pesticides

Sister-chromatid exchanges (SCE) and chromosomal aberrations were studied in a population of floriculturists occupationally exposed to organophosphorus, carbamate and organochlorine pesticides. Blood samples from 36 individuals from a community of 154 persons of asiatic origin were obtained. Among the group sampled, 21 individuals exhibited at least one symptom of chronic intoxication. SCE analysis was performed in 14 symptomatic and 13 asymptomatic persons. The asymptomatic group showed a SCE frequency of 5.47 +/- 1.03 and the symptomatic group a frequency of 6.45 +/- 1.19. Comparison between both groups with the Mann-Whitney 'U' test revealed a significant difference (p 0.0409). Case-control analysis of 9 pairs matched by sex and age also showed significant differences between both groups (p 0.0104). In contrast, the frequencies of chromosomal aberrations were not correlated with intoxication symptomatology, though a significant increment of exchange-type aberrations in relation to a group of non floriculturists was observed in the population studied.     

Sister-chromatid exchange in childhood in relation to age and sex

Small children have been found to have a lower SCE/cell than adults and in recent reports females have had higher SCEs/cell than males. We here describe the relationship between SCE/cell and age and sex in 46 girls and 39 boys with an age range of 1.4-19.2 years and 2.6-18.7 years, respectively. For the calculation a transformation y = (sum SCE)1/2 + (sum SCE + 1)1/2 was used. The best fit to our material was represented by the equation y = b0 + b1 X log age. A common slope (b1) could be used for the boys and girls. This slope was significantly different from zero (P less than 0.0005). The levels of the regression lines for the two sexes were different (P = 0.0006). The girls had a 0.55-0.7 higher SCE/cell than the boys, depending on age. The following equations were found: Girls: y = 22.49 + 6.53 X log age. Boys: y = 21.11 + 6.53 X log age. By this model 43% of the variation in y could be explained. As a consequence of the result it is absolutely essential, when planning studies of children, to use age-matched groups to decrease the variability of the test system.     

Sister-chromatid exchanges in lymphocytes of workers exposed to trichloroethylene

To detect mutagenic effects of trichloroethylene (TCE) on humans, sister-chromatid exchanges (SCEs) were analyzed in lymphocytes of 22 workers occupationally exposed to TCE and 22 matched controls. Although urinalysis in the workers revealed their obvious exposure to TCE, no increase in SCE frequencies was found in lymphocytes of the workers. SCE analysis in lymphocytes could not detect mutagenic effects by occupational exposure to TCE on humans.     

A statistical evaluation of the reproducibility of micronucleus, sister-chromatid exchange, thioguanine-resistance and ouabain-resistance assays in V79 cells exposed to ethyl methanesulfonate and 7,12-dimethylbenz[a]anthracene

In contrast to the "validation" of short-term in vitro genotoxicity assays by concordance with the rodent cancer bioassay, the present report describes the multiple replication of 4 short-term tests with V79 cells (micronucleus assay, MN; sister-chromatid exchange, SCE; ouabain resistance. OUR; and thioguanine resistance, TGR) within the same assay system following exposure to each of two genotoxins, ethyl methanesulfonate (direct acting) and 7,12-dimethylbenz[a]anthracene (indirect acting). Reproducibility, proportion of genotoxins correctly identified, and proportion of non-genotoxins correctly identified by each test were each determined statistically. Decision rules were formulated to declare a positive response in each assay, and overall accuracy of each was determined. Statistical analysis of the data, obtained under standardized test conditions, showed that for these two chemicals SCE identified 100% of genotoxins and 86% of non-genotoxins, with overall accuracy of prediction of 93%; TGR identified 98% of genotoxins and 74% of non-genotoxins, with overall accuracy of 86%; MN identified 78% of genotoxins and 84% of non-genotoxins, with overall accuracy of 81%; while OUR indicated 100% of genotoxins, but only 50% of non-genotoxins, and only 76% overall accuracy. The results suggested that the best overall accuracy of classification with the V79 assay system could be achieved by measurement of SCE in combination with thioguanine resistance.     

Some electrical properties of murine thymocytes, and B- and T-lymphocytes.

The electrical properties of the peripheries of mouse thymocytes, B-lymphocytes and T-lymphocytes have been examined in the fresh and glutaraldehyde-fixed states by electrophoresis, and in the fixed state by electron microscopic observation of the densities of binding of positively charged, colloidal iron hydroxide (CIH) particles. On the one hand, the electrophoretic mobilities of the T-lymphocytes were higher than those of either the B-lymphocytes or the thymocytes, in both the fresh and fixed states. On the other hand, the densities of CIH particles binding to the B-lymphocytes were higher than those on either the T-lymphocytes or the thymocytes. It is suggested that the disproportions between the electrokinetic and electron microscopic observations in the three classes of cells, are due to different (average) numbers of anionic sites, in the clusters of these sites marked by each CIH particle.     

Mutational specificities of 1'-acetoxysafrole, N-benzoyloxy-N-methyl-4-aminoazobenzene, and ethyl methanesulfonate in human cells

We have used an oriP-tk shuttle vector to determine the types of mutations induced in human cells by ethyl methanesulfonate (EMS), 1'-acetoxysafrole (AcOS), and N-benzoyloxy-N-methyl-4-aminoazobenzene (BzOMAB). Plasmid DNA was treated in vitro with mutagen and electroporated into human lymphoblastoid cells. After replication of the vector in human cells, plasmids were analyzed for mutations in the herpes simplex virus type 1 thymidine kinase gene. Ethyl methanesulfonate induced predominantly GC----AT transition mutations. Treatment of the shuttle vector with AcOS induced 5 of the 6 possible base substitution mutations, including GC----AT (32%) and AT----GC (14%) transition mutations, GC----TA (9%), GC----CG (18%), and AT----TA (14%) transversion mutations, as well as a low frequency (9%) of -1 frameshift mutations at GC base pairs. Replication in human cells of DNA modified with BzOMAB yielded a significant increase (17-fold) in the frequency of deletion mutations relative to solvent-treated DNA. A majority (94%) of the point mutations induced by BzOMAB occurred at GC base pairs and were predominantly GC----AT transitions (33%) and -1 frameshift (22%) mutations, with the remainder consisting mainly of transversions at GC base pairs (28%). The broad spectrum of base substitution mutations observed for AcOS and BzOMAB may indicate the frequent insertion of a variety of bases during replicative bypass of aralkylated bases in human cells.     

Combined mutagenicity of methyl methanesulfonate and ethyl methanesulfonate in Chinese hamster V79 cells.

The combined effects of methyl methanesulfonate (MMS) and ethyl methanesulfonate (EMS) on the induction of 6-thioguanine (6TG)-resistant mutants and chromosome aberrations were examined in Chinese hamster V79 cells. Cells were simultaneously treated with EMS at a concentration of D20 and MMS at various concentrations for 3, 6 or 9 h. In other experiments cells were simultaneously treated with MMS at a concentration of D20 and EMS at various concentrations for 3, 6 or 9 h. The mathematical analysis of the combined effects of both chemicals for cell killing (cytotoxicity) and 6TG-resistant mutations indicates that synergistic interactions were observed for both cell killing and mutations induced by MMS and EMS. The frequency of chromosome aberrations induced by simultaneous treatment with MMS at a concentration of D20 and EMS at various concentrations for 3 h was additive. However, the frequency of chromosome aberrations induced by EMS at a concentration of D20 and MMS at various concentrations for 3 h was not significantly different from those induced by MMS alone.