Analysis of the frequency and distribution of sister chromatid exchanges in cultured human lymphocytes

Summary Lymphocytes from 20 normal subjects (11 male and 9 female) were examined for the frequency and location of sister chromatid exchanges (SCE) by the BrdU—Giemsa method. The mean frequency of SCE was 6.37 with little significant variation. One subject had a high number of exchanges in chromosome 1 while the remainder showed a random distribution of exchanges between chromosomes. The frequency of exchanges generally increased with chromosome length. However, chromosome 1, 2 and the B group had more exchanges than expected while the E, F and G groups had less than expected. The distribution of exchanges in chromosomes 1, 2 and the B group was non-random with a concentration of exchanges below the centromere and to a lesser extent on the distal portion of the long arm. The majority of exchanges appeared to occur at the junction between the dark and light G bands. It is suggested that the concentration of exchanges may reflect differences in BrdU incorporation along the length of the chromosome.     

Non-uniform distribution of sister chromatid exchanges in human lymphocytes

To test whether sister chromatid exchange (SCE) scores on human chromosomes have a uniform distribution, simulated SCE scores were generated and compared with observed scores using log-linear models. The analysis was performed at the level of the chromosome groups. Using this method we first tested whether the number of SCEs was distributed uniformly, i.e. proportional to the relative length of the chromosomes. Refinements of this hypothesis were made by considering a variable region around a first SCE to be inert for other SCEs and by making the occurrence of an SCE on a chromosome dependent on the occurrence of another SCE on the same chromosome. In further analyses it was tested whether the number of SCEs was proportional to the number of G bands on a chromosome, or to the DNA content of the chromosomes. None of the tested hypotheses fitted the observed data, establishing the non-uniform distribution of these events.     

Ultrasonic induction of sister chromatid exchanges in human lymphocytes

Summary We analyzed sister chromatid exchange (SCE) frequencies as an indicator of DNA damage induced in human lymphocytes by real-time ultrasound. A range of exposure times and intensities was tested in a series of blind, randomized, in vitro experiments under spatial and sonographic conditions simulating exposure of a gravid abdomen and uterus. Our studies showed small but consistent effects of ultrasound on SCE frequencies, for each experiment. Differences between matched control and exposed means were significantly different from zero. X ² tests for homogeneity indicated no significant differences among either the means or the total distributions of the controls, nor among each of the separate dose levels. Consequently, experiments were pooled, and X ² analysis indicated significant differences both among distributions and among means of SCE frequencies for controls versus exposed cells (P(0.001). The pooled control mean was also significantly different from each of the pooled dose means. Correcting for multiple comparisons gave identical results for the paired comparisons of means except for the 20-min level which was borderline (0.025P(0.01). We conclude that the well-established value of clinical ultrasonography warrants its continued use; however, minimizing the numbers and lengths of exposure per patient would seem prudent, pending further information on clinical implications of our results.Supported in part by NIH-HD82855 and HD 11021 and a National Foundation Summer Science Research Grant for Medical Students, 8-80-22     

Reduced frequency of baseline sister chromatid exchanges in lymphocytes grown in antibiotics and serum-excluded culture medium

Peripheral venous blood from man, muntjac, and cattle were grown in medium (1) containing different serum (human AB+/FCS/autologous), (2) without serum or antibiotics (penicillin and streptomycin), or (3) without both serum and antibiotics to investigate to what extent certain essential culture components used in in vitro mammalian cell cultures affect the incidence of spontaneous sister chromatid exchanges (SCEs). The observation that exclusion of only serum from culture medium enhanced the frequency of SCEs whereas exclusion of both serum and antibiotics, which influence the cell cycle kinetics to a great extent, exhibited the lowest frequency of SCEs reported so far for lymphocyte cultures, indicates that the frequency of so-called spontaneous SCEs usually observed in normal lymphocyte cultures grown in medium supplemented with serum and antibiotics is in fact an elevated frequency.     

Analysis of sister chromatid exchanges in lymphocytes cultured from 71 healthy men

Sister chromatid exchanges (SCE) were analyzed in peripheral blood lymphocytes from a select group of 71 healthy men, 56 nonsmokers and 15 cigarette smokers. In addition to estimating baseline SCE, data were examined to seek relationships of SCE frequencies to age and smoking. The baseline value of 7.53 SCE per cell from the 56 nonsmokers was within the range (5.60 to 9.10 SCE/cell) reported for other human populations. No relationship was found between the mean SCE frequency per cell and age. However, a significant increase in the SCE mean value was observed in smokers as compared to nonsmokers. The results of this study are compared with those of other reports on SCE effects of age and smoking.Abbreviations BUdR 5-bromo,2-deoxyuridine - SCE sister chromatid exchange     

The effects of boric acid on sister chromatid exchanges and chromosome aberrations in cultured human lymphocytes

The aim of this study was to determine the possible genotoxic effects of boric acid (BA) (E284), which is used as an antimicrobial agent in food, by using sister chromatid exchange (SCEs) and chromosome aberration (CAs) tests in human peripheral lymphocytes. The human lymphocytes were treated with 400, 600, 800, and 1000 μg/mL concentrations of BA dissolved in dimethyl sulfoxide (DMSO), for 24 h and 48 h treatment periods. BA did not increase the SCEs for all the concentrations and treatment periods when compared to control and solvent control (DMSO). BA induced structural and total CAs at all the tested concentrations for 24 and 48 h treatment periods. The induction of the total CAs was dose dependent for the 24 h treatment period. However, BA did not cause numerical CAs. BA showed a cytotoxic effect by decreasing the replication index (RI) and mitotic index (MI). BA decreased the MI in a dose-dependent manner for the 24 h treatment period.     

Micronuclei and sister chromatid exchanges induced by capsaicin in human lymphocytes

Escherichia coli has provided an important model system for understanding the molecular basis for genetic instabilities associated with repeated DNA. Changes in triplet repeat length during growth following transformation in E. coli have been used as a measure of repeat instability. However, very little is known about the molecular and biological changes that may occur on transformation. Since only a small proportion of viable cells become competent, uncertainty exists regarding the nature of these transformed cells. To establish whether the process of transformation can be inherently mutagenic for certain DNA sequences, we used a genetic assay in E. coli to compare the frequency of genetic instabilities associated with transformation with those occurring in plasmid maintained in E. coli. Our results indicate that, for certain DNA sequences, bacterial transformation can be highly mutagenic. The deletion frequency of a 106 bp perfect inverted repeat is increased by as much as a factor of 2 x 10(5) following transformation. The high frequency of instability was not observed when cells stably harboring plasmid were rendered competent. Thus, the process of transformation was required to observe the instability. Instabilities of (CAG).(CTG) repeats are also dramatically elevated upon transformation. The magnitude of the instability is dependent on the nature and length of the repeat. Differences in the methylation status of plasmid used for transformation and the methylation and restriction/modification systems present in the bacterial strain used must also be considered in repeat instability measurements. Moreover, different E. coli genetic backgrounds show different levels of instability during transformation.     

The frequency and distribution of sister chromatid exchanges in human chromosomes

Summary A detailed procedure is described for a rapid detection of phosphoglucomutase-2 (=phosphopentomutase; PGM-2) on Cellogel following electrophoresis of extracts of human red blood cells and other tissues, including cultured fibroblasts and various types of primate-rodent somatic hybrid cells.The present study indicated that there is only one locus for phosphopentomutase in man. The data from a selected panel of 20 independent clones of man-mouse somatic cell hybrids, investigated for the presence of human chromosomes and for the presence or absence of human PGM-2 favored the assignment of the human PGM-2 locus to chromosome 4.     

A schedule to demonstrate radiation-induced sister chromatid exchanges in human lymphocytes

Summary The reciprocal interchange between the chromatids of a chromosome, termed sister chromatid exchange (SCE), is considered to be one of the most sensitive and accurate cytogenetic parameters and respond to toxic chemicals at very low doses. But the response of SCE to ionizing radiation is very poor. Human lymphocytes fail to give SCE response when irradiated at G₀. Probably the primary lesions induced at G₀ do not remain available long enough to find expression as SCEs. Based on this assumption a schedule was developed using caffeine to demonstrate radiation induced SCEs. Following this schedule a dose-dependent increase in the frequency of radiation induced SCEs has been observed.     

Chromosome aberrations and sister chromatid exchanges in cultured human lymphocytes treated with sodium metabisulfite, a food preservative

The aim of this study was to investigate the ability of sodium metabisulfite (SMB) which is used as an antimicrobial substance in food, to induce chromosome aberrations (CA) and sister chromatid exchanges (SCE) in human lymphocytes. SMB-induced CAs and SCEs at all concentrations (75, 150 and 300 microg/ml) and treatment periods (24 and 48h) dose-dependently. However, SMB decreased the replication index (RI) and the mitotic index (MI) at the concentrations of 150 and 300 microg/ml for 24 and 48h treatment periods. This decrease was dose-dependent as well.     

Chromosome aberrations and sister chromatid exchanges in cultured human lymphocytes treated with sodium metabisulfite, a food preservative

The aim of this study was to investigate the ability of sodium metabisulfite (SMB) which is used as an antimicrobial substance in food, to induce chromosome aberrations (CA) and sister chromatid exchanges (SCE) in human lymphocytes. SMB-induced CAs and SCEs at all concentrations (75, 150 and 300 μg/ml) and treatment periods (24 and 48 h) dose-dependently. However, SMB decreased the replication index (RI) and the mitotic index (MI) at the concentrations of 150 and 300 μg/ml for 24 and 48 h treatment periods. This decrease was dose-dependent as well.     

5-Bromodeoxycytidine in the study of sister chromatid exchanges in human lymphocytes

Summary When [³H]dC was added with a high dose (4x10^-1 mM) of dT to human blood lymphocyte cultures, much heavier labeling of interphase nuclei and metaphase chromosomes was observed compared with that in cultures treated with [³H]dC alone. This observation indicates that in the presence of excess dT, exogenous dC is included into cytosine bases of DNA, releasing the cells from the thymidine block.BrdC 5x10^-2 mM added with a high dose of dT (4x10^-1 to 1.0 mM) to the cultures did not relieve the thymidine block as determined from the percentage of metaphases of the first to third divisions. It is concluded that BrdC, in contrast to dC, is not utilized as a cytosine DNA precursor even in the presence of high concentrations of dT.The frequency of SCEs per cell was the same when studied with the aid of BrdC and BrdU used under similar conditions. The distribution of SCEs among chromosomes was also identical for both analogues: The number of SCEs was significantly higher than expected in chromosomes of group B and lower than expected in chromosomes of groups E, F, and G.     

Increased frequency of sister chromatid exchanges in peripheral lymphocytes of alcoholics and cigarette smokers

Sister chromatid exchange (SCE) is a sensitive indicator of genotoxicity. In this study we investigated the effects of alcohol consumption and cigarette smoking on the frequency of SCE in cultures of peripheral lymphocytes. The rate was higher in alcoholics who smoked (10.89+/-2.46) and in smokers (positive controls) (7.64+/-1.01) than in healthy non-smokers (negative controls) (6.96+/-2.18). Statistical analysis suggested that the increases were related to alcohol consumption and cigarette smoking (p<0.05).     

Benzpyrene-Induced sister chromatid exchanges in lymphocytes of patients with lung cancer

Summary The effect of benzpyrene on sister chromatid exchange was determined in PHA-stimulated lymphocytes of 18 patients with lung cancer and 11 controls without cancer or bronchopulmonary diseases. Patients and controls did not differ either with respect to the spontaneous rate of sister chromatid exchanges or in their response to the carcinogen. We conclude that individual susceptibility to lung cancer cannot be detected by an individual response to benzpyrene, at least in lymphocytes and at the chromosomal level.     

UV-light induced sister chromatid exchanges in xeroderma pigmentosum lymphocytes

Summary Cultured lymphocytes from 9 patients with clinically different types of xeroderma pigmentosum were exposed to ultraviolet light at 24 h. An increased rate of sister chromatid exchanges was observed in 6 patients (128–148% increase in three, 34–51% in three), but not in three patients with deSanctis-Cacchione syndrome (xeroderma pigmentosum with mental defect), compared to simultaneously cultured controls. A positive result could be useful as preliminary cytogenetic diagnostic test. The results are interpreted as an expression of UV-light induced chromosomal instability due to impaired DNA repair.     

Frequency of sister chromatid exchanges in human lymphocytes at different times of day

Analysis of the frequency of sister chromatid exchanges in the human blood taken from normal female donors was made at 9 a. m. and 9 p. m. The morning rate of exchange is shown to be less than the evening one. The influence of physiological state of the organism on the sister chromatid exchange is suggested.     

Interaction of nickel with mutagens in the induction of sister chromatid exchanges in human lymphocytes

In this study, individual treatments of human lymphocytes with Ni(II) [0.5–25 μM], Cr(VI) [0.65–1.30 μM], UV-light or X-rays induced SCEs in a dose-dependent fashion, and combined treatments of Ni(II) with Cr(VI), UV-light or X-rays interacted antagonistically. Nickel, at environmentaly relevant exposure levels, cna have the effect in complex mixtures of reducing an otherwise positive SCE response and could lead to underestimating human exposures to certain classes of chemicals or radiation. Furthermore, our data indicate that antagonism may occure when human lymphocytes are exposed simultaneously to Ni(II) and Cr(VI), suggesting an explanation for epidemiological studies reporting conflicting results for cytogenetic effects in lymphocytes of workers exposed to chromium and nickel.     

Adaptation-like response to the chemical induction of sister chromatid exchanges in human lymphocytes

Summary Experiments have been performed to determine whether human lymphocytes in primary cultures can show an adaptive response to the induction of cellular lesions (manifested as a production of sister chromatid exchanges, SCEs) as previously found in bacteria and established human and mammalian cell lines. Human lymphocytes were pretreated with various subtoxic concentrations (5–50ng/ml) of N-methyl-N-nitro-N-nitrosoguanidine (MNNG) once every 6h for 72h, and subsequently challenged by a high dose (4g/ml) of MNNG. The lymphocytes in MNNG-challenged cultures had the lowest frequency of SCEs when pretreated with 10ng/ml MNNG. Further cross-resistance study revealed that repeaied pretreatments of lymphocytes with 10ng/ml MNNG for 72h can render the cells resistant to the induction of SCEs by the following challenge with a high dose of MNNG, but not of mitomycin C or ethyl nitrosourea. The data also suggest variations in the degree of the adaptation-like response among individuals.     

Effect of deoxycytidine on the frequency of sister chromatid exchanges in human lymphocytes detected by using 5-bromdeoxyuridine

The frequency of sister chromatid exchanges (SCE) was studied in cultivated blood lymphocytes of three normal individuals under elimination of DNA synthesis inhibiting action of 5-bromodeoxyuridine (BrdUrd 0.05 mM) with deoxycytidine (Cdr 0.1 and 0.01 mM). The frequency of SCE was significantly increased in the presence of 0.1 mM Cdr. In parallel with SCE frequency, Cdr elevated the percentage of metaphases of the second division. The increase of SCE in the presence of Cdr may be connected with normalization of the DNA replication under its action.     

Effect of incubation temperature on the frequency of sister chromatid exchanges in Bloom's syndrome lymphocytes

Summary The effect of incubation temperature on the frequency of sister chromatid exchange (SCE) has been studied in blood cultures from three Bloom's syndrome (BS) patients, three controls, and three BS heterozygotes. All cell types show slight increases of SCE at 39°C while at 35°C and 32°C, SCE is reduced considerably in BS and slightly increased in normal cells. Prolonging lymphocyte culture to 140 h and adding BUdR for the last two S periods causes a similar decrease in the percentage of SCE in normal and BS cells but, while the latter show a further reduction if they are incubated at 32°C during BUdR labelling, the normal cells show an increase. Therefore, BS and control lymphocytes respond similarly to changes in incubation time and differently to changes in incubation temperature. The possibility that the discrepant behaviour of the BS and control cultures may be due to different growth kinetics of their B and T lymphocytes has been discussed but considered unlikely. Since low temperature lengthens the cell cycle, it has been suggested that our findings and those published by others on co-cultivation experiments (except those of Tice et al. 1978) can be explained by assuming that slow growth reduces SCE in BS cells. This, and unpublished observations (Giannelli et al. 1981), suggest that some imbalance in the factors responsible for DNA replication may exist in BS and possibly account for the high level of SCE.