Induction of sister chromatid exchanges by UV light and its inhibition by caffeine

Sister chromatid exchanges in Chinese hamster chromosomes were studied after pulse-labeling cells with ³H-thymidine at various concentrations. Whereas the frequency of chromatid aberrations varied widely, depending upon tritium dose, there was no significant change in the sister chromatid exchange frequency, even with a 40-fold range of variation in the tritium concentration in the medium. When cells were exposed immediately after labeling to UV light at 40 erg/mm² and examined at the second mitosis, the frequency of sister chromatid exchanges was found to be 4 times higher than that of the unirradiated controls. A synchronization treatment utilizing 2 mM thymidine also caused a two-fold rise in the exchange frequency above the control level. Furthermore, when synchronized cells were irradiated with UV light at a dose of 40 erg/mm², the exchange frequency exceeded 5 times that of the untreated controls. However, this effect was detectable only when cells were irradiated at the earlier part of the S phase, while no change was detected when irradiated at the late S or G2 phase. A post-treatment of irradiated cells with caffeine caused a remarkable decrease in the frequency of sister chromatid exchanges. On the other hand, the frequency of chromatid aberrations of the deletion type increased strikingly after the same treatment. The results appear to suggest a certain correlation between the mechanism involved in the induction of sister chromatid exchanges and a post-replication repair of DNA damage.     

Sister chromatid exchanges induced by sarcolysine in human lymphocytes in vitro

The effect of three concentrations of sarcolysine (0.5 micrograms/ml, 1 microgram/ml and 2 micrograms/ml) on the sister chromatid exchanges (SCE) was investigated in human lymphocytes in vitro. A dose related increase in SCEs frequencies was observed after sarcolysine administration and also a delayed development of cell cycle has been induced by the two last concentrations. The variation range of SCEs per cell was dose-dependent and it was considered to represent the acquired genetic instability induced by the drug.     

Induction of sister chromatid exchanges by chemical mutagens and its possible relevance to DNA repair

Several chemical mutagens were found to induce sister chromatid exchanges in Chinese hamster chromosomes. Among them, effects of 4NQO and MMC were very similar to those of UV light in that the exchange frequency increased with increasing dose of chemicals and that it was markedly lowered in the presence of 1 mM caffeine during a post-treatment period. The frequency of proflavin-induced sister chromatid exchanges was also found to be dose dependent, but it was insensitive to the caffeine post-treatment. On the other hand, no appreciable increase was detected in the incidence of sister chromatid exchanges in MNNG-treated cells over a 100-fold range of variation in chemical dose. Caffeine by itself raised the exchange frequency only slightly over a control level. It was found that 4NQO and MMC exerted remarkable delayed effects on the exchange induction, whereas proflavin did not. This seems to suggest that the lesions caused by the former mutagens would be long-lived and repeatedly provoke sister chromatid exchanges. These data imply that there are several possible ways in which the initial DNA lesions ultimately lead to the formation of sister chromatid exchanges, and that at least UV-, 4NQO- and MMC-induced sister chromatid exchanges would have evolved through a caffeine sensitive repair process, probably related to a post-replication repair of DNA damage.     

Genotoxic effect of benzene hexachloride in cultured human lymphocytes

Summary Chromosomal aberrations, sister chromatid exchanges, mitotic index and cell kinetics were observed in human peripheral lymphocytes after treatment with four different concentrations (0.0125, 0.025, 0.05 and 0.1 g/ml) of benzene hexachloride (BHC), an organochlorine pesticide. Cells were treated with BHC for 24, 48 and 72h. There was a dose-dependent increase in the frequency of chromosomal aberrations and sister chromatid exchanges. A significant decrease in mitotic index was observed at all concentrations and times of exposure. BHC did not show a significant effect on cell kinetics.     

The effect of human alpha leukocyte interferon (IFN) on the frequency of sister chromatid exchanges (SCE) in the KB cell line

Concentrations of 50, 500 and 5,000 iu/ml of natural human alpha leukocyte interferon (IFN) were added into the culture medium of KB cells, 4 h after serial passage, in the presence of 5-bromo-desoxyuridine (Brdu) at 10 micrograms/ml. Similar cultures without IFN were set up as controls. After 72 h of incubation, the harlequin technique (differential staining of sister chromatids) was applied in order to discriminate among the metaphases of different generations and to appreciate the frequency of sister chromatid exchanges (SCE) in the second generation cells. The incidence of sister chromatid exchanges was slightly increased following IFN treatment but no dose-effect relationships were observed. At the same time, cell cycle kinetics estimated as replication index (RI) and average generation time (AGT) was not modified in IFN-treated cells as against the controls.     

THE FREQUENCY OF SISTER CHROMATID EXCHANGES FOLLOWING EXPOSURE TO VARYING DOSES OF H3-THYMIDINE OR X-RAYS

In the Chinese hamster cell line CHEF-125, sister chromatid exchanges occurred at a rate of a little higher than one per three chromosomes for each cell cycle. The exchanges were detectable by labeling with H³-thymidine and autoradiographic analyses of chromosomes at the second and subsequent metaphases after labeling had occurred. To test the hypothesis that sister chromatid exchanges are caused by radiation, cells were incubated in media with different amounts of H³-thymidine. No statistically significant change in the exchange rate was detected over 100-fold range of variation in the amount of incorporated H³-thymidine (determined by grain counts of autoradiographs). We have concluded that sister chromatid exchanges are not caused by tritium radiation and therefore are spontaneous events. Cultures were also irradiated with acute doses of x-rays up to 200 r and scored for sister chromatid exchanges. Between zero and 50 r there was a statistically significant increase in the rate of exchanges. This is interpreted as evidence that x-rays can induce some exchanges, although the majority of these events are probably spontaneous.     

The fragile site (16)(q22)

Summary In the lymphocytes of heterozygous carriers of the rare autosomal fragile site (16)(q22) an exceptionally high frequency of sister chromatid exchanges was demonstrated at the induced fragile site by means of simultaneous berenil and BrdU treatment of the cultures. The rate of sister chromatid exchanges at q22 is also increased in the fragile chromosome 16 by treating the cells with BrdU alone. The possible reasons for the preferential occurrence of induced and spontaneous sister chromatid exchanges at fra (16)(q22) are discussed.     

Increased rates of sister chromatid exchanges induced by the herbicide 2,4-D

The potential for genetic damage from widely used hormonic herbicides, such as 2,4-dichlorophenoxyacetic acid (2,4-D), continues to be of serious concern. The mutagenic effect as reflected by the rates of sister chromatid exchanges (SCE) was determined in cultured human lymphocytes. Data were based on the analysis of 50 cells for the control and each of the three treatments. A 50 micrograms/ml dosage caused a highly significant increase in SCE. Dosages of 100 and 250 micrograms/ml elevated the rate of SCE, but not significantly. Since 2,4-D biodegrades rapidly in soil and water, its continued use is not in serious question until safer compounds are available. However, the results of this study suggest that the danger of genetic damage from direct exposure to commercial samples of 2,4-D should not be ignored.     

Induction of sister chromatid exchanges (SCE) in G0 lymphocytes by plutonium-238 alpha-particles

Irradiation of human G0 lymphocytes with plutonium-238 alpha-particles and X-rays was performed to investigate the production of sister chromatid exchanges (SCE). Alpha-particles produce a significant increase in SCE and this elevation is more significant when separated lymphocytes are irradiated. X-ray irradiation did not induce any significant increase in SCE. Therefore the relative biological effectiveness (RBE) for the induction of SCE by alpha-particles in this system is undefined and effectively infinite.     

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.     

Comparison of chromosome aberrations, sister chromatid exchanges and unscheduled DNA synthesis in the evaluation of the mutagenicity of environmental factors

Mutagenic character of formaldehyde in vivo was estimated by determining the level of chromosomal aberrations, sister chromatid exchanges and unscheduled DNA synthesis in human lymphocytes. It was found that in case of occupational exposure to formaldehyde the unscheduled DNA synthesis after thiophosphamide treatment in vitro was inhibited and spontaneous level of chromosomal aberrations increased. A negative correlation observed between the unscheduled DNA synthesis and sister chromatid exchanges indirectly confirmed a connection of these exchanges with the DNA repair. The comparison of the results obtained from evaluation of chromosomal aberrations, sister chromatid exchanges and unscheduled DNA synthesis permits suggesting that these methods estimate different sides of the mutagen interaction with a cell and should be considered as mutually complementary methods but not as interchangeable ones.     

Analysis of low concentrations of 5-bromo-2-deoxyuridine on sister chromatid exchanges in human lymphocytes

To determine a concentration of 5-bromo-2-deoxyuridine (BrdU) sufficient for sister chromatid differentiation (SCD), and yet having a minimal effect on the number of sister chromatid exchanges (SCEs), we assessed the effect produced on the number of SCEs by low concentrations (1, 3, and 10 micrograms/mL) of BrdU. SCD was not obtained in 19% of the 31 subjects with 1 microgram/mL of BrdU, while the differentiation was adequate for all samples treated with 3 and 10 micrograms/mL. We statistically analysed the effects of these three different doses and found no significant difference in the number of SCEs obtained with the doses of 1 and 3 micrograms/mL, but a significant difference was observed between these two concentrations and 10 micrograms/mL. We therefore suggest that the dose of 3 micrograms/mL, while sufficient to produce reliable differential staining, still permits an adequate evaluation of the base line of SCEs and appears to enhance the sensitivity of the test to evaluate between-individual variations. Our experiments also underline that SCE counts should include the centromere exchanges.     

Clastogenic effects of the fasciolicide drug fasinex on river buffalo lymphocyte cultures in vitro

Fasinex (triclabendazole) has been reported to be an active fasciolocidal agent used in humans and in farm animals. The clastogenic effects of fasinex were tested in lymphocyte cultures of the river buffalo at three final concentrations: 25, 50 and 100 microg/ml. Chromosomal aberrations, sister chromatid exchanges and micronucleus formation are the three cytogenetic parameters used in this study.The results demonstrated that the number of cells with different types of chromosomal aberrations, including chromatid breaks and gaps, isochromatid breaks and gaps and polyploidy, was increased significantly in cultures treated with different doses of fasinex compared to the control. This increase was dose-dependent where there was a positive correlation between increased drug concentration and induction of chromosomal aberrations.The frequency of sister chromatid exchanges and the formation of micronuclei in all lymphocyte cultures treated with different doses of fasinex were increased significantly compared to the control; these increases were also dose-dependent.In conclusion, the three cytogenetic parameters used to evaluate the effect of fasinex revealed that the drug has a strong clastogenic effect on river buffalo lymphocytes in vitro.     

Psoralen/UVA treatment and chromosomes

Summary Treatment of human lymphocytes in vitro with trimethylpsoralen or 8-methoxypsoralen and UVA irradiation (PUVA) induced chromosome damage, mainly constrictions and gaps, but also breaks and exchanges, and increased the frequency of sister chromatid exchange (SCE). The localization of the chromosome aberrations was nonrandom. The coincidence of many PUVA hits with mercaptoenthanol hits suggests that PUVA may have other targets in the cell than the DNA, perhaps the folding proteins of the chromosomes and the nuclear membrane/chromatin attachment organelles.Caffeine increased in a synergistic way the chromosome aberration yield if added after PUVA treatment, but there was no effect when caffeine was present before and during PUVA treatment. The SCE frequency was increased in the presence of caffeine.     

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.     

Automatic measurement of sister chromatid exchange frequency.

An automatic system for detecting and counting sister chromatid exchanges in human chromosomes has been developed. Metaphase chromosomes from lymphocytes which had incorporated 5-bromodeoxyuridine for two replication cycles were treated with the dye 33258 Hoechst and photodegraded so that the sister chromatids exhibited differential Giemsa staining. A computer-controlled television-microscope system was used to acquire digitized metaphase spread images by direct scanning of microscope slides. Individual objects in the images were identified by a thresholding procedure. The probability that each object was a single, separate chromosome was estimated from size and shape measurements. An analysis of the spatial relationships of the dark-chromatid regions of each object yielded a set of possible exchange locations and estimated probabilities that such locations corresponded to sister chromatid exchanges. A normalized estimate of the sister chromatid exchange frequency was obtained by summing the joint probabilities that a location contained an exchange within a single, separate chromosome over the set of chromosomes from one or more cells and dividing by the expected value of the total chromosome area analyzed. Comparison with manual scoring of exchanges showed satisfactory agreement up to levels of approximately 30 sister chromatid exchanges/cell, or slightly more than twice control levels. The processing time for this automated sister chromatid exchange detection system was comparable to that of manual scoring.     

Mutagenic and genotoxic evaluation of bisphenol F diglycidyl ether (BFDGE) in prokaryotic and eukaryotic systems

The epoxy resin bisphenol F diglycidyl ether (BFDGE), was examined for its mutagenicity in prokaryotic assays (Salmonella typhimurium His(-) and Escherichia coli Trp(-) tests) and its genotoxicity in eukaryotic systems (sister chromatid exchange (SCE) and micronucleus tests in human lymphocytes), in the presence or absence of an exogenous metabolizing system (S9 from rat liver). In the prokaryotic tests, the concentrations of BFDGE ranged between 100 and 5000 micro g per plate, and in the eukaryotic assays from 12.5 to 62.5 micro g/ml. The compound is able to induce mutagenic effects in bacterial strains TA100, TA1535, WP2uvrA and IC3327, as revealed by the increase observed in the number of induced revertants. With respect to the genotoxicity assays, BFDGE induces an increase in the frequency of sister chromatid exchanges and micronuclei in human peripheral blood lymphocytes.     

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.     

Genotoxicity evaluation of norethisterone acetate

Genotoxic evaluation of a commonly used progestogen, norethisterone acetate, was undertaken using a combination of short-term in vitro and in vivo assays. The clastogenic potentiality of norethisterone acetate was evident from the chromosome aberrations and sister chromatid exchanges induced both with and without S9 mix in cultured human lymphocytes and also from the increased frequency of micronuclei formation and sister chromatid exchanges in mice. However, in the Ames Salmonella assay, both with and without S9 mix and in host-mediated assay, norethisterone acetate was unable to cause any significant increase/decrease in the His⁺ revertants/plate.     

The detection of high-risk groups among workers in contact with heavy metals based on an analysis of chromosome aberrations and sister chromatid exchanges

Heavy metal salts, the workers from molybdenum, tungsten and cobalt plants to make in contact with, reveal their mutagenic activity. Individual sensitivity to heavy metal salts has been analyzed through the example of molybdenum. Chromosome aberrations and sister chromatid exchanges have been studied for the regularities of their formation in lymphocytes of workers depending on the length of service. Sensitivity of tests of chromosome aberrations and sister chromatid exchanges has been compared to reveal genetic consequences of these types of the effects.