Increased sister chromatid exchange in human lymphocyte cultures infected with mycoplasma

The effects of fermenting, poorly arginine-utilizing Mycoplasma fermentans and arginine-utilizing Mycoplasma salivarium on the frequency of sister chromatid exchange (SCE) in cultured human lymphocytes were examined. M. fermentans caused no apparent mitosis inhibition of lymphocytes and the increase in SCE frequency was dependent on the inoculum size of the mycoplasma. An evident increase in SCE frequency was observed in lymphocytes infected with smaller inoculum sizes of M. salivarium whereas there was mitosis inhibition of lymphocytes infected with larger inoculum sizes of the mycoplasma. In lymphocyte cultures infected with M. salivarium, the addition of arginine to the culture medium reduced mitosis inhibition but did not diminish the increase in SCE frequency, indicating that arginine depletion was not involved in causing the induction of SCEs in mycoplasma-infected lymphocytes. With regard to the genetic effectiveness of SCE, these results suggested that mycoplasmas are capable of inducing cytogenetic changes in infected host cells.     

Enhanced assays detect increased chromosome damage and sister-chromatid exchanges in heroin addicts

To refine previous studies of chromosome damage (CD) and sister-chromatid exchanges (SCE) in heroin addicts, we applied new methods developed in our laboratory to enhance detection of the cytogenetic effects of low-level radiation exposure in hospital workers. For CD analysis, we applied our thymidine-fluorodeoxyuridine-caffeine (TFC) enhancement procedure in which cells at setup receive 1 x 10(-7) M fluorodeoxyuridine to inhibit thymidylate synthetase and 4 X 10(-5) M thymidine to satisfy the induced requirement, and then in G2 receive 2.2 mM caffeine to modulate DNA repair. For SCE enhancement, caffeine treatment was initiated in G1 at 19 h before harvest. Using both standard and enhanced procedures for CD and SCE analysis, blood samples were evaluated from 20 street heroin addicts and 22 controls. Standard 2-day CD and 3-day SCE assays showed small, insignificant genotoxic increases in addicts while the enhanced CD and SCE assays showed highly significant increases. Most CD events were in the form of chromatid and chromosome breaks. There were no rings and only a few dicentrics were observed in the TFC-enhanced cultures. Although quadriradials are rare, 10 were found in addict TFC-cultures and 3 in control TFC-cultures. With the standard CD assay, the mean number of chromosome breaks per 100 cells was 0.727 for controls and 1.056 for addicts (not significant). With the TFC-enhanced assay, the same measure showed 1.483 chromosome breaks for controls and 5.143 for addicts (highly significant, ANOVA: p less than 0.0001). A highly significant difference was also observed for chromatid-type damage with the TFC-enhanced assay (chromatid breaks per 100 cells: 16.793 for controls; 48.191 for addicts). The SCE data also showed significant differences with the enhanced assay. Scoring 25 cells/condition, standard SCE cultures showed 10.892 SCE/cell for controls and 11.732 SCE/cell for addicts (not significant). With CAF enhancement there were 13.08 SCE/cell for controls and 17.05 SCE/cell for addicts (ANOVA: p less than 0.008). These findings indicate that detection of CD and SCE effects can be significantly enhanced by the use of these new procedures. The finding of greatly increased chromatid damage in the addicts with the TFC procedure suggests that at least part of the CD detected occurred in vitro and is not a product of prior in vivo damage. Therefore exposure to this drug and perhaps other environmental agents may not only leave a residue of DNA or chromosome damage but may also induce a sensitivity to further genotoxic damage that is revealed by using the enhanced procedures.     

Relationship between cytotoxicity and induction of sister-chromatid exchanges in mouse foetal cells exposed to several doses of carcinogenic and non-carcinogenic chemicals.

The increase in sister-chromatid exchanges induced by 5 chemicals, with different DNA damaging and carcinogenic activities, was studied in short-term foetal-mouse cultures. A significant increase in SCE was induced by N-methyl-N'-nitro-N-nitrosoguanidine, N-diazoacetylglycine-amide, azaserine and methotrexate. k-Strophantin, on the contrary, was totally inactive. On a molar basis, MNNG was the most active chemical followed by MTX, AZS and DGA, in that order. At equitoxic concentrations (D37), the order of SCE-inducing abilities was MNNG, DGA, AZS and MTX. Compared with previous data, at equitoxic concentrations, the most DNA-damaging agents were also the most effective in inducing SCE. The SCE increase seems to correlate not with unspecific cytotoxicity but more with DNA damage or other damage at the genome level. MTX, a non-mutagen, which induced SCE only at toxic levels, could be considered a false positive because this positivity may reflect an enhancement of incorporation of 5-BrdUrd into DNA. The positive results obtained with AZS suggest a sufficient sensitivity of the method for detecting relatively weak carcinogens.     

Endogenous sex hormones affect the mutagen-induced chromosome damage by altering a caffeine-sensitive checkpoint

In the present study we analysed the effect of endogenous sex hormones on the SCE frequencies induced in vitro by mitomycin C (MMC), a bifunctional alkylating agent producing high chromosome damage and mitotic arrest. The analysis has been performed on lymphocytes obtained at three different phases of menstrual cycle, from women with regular cycle and hormones dosage. At all phases we further analysed the effect of a post-treatment with caffeine, an agent that it is known to overrride the DNA damage checkpoints.

After MMC, the cultures obtained at ovulation and luteal phases have SCE frequencies statistically higher than the cultures obtained at the progestogenic phase, showing increases of 15 and 25%, respectively. After caffeine, the MMC treated cultures which were set up at the progestogenic phase show a high potentiation of SCE frequencies (28%) whereas the treated cultures set up at ovulatory and luteal phases show little or no potentiation.

These findings demonstrate that the endogenous hormones greatly modulate the SCE frequencies induced by the mutagen; they also indicate that hormones action competes with the caffeine effect. Caffeine acts by abrogating the mitotic arrest produced by DNA damage and induced cells with a higher chromosome damage into a premature mitosis. Our findings suggest that endogenous hormones could overcome the checkpoint controls activated in cells after mutagenic exposure. This action may be an epigenetic mechanism relevant in hormone carcinogenesis.     

Genetic toxicology evaluation of commercial beers, III. SCE, chromosome aberrations, and forward mutation (HGPRT) of commercial beer products in CHO cells.

Concentrated organic residues extracted from 5 blended aliquots of commercial beers were evaluated for their ability to induce sister chromatid exchange (SCE), chromosomal aberrations and forward mutation in Chinese hamster ovary (CHO) cells. Each extract was prepared by blending 4 commercial beers of similar ingredients and brewing method, passing the beer pool over XAD-2 resin, extracting the resin and concentrating the extract. Studies were performed both with and without metabolic activation using variable amounts of reconstituted residues from 225-fold concentrates of the blended samples. CHO cultures were treated with 0.75 microliters/ml through 10.0 microliters/ml of the concentrates in the SCE assays, 1.0 microliters/ml through 10.0 microliters/ml of the extracts in the aberration assays and 2.5 microliters/ml up to 20 microliters/ml for forward mutation assays. In preliminary screening for SCE as an indicator of potential DNA damage, a significant increase was observed for 3 of 5 concentrated samples; however, no increase in SCE was induced by any of the 5 samples when S9 was added as a source of exogenous metabolic activation. More definitive tests for induction of genetic events, i.e., chromosome aberrations and forward HGPRT mutations, were negative for all 5 extracts whether or not S9 mix was present. Since SCE were not induced in tests with metabolic activation and since there was no concordant aberration or point mutation induction, the preliminary indication of potential DNA damage shown by elevated SCE under conditions without metabolic activation appears to have little biological significance.     

Aging and sister chromatid exchange : II. The effect of the in vitro passage level of human fetal lung fibroblasts on baseline and mutagen-induced sister chromatid exchange frequencies

The frequencies of baseline and mutagen-induced sister chromatid exchanges (SCE) were examined in human fetal lung fibroblasts (IMR-90, WI38) as a function of in vitro serial passage (in vitro aging). Although baseline SCE levels remained relatively constant throughout the in vitro lifespan of these cell cultures, a significant decline was observed at middle and late passage in the levels of SCE induced by mitomycin-C, ethyl methane-sulfonate and N-acetoxy-2-acetylaminofluorene. These findings indicate that cellular aging results in an altered response to certain types of induced DNA damage.     

Hypotonic treatment leads to chromosomal aberrations but not to sister-chromatid exchanges in human lymphocytes

Human peripheral lymphocytes were isolated from whole blood and exposed to culture medium of reduced osmolality. This hypotonic treatment led to a significant increase in the frequencies of chromosomal aberrations when the osmolality was reduced to 60 mOsm/kg H2O and below. Maximum damage occurred when the hypotonic treatment was done 27 or 30 h after starting the cultures. We also looked for the induction of sister-chromatid exchanges (SCE) by hypotonic culture conditions, but the SCE frequencies were not influenced.     

Effects of caffeine-induced defective DNA replication on SCE and chromosome aberrations produced by alkylating agents

The effect of caffeine (CAF) pretreatment (during the first cell cycle) on the frequency of sister-chromatid exchanges (SCE) and chromosome aberrations induced by bifunctional(MC)- and monofunctional(M-MC)-mitomycin C, 4-nitroquinoline N-oxide (4NQO) and ethyl methanesulphonate (EMS) were examined by using a BrdU—Hoechst staining technique. When CAF was added to the cultures during the first cell cycle in the presence of BrdU and then the cultures treated with MC, M-MC, 4NQO or EMS during the second cell cycle, the effect of the CAF was synergistic, i.e., the SCE level achieved was much higher than that expected from a simple additive effect of the agents and CAF. These results do not support the concept that the process of SCE is a manifestation of CAF-sensitive post-replication repair of DNA damage (single-strand exchanges), but, instead, point to exchanges between the double-strands of the DNA duplex present in each chromatid. CAF at certain concentrations is known to significantly slow down the rate of DNA-chain growth, but not appreciably induce strand breaks. Inasmuch as CAF alone induced only a small increase in SCE rates, possible mechanisms which may induce SCE are not only related to the slowing down of the rate of DNA-chain growth, but may also involve breaks in the template strand permitting double-strand exchanges to occur. The mechanisms responsible for chemically induced SCE are also discussed.     

Chromosome studies on lymphocytes of patients under cytostatic therapy

Summary Lymphocyte cultures from the peripheral blood of 38 patients undergoing a cytostatic interval therapy with a regimen of methyl-CCNU (1-[2-chloroethyl-3-(4-methyl-cyclohexyl)]-1-nitrosourea), 5-fluorouracil, and vincristine (each 5-day course of therapy was followed by a therapy interval of 4 weeks) were supplied with 5-bromodeoxyuridine (BUDR) for the whole culture time to determine the sister chromatid labelling pattern. From a total of 92 individual blood samples sister chromatid exchange (SCE) studies were performed including analyses before the start of the therapy, and immediately and 4 weeks after each course of therapy. In addition, the frequency of first, second, and third metaphases in the 72-h cultures was estimated using the characteristic labelling patterns.A distinct increase of SCE frequency over the control level (i.e., lymphocyte cultures of patients before the start of therapy) was observed at all phases of therapy. It was clearly correlated with the number of courses of therapy up to course 7, later on the SCE rate remained more or less at the level reached. The influence of the composition of each drug regimen on the SCE rate was less pronounced than it was on the breakage rate. Moreover, although a clear correlation existed between the individual rates of breakage and SCE, the formation of the latter appeared to reflect a long-term effect of the therapy rather than did the formation of break aberrations. In addition, as the intercellular variability of the number of SCEs per cell was much higher than that of breaks, the interindividual variability (variation of the mean values for each patient) was small compared to the respective variability of breakage rates.The proportion of first, second, and third metaphases present in 72-h cultures evidently was influenced by single courses of therapy. The observed delay of proliferation was also reflected in different amounts of chromosome damage. Although the BUDR treatment enhanced the cytostatic effect of the therapy on the lymphocytes in culture rendering SCE analysis rather difficult in several cases, the other data of this study and in particular the experiences with the long-term effect make it imperative to include BUDR-labelling in further cytogenetic studies in subjects with exceptional exposure to chemicals. However, the SCE method can by no means, replace the classic cytogenetic analysis.     

Effects of UV-irradiation on the SCE frequency in human lymphocyte cultures

UV-irradiation (254 nm) was found to induce a smaller increase of SCE in human lymphocytes than in human fibroblasts and CHO cells. The UV-induced SCE frequency in human lymphocytes was not influenced by the duration between irradiation and the subsequent S-phase. UV-irradiated lymphocytes showed a slightly more than additive response to the SCE-inducing effect of HN2 and acetaldehyde in comparison with non-irradiated cells. The UV-induced SCE frequency was similar in lymphocyte cultures containing 20 and 100 microM of BrdUrd. The results suggest that human lymphocytes are relatively insensitive to the SCE-inducing effect of UV-irradiation, and that SCE-inducing damage caused by UV is not removed during the G1 phase in these cells.     

Biomarkers of genotoxicity and genomic instability in a non-human primate, Cebus libidinosus (Cebidae, Platyrrhini), exposed to nitroimidazole derivatives

The genotoxicity of two nitroimidazole derivatives, ornidazole (ONZ) and metronidazole (MTZ) in the peripheral blood lymphocytes of Cebus libidinosus (CLI) (Primates, Cebidae) was assessed. Endpoints measured included sister chromatid exchange (SCE) frequency, cell proliferation kinetics (CPK), replication index (RI), mitotic index (MI), and damage incidence in or near CLI heterochromatin regions. MI and SCE values following ONZ or MTZ treatments were significantly different (p<0.001) from control. SCE frequency per chromosome was not proportional to chromosome length. The chromosomes most affected for SCE were 1, 2, 4, 6, 11-13, 17, and 18, many of which possess interstitial or terminal heterochromatin. In the CLI genome, chromosomes 11 and 17 showed higher susceptibility to damage RI was the only biomarker that did not show statistically significant differences between control and treated cultures. C. libidinosus bands 11q1.4 and 11q1.5 may be hot-spots in the context of nitroimidazole exposure.     

Enhanced cytogenetic detection of previous in vivo exposure to mutagens in human lymphocytes after treatment with inhibitors of DNA synthesis and DNA repair in vitro.

To increase the sensitivity of cytogenetic surveillance of exposure to mutagens in the peripheral lymphocyte assay, structural chromosome aberrations (CA) were studied after inhibition of DNA synthesis and DNA repair with hydroxyurea and caffeine in culture 3 h prior to harvesting. CA and sister-chromatid exchanges (SCE) from conventional cultures from the same subjects were used for comparison. Smoking was used as exposure parameter. Thirty-two smokers and 35 nonsmokers were studied. In the inhibited cultures a significantly higher number of aberrations was found in lymphocytes from smokers than nonsmokers: chromatid breaks (20.4 vs. 11.8, p = 0.0002), chromosome breaks (4.5 vs. 1.7, p = 0.0003), and the number of cells with aberrations (18.9 vs. 12.4, p = 0.0001), when 50 cells per subject were analyzed. In conventional cultures no increase in gaps, chromatid and chromosome breaks or number of cells with aberrations was found in smokers when 100 cells from each subject were studied. Smokers showed an increased number of SCE (6.8 vs. nonsmokers 5.9, p = 0.02). A significant positive linear correlation (r = 0.39, p = 0.01) was seen between SCE and the number of cells with chromatid breaks from inhibited cultures. The present results indicate that adding hydroxyurea and caffeine to lymphocyte cultures for the last 3 h prior to harvesting may enhance the detection of cytogenetic damage from previous in vivo exposure to mutagens.     

The genotoxic and cytotoxic effects of ribavirin in rat bone marrow

The genotoxicity of the 2-furylethylene derivative 1-(5-bromofur-2-yl)-2-nitroethene (2-betaNF) has been evaluated in cultured human peripheral blood lymphocytes at concentrations ranging from 0.5 to 15microg/ml. The frequencies of micronuclei (MN) and sister-chromatid exchanges (SCEs) were used and scored as indicators of genetic damage. To asses the role of the metabolism mediated by the enzymes present in the S9 mix, over the possible genotoxic potential of the test agent, the cultures for MN and SCE demonstrations were treated for 3h in presence and in absence of rat liver microsomal fraction. The results indicate that, under the experimental conditions used, the test agent does not induce significant increases in the frequency of micronucleated cells, irrespective of the presence/absence of metabolic fraction. Nevertheless, a slight increase in the SCE frequency was observed in those cultures treated without the S9 mix; although this slight increase disappeared in the experiments carried out with the microsomal fraction. In addition, cytotoxic/cytostatic effects of (2-betaNF) were observed mainly in the cultures treated without the S9 fraction.     

Effect of tumor promoter 12-O-tetradecanoyl-phorbol 13-acetate on induction of sister-chromatid exchanges in Chinese hamster V79 cells treated with mutagens

The effect of a tumor promoter, 12-O-tetradecanoyl-phorbol 13-acetate (TPA) alone and in combination with mitomycin C (MMC) or cyclophosphamide (CPP) on the induction of sister-chromatid exchanges (SCE) in Chinese hamster V79 cells was investigated. TPA alone at various doses and durations caused no increase of SCE frequency. MMC either at the dose of 0.03 or 0.003 μg/ml alone or in combination with TPA (2 μg/ml) all caused a significant increase of SCE frequencies. There was no difference in SCE frequencies between the cultures treated with MMC alone at 0.03 μg/ml and those treated with MMC plus TPA. However, cultures treated with MMC at 0.003 μg/ml plus TPA had significantly and consistently higher SCE frequencies than those treated with MMC alone at all durations. Treatment of CPP at 1 μg/ml activated by S9 mix caused significant increase of SCE frequencies. Surprisingly, the cultures treated with CPP, S9 mix plus TPA (2 μg/ml) caused a drastic reduction of SCE frequencies as compared to those treated with CPP and S9 mix only at all durations. These results indicate that TPA alone had no effect on SCE in V79 cells. TPA enhanced the SCE induction in V79 cells treated with MMC at a low dose, i.e. 0.003 μg/ml, but it inhibited SCE induction in cultures treated with the indirect mutagen CPP. Thus, TPA has no direct effect on genetic materials but it may indirectly alter the effects of a mutagen.     

Genotoxic activity of the commercial herbicide containing bifenox in bovine peripheral lymphocytes

The commercial herbicide with active element bifenox (principal tradename Modown) was tested for the evaluation of genotoxicity in cultured cow peripheral lymphocytes in vitro. Several cytogenetic endpoints as chromosome aberrations (CA), sister chromatid exchanges (SCE), mitotic (MI) and proliferation (PI) indices were investigated in different sampling times. To detect possible metabolic modifications in herbicide genotoxicity, the cultures for SCE determination were also treated with S9 fraction. Cultures of lymphocytes were exposed to the herbicide at concentrations of 25, 50, 250, 500 and 1000 microg/ml. A slight increase of CAs was found after exposure of this agent to doses ranging from 25 to 250 microg/ml for 24 h. In the CA assay no statistical significance was seen. Both higher doses (500 and 1000 microg/ml) caused a decrease of chromosome damage in comparison to the last active dose or control values correlated to induced cytotoxicity. Four concentrations (all except the highest one) of the herbicide were applied into cultures in SCE assays both with and without metabolic activation. Significant elevations of SCE were observed after applications of herbicide tested at doses of 250 and 500 microg/ml in each donor (P<0.001 and P<0.05, respectively) for 24 h. These concentrations also caused a statistically significant decrease in the MI and PI. Treatment for 48 h provided inadequate evidence for the genotoxic activity of the herbicide.     

Implications of an elevated sister-chromatid exchange frequency in rat lymphocytes cultured in the absence of erythrocytes

One important variable in complex culture systems such as whole blood is the interaction of the cell types present. To investigate the effects of erythrocytes (RBCs) and monocytes on the sister-chromatid exchange (SCE) frequency, Ficoll-Hypaque-separated Fischer-344 rat leukocytes were added to 1.9 ml of culture medium containing either 4 micrograms phytohemagglutinin or 4-8 micrograms concanavalin A/ml. Bromodeoxyuridine (BrdU;2 microM) was added at 24 h, and the cultures were harvested at 54 or 72 h. SCE frequencies in the mononuclear leukocyte cultures were consistently about 1.5- to 2-fold higher than in the whole-blood cultures. The titration of rat or human RBCs (0.05-2.5 X 10(9)) into purified rat leukocyte cultures reduced the SCE frequency to that of whole-blood cultures. Monocyte depletion decreased the elevated SCE frequency by approximately 50%. Scintillation counting of [14C]BrdU uptake in isolated RBCs revealed that less than 8% of the total amount of BrdU was sequestered. Also, BrdU induced a concentration-dependent increase in SCE in purified leukocytes, but the absolute increase was no greater than in whole-blood lymphocytes. Thus, BrdU had a minor role in the elevated SCE frequency in purified lymphocytes. Neither anti-oxidant enzymes such as catalase and superoxide dismutase nor the hydroxyl radical scavenger, dimethyl sulfoxide, decreased the SCE frequency. Although purified human lymphocytes had a small, but significant increase in SCE compared to whole blood, the magnitude of the dichotomous response between man and rat may represent a fundamental species difference.     

Physical,chemical, and biological factors affecting sister-chromatid exchange induction in human lymphocytes exposed to mitomycin C prior to culture

In experiments to assess the effects of several biological, chemical, and physical variables on sister-chromatid exchange (SCE) induction in cultured lymphocytes exposed to mitomycin C (MMC) before PHA stimulation we observed: (1) high SCE frequencies in female cells, and normal SCE frequencies in Y-bearing metaphases in mixed cultures containing equal numbers of MMC-treated female lymphocytes and untreated male lymphocytes; (2) small, but statistically significant, decreases in SCEs with increasing pH after G₀ exposure in the pH range 6.6–7.6; (3) pronounced reductions in MMC-induced SCEs in lymphocytes exposed at 4°C vs. 37°C. In other studies, SCE induction was evaluated in cultures exposed during G₀ to MMC concentrations ranging from 0.25 to 2.5 μg/ml for varying time intervals ranging from 5 min to 24 h. For all concentrations tested SCE induction varied as a linear function of G₀ exposure time. To compare SCE induction between cultures, we calculated the mean frequencies of SCEs induced per metaphase/unit dose MMC/unit G₀ exposure time (SCE/μg/h). A mean frequency of 20.7 ± 4.8 SCE/μg/h was observed for 41 lymphocyte cultures suggesting that a single term adequately describes the rate of SCE induction following G₀ exposure to a 10-fold range in concentration of MMC for time intervals of 30 min to 24 h.     

Quantitative analyses of the induction of chromosome aberrations and sister-chromatid exchanges in human lymphocytes exposed to gamma-rays and mitomycin-C in combination

Most chemicals are S-dependent and are potent inducers of SCE, but do not produce chromosome-type aberrations in the first metaphases after exposure. Ionizing radiation, which is an S-independent agent, produces chromosome-type aberrations, especially dicentrics and rings, but inefficiently produces chromatid-type aberrations. A series of experiments has been performed to investigate whether cytogenetic damage induced by ionizing radiation (gamma-rays) might be assessed separately from that induced by the alkylating chemical, mitomycin C (MMC), when human lymphocytes were exposed to these 2 agents in combination. Whole-blood cultures of human lymphocytes in G0 phase were exposed to gamma-rays and MMC in combination or separately. Cytogenetic analyses were done for both chromosome aberrations (CA), analyzed in cultures incubated for 56 h without BrdUrd, and sister-chromatid exchanges (SCEs) in cultures incubated for 72 h with BrdUrd. The frequency of chromosome-type aberrations (dicentrics and rings) increased with increasing doses of gamma-rays from 0.5 to 4.0 Gy. The dose-response relationships were the same with or without concomitant treatment with MMC (10(-6) M). Although the SCE frequency increased with increasing doses of MMC, the increase was nearly the same as when cells were treated with both MMC and gamma-rays (2 Gy). There was no interaction between MMC and gamma-rays concerning these 2 endpoints.     

Enhanced cytogenetic detection of previous in vivo exposure to mutagens in human lymphocytes after treatment with inhibitors of DNA synthesis and DNA repair in vitro

To increase the sensitivity of cytogenetic surveillance of exposure to mutagens in the peripheral lymphocyte assay, structural chromosome aberrations (CA) were studied after inhibition of DNA synthesis and DNA repair with hydroxyurea and caffeine in culture 3 h prior to harvesting. CA and sister-chromatid exchanges (SCE) from conventional cultures from the same subjects were used for comparison. Smoking was used as exposure parameter. Thirty-two smokers and 35 nonsmokers were studied. In the inhibited cultures a significantly higher number of aberrations was found in lymphocytes from smokers than nonsmokers: chromatid breaks (20.4 vs. 11.8, p = 0.0002), chromosome breaks (4.5 vs. 1.7, p = 0.0003), and the number of cells with aberrations (18.9 vs. 12.4, p = 0.0001), when 50 cells per subject were analyzed. In conventional cultures no increase in gaps, chromatid and chromosome breaks or number of cells with aberrations was found in smokers when 100 cells from each subject were studied. Smokers showed an increased number of SCE (6.8 vs. nonsmokers 5.9, p = 0.02). A significant positive linear correlation (r = 0.39, p = 0.01) was seen between SCE and the number of cells with chromatid breaks from inhibited cultures. The present results indicate that adding hydroxyurea and caffeine to lymphocyte cultures for the last 3 h prior to harvesting may enhance the detection of cytogenetic damage from previous in vivo exposure to mutagens.     

A decrease in sister chromatid exchange frequency during the prolonged cultivation of human lymphocytes

Sister chromatid exchange (SCE) frequency at different times of fixation was studied in human lymphocyte cultures obtained from 6 donors. No differences were found in the SCE frequency between human lymphocyte cultures fixed at 72 and 96 hours of incubation (10.61 +/- 0.85 and 10.15 +/- 0.81 SCE per cell, respectively). However, a decreased SCE frequency (8.11 +/- 0.36 SCE per cell) was observed in cultures fixed at 120 hours of incubation. For a more detailed studies, one lymphocyte culture was fixed at different times of incubation (from 56 to 128 hours, at each a 8 hours). A slight increase in SCE frequencies was found at the interval between 56 and 88 hours of incubation, while starting from 104 hours of incubation a marked decrease in the SCE frequency was observed. Time-dependent changes in the SCE frequency may be described by the equation y = -1.8614 + 0.3922x - (2.5183 x 10(-3))x2, where y is the number of SCEs per cell, and x--the duration of culture incubation in hours. The observed phenomenon may be associated with changes in proportion of T and B lymphocytes, or with heterochromatization of chromosomes during a prolonged cultivation, or with an early in vitro stimulation of the in vivo long-lived lymphocytes that may be more damaged than the in vivo short-lived and the in vitro late-stimulating ones.