The effects of busulphan on the chromosomes of normal human lymphocytes

In vitro exposure of human lymphocytes to busulphan (BUS) produced an increase in chromosome aberrations and in sister-chromatid exchange (SCE) frequency. The distribution of chromosome breaks throughout the karyotype was non-random and they occurred mainly in the G-negative bands. Certain bands had a marked susceptibility to BUS and comparisons with the human chromosome-break distributions reported for a number of drugs revealed that some of these bands were equally susceptible to other alkylating agents. Both the number of chromosome gaps and breaks and the SCE frequency increased with BUS concentration, but only the SCE dose--response was a clearly defined linear relationship. Therefore a standard SCE dose--response curve was constructed for future comparison with the results of similar investigations of patients on BUS therapy.     

The effects of busulphan on the chromosomes of normal human lymphocytes

In vitro exposure of human lymphocytes to busulphan (BUS) produced an increase in chromosome aberrations and in sister-chromatid exchange (SCE) frequency. The distribution of chromosome breaks throughout the karyotype was non-random and they occurred mainly in the G-negative bands. Certain bands had a marked susceptibility to BUS and comparisons with the human chromosome-break distributions reported for a number of drugs revealed that some of these bands were equally susceptible to other alkylating agents. Both the number of chromosome gaps and breaks and the SCE frequency increased with BUS concentration, but only the SCE dose-response was a clearly defined linear relationship. Therefore a standard SCE dose-response curve was constructed for future comparison with the results of similar investigations of patients on BUS therapy.     

Increased sister chromatid exchange in bone marrow and blood cells from Bloom's syndrome.

Bone-marrow cells from a patient with Bloom's syndrome cultured for 48 h in the presence of BudR exhibited a striking increase in the number of sister chromatid exchanges (SCEs) in comparison to that in the marrow cells of a patient with treated polycythemia vera (PV). Thus, it appears that an increased incidence of SCE in Bloom's syndrome occurs in various differentiated types of cells, not just blood lymphocytes, and constitutes the syndrome's most characteristic cytogenetic feature. In contrast, the incidence of SCE was not increased in marrow cells and lymphocytes of the particular PV patient studied here, whose cells did exhibit increased numbers of chromatid and chromosome gaps and breaks, presumably as result of the patient's earlier treatment. An increased frequency of SCE was demonstrated in Bloom's syndrome lymphocytes using both a technique based on BudR incorporation and one based on labeling with tritated deoxycytidine. This observation constitutes evidence against the increase of SCE being due to an unusual reaction to BudR. By conventional cytogenetic techniques, chromosome instability, including chromatid and chromosome breaks, but no homologous chromatid interchanges were also recognized in Bloom's syndrome bone-marrow cells incubated in vitro (without BudR) for either 1.k or 16 h. This observation points to the existence of chromosome instability in vivo.     

Increased frequency of sister-chromatid exchange and chromatid breaks in lymphocytes after treatment of human volunteers with therapeutic doses of paracetamol

Paracetamol was given to 10 healthy human volunteers in 3 doses of 1 g each during a period of 8 h. Blood samples for lymphocyte cultures were taken before and 24 h after paracetamol administration. A small but significant increase was found in the frequency of sister-chromatid exchanges (SCE) after intake of paracetamol (0.187 +/- 0.030 per chromosome before and 0.208 +/- 0.024 per chromosome after). After exposure the mean frequency of chromatid breaks per 100 cells was significantly increased (2.16 +/- 1.33 versus 0.33 +/- 0.50 before exposure). Exposure of human lymphocytes in vitro showed that concentrations of paracetamol above 0.1 mM induced inhibition of replicative DNA synthesis. Increased SCE was found in lymphocytes exposed to 1-10 mM paracetamol for 2 h. Furthermore, 0.75-1.5 mM paracetamol exposure for 24 h increased the frequency of chromatid and chromosome breaks in the lymphocytes. The paracetamol-induced SCE and chromosome aberrations may be secondary effects of paracetamol-induced inhibition of DNA synthesis or due to covalent binding of paracetamol metabolite(s) to DNA.     

Effects of alkylating agents on lymphocytes from controls and from patients with Fanconi's anemia

Summary The frequency of sister chromatid exchanges (SCE) and chromosome aberrations and the dynamics of cell division in peripheral blood lymphocytes of four patients with Fanconi's anemia were studied after in vitro exposure to alkylating agents TEPA and mitomycin.SCE frequency was significantly increased even after very low doses of mutagens, while chromosome aberrations were significantly increased only after high doses (0.160 g/ml mitomycin and 10^-5 M TEPA). The responses of Fanconi's anemia cells and control cells did not differ significantly. The increased frequency of both SCE and chromosome aberrations was accompanied by gradual delay of cell division, which was most conspicuous in cells from patients with Fanconi's anemia.     

Effects of caffeine on chromosome aberrations and sister-chromatid exchanges induced by mitomycin C in BrdU-labeled human chromosomes.

The BrdU-Hoechst staining technique has been used in analyzing the effect of caffeine (CAF) on chromosome aberrations and sister-chromatid exchanges (SCEs) induced by mitomycin C (MC). CAF increased the frequency of SCE in MC-treated chromosomes in all specimens. The combination of MC and CAF caused a remarkable increase in all types of chromosome aberrations, but the most startling effect was the appearance of many cells with multiple aberrations (shattered chromosomes). The BrdU-Hoechst technique showed that the shattered chromosomes did not appear in cells that had replicated only once, but did occur in cells which replicated twice in the presence of MC and CAF. The large majority of chromatid breaks observed did not involve areas common to SCE; and the SCE frequency significantly increased in spite of the existence of multiple breaks. This indicates that very few of the breaks are incomplete exchanges and that the mechanism for formation of SCE might be different from that of chromosome breaks. In another experiment, monofunctional-MC (M-MC) had a small effect on SCE rates, though it induced shattered chromosomes with CAF post-treatment. Possible differences in the mechanisms leading to SCE and chromosome breaks are discussed.     

Cytogenetic study of <Emphasis Type="Italic">Ascaris</Emphasis> trypsin inhibitor in cultured human lymphocytes with metabolic activation

The trypsin inhibitor (ATI) isolated from gastrointestinal nematode Ascaris suum was tested in vitro for induction of chromosome aberrations and sister chromatid exchanges (SCE). Genotoxicity assessment of purified ATI was carried out on metaphase plates received from peripheral blood lymphocyte macroculture (48 h test of structural chromosome aberrations and 72 h test of SCE) with exogenous metabolic activation. ATI was tested in dose of 25, 50 and 100 μg per ml of culture. Kinetics of cell divisions were determined by the replication index (RI). The mitotic index (MI) was expressed as a number of metaphases per 1000 nuclei analysed. Analysis of chromosome aberrations showed that higher doses of ATI (50 and 100 μg/ml) significantly increased the frequency of chromosome aberrations (mainly of chromatid gaps and breaks) compared to the negative control. All concentrations of ATI caused a statistically significant reduction in the MI and RI. In comparison with the negative control, a significant increase in the SCE frequency was observed in all applied doses of ATI. Thus, in the presence of S9 activation, the Ascaris trypsin inhibitor showed potential clastogenic activity and inhibition of the dynamics of lymphocyte divisions.     

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.     

Induction of sister chromatid exchanges and chromosome aberrations in cho cells arrested in the cell cycle by arginine deprivation

Summary Sister chromatid exchanges (SCE) and chromosome aberrations were induced in nondividing CHO cells that had been arrested in their cell cycle by deprivation of the essential amino acid arginine. Cells arrested in arginine-deficient medium (ADM) were treated with one of the mutagenic agents N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) or mitomycin C (MMC) and refed with complete medium; the recovering cell population was sampled at various intervals thereafter and mitotic cells analyzed for the presence of chromosome aberrations and SCE. Both chemicals were observed to cause delays in the cell cycle of recovering cells and to induce, chromosome aberrations and SCE at low doses. We have described the variation in the incidence of chromosome aberrations and SCE with respect to sampling time and the number of cell cycles traversed. When ADM-arrested CHO cells were treated with three mutagens at various intervals either before or after release from ADM, it was observed that: (a) UV light induced the greatest number of SCE when applied to cells undergoing DNA synthesis, and SCE yeilds induced by this agent could be reduced by postirradiation incubation in ADM; (b) MNNG induced fewer SCE when applied to cells undergoing DNA synthesis, and SCE yields induced by this agent could not be reduced by posttreatment incubation in ADM for 24 hr. (c) MMC induced the same level irrespective of the time of exposure, and SCE yields induced by this agent could not be reduced by posttreatment incubation in ADM for 24 hr. This work was supported by grants from the British Columbia Foundation for Non-Animal Research (to W. D. M.), and the National Cancer Institute of Canada and the National Research Council of Canada (to H. F. S.). Professor H. F. Stich is a Research Associate of the NCI.     

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.     

Effects of mitomycin C on sister chromatid exchange in normal and Bloom's syndrome cells

Bloom's syndrome lymphocytes, which are characterized by a high incidence of sister chromatid exchanges (SCE: 80.6 per cell), were treated with mitomycin C (MMC) and the effect of the chemical on SCE frequency compared with that in normal cells. Raising the concentration of MMC from 1 X 10(-9) to 1 X 10(-7) g/ml led to about 10-fold increase (61.7 SCE per cell) in the SCE frequency over the base line in normal lymphocytes (6.4 SCE per cell), though chromosome aberrations remained at a relatively low frequency. MMC caused about a two-fold rise in SCE in cells of Bloom's syndrome (128.8 SCE at 10(-9) g/ml; 139.3 SCE at 10(-8) g/ml). The frequency of chromosome aberrations in Bloom's syndrome cells at concentrations of MMC of 1 X 10(-9) and 1 X 10(-8) g/ml was 0.350 and 0.825 per cell, respectively, and low when compared to the increased number of SCE. The increased frequency of SCE in normal and Bloom's syndrome cells is in contrast to the reported findings with cells from Fanconi's anemia and xeroderma pigmentosum. The distribution of SCE in MMC-treated normal cell correlates with that of spontaneous SCE in cells of Bloom's syndrome.     

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.     

Induction of sister-chromatid exchanges by DNA-damaging agents and 12-O-tetradecanoyl-phorbol-13-acetate (TPA) in synchronous Chinese hamster ovary (CHO) cells

Synchronous CHO cells were obtained by mitotic selection; synchrony was maintained up to the 5th cell cycle. The mitotic cells were seeded into T-25 flasks or P-60 plastic petri dishes, and cultured for 1 h at 37 degrees C, then the cells were treated by X-ray, UV light, and mitomycin C. The cells were then cultured for 2 cell cycles with TPA and BrdUrd and sister-chromatid exchanges (SCE) analyzed by the FPG method. Following X-irradiation, the frequency of induced SCE increased linearly with dose reaching a maximum of 19.8 times the control frequency after 200 rad. With higher doses, the SCE frequency declined. In the presence of TPA, SCE frequencies were 1.8 times control levels for all X-ray doses studied (0-800 rad), the frequency seen in non-irradiated cultures treated with TPA. The induced SCE frequency also increased linearly following treatment with UVL and mitomycin C, reaching levels higher than 1.8 times controls with doses exceeding 2.5 J/m2 UVL or mitomycin C (30 min). In the presence of TPA, the SCE frequencies increased to 1.8 times controls following low UVL and mitomycin C doses, but were not influenced by TPA in the higher dose range (above 2.5 J/m2 or 10(-10) M mitomycin C. Most of the SCE were induced by X-rays during the first S phase after treatment. Following higher UVL doses (5 J/m2), however, the SCE frequency remained elevated (1.5 times controls) for 4 cell cycles after exposure.     

Frequency of sister chromatid exchanges in oncological patients during treatment with cyclophosphane

The sister chromatid exchange (SCE) level in patients with cancer of lungs before treatment did not differ from the mean number of SCE in healthy donors. In the process of a cyclophosphane treatment the SCE frequency in the patient lymphocyte culture increased with the drug doses of 4 and 5 g. Analysis of the ratio of cells in mitoses I, II and III revealed no differences in the cell cycle rate for groups of healthy donors, patients before the treatment, and treated patients.     

Human lymphocytes exposed to low doses of ionizing radiations become refractory to high doses of radiation as well as to chemical mutagens that induce double-strand breaks in DNA

Human lymphocytes exposed to low doses of ionizing radiation from incorporated tritiated thymidine or from X-rays become less susceptible to the induction of chromatid breaks by high doses of X-rays. This response can be induced by 0.01 Gy (1 rad) of X-rays, and has been attributed to the induction of a repair mechanism that causes the restitution of X-ray-induced chromosome breaks. Because the major lesions responsible for the induction of chromosome breakage are double-strand breaks in DNA, attempts have been made to see if the repair mechanism can affect various types of clastogenic lesions induced in DNA by chemical mutagens and carcinogens. When cells exposed to 0.01 Gy of X-rays or to low doses of tritiated thymidine were subsequently challenged with high doses of tritiated thymidine or bleomycin, which can induce double-strand breaks in DNA, or mitomycin C, which can induce cross-links in DNA, approximately half as many chromatid breaks were induced as expected. When, on the other hand, the cells were challenged with the alkylating agent methyl methanesulfonate (MMS), which can produce single-strand breaks in DNA, approximately twice as much damage was found as was induced by MMS alone. The results indicate that prior exposure to 0.01 Gy of X-rays reduces the number of chromosome breaks induced by double-strand breaks, and perhaps even by cross-links, in DNA, but has the opposite effect on breaks induced by the alkylating agent MMS. The results also show that the induced repair mechanism is different from that observed in the adaptive response that follows exposure to low doses of alkylating agents.     

Sister chromatid exchange assessment by chromosome orientation-fluorescence in situ hybridization on the bovine sex chromosomes and autosomes 16 and 26

Mammalian genome replication and maintenance are intimately coupled with the mechanisms that ensure cohesion between the resultant sister chromatids and the repair of DNA breaks. Although a sister chromatid exchange (SCE) is an error-free swapping of precisely matched and identical DNA strands, repetitive elements adjacent to the break site can act as alternative template sites and an unequal sister chromatid exchange can result, leading to structural variations and copy number change. Here we test the vulnerability for SCEs of the repeat-rich bovine Y chromosome in comparison with X, 16 and 26 chromosomes, using chromosome orientation-fluorescence in situ hybridization. The mean SCE rate of the Y chromosome (0.065 ± 0.029) was similar to that of BTA16 and BTA26 (0.065, 0.055), but was only approximately half of that of the X chromosome (0.142). As the chromosomal length affects the number of SCE events, we adjusted the SCE rates of the Y, 16, and 26 chromosomes to the length of the largest chromosome X resulting in very similar adjusted SCE (SCE(adj)) rates in all categories. Our results - based on 3 independent bulls - show that, although the cattle Y chromosome is a chest full of repeated elements, their presence and the documented activity of repeats in SCE formation does not manifest in significantly higher SCE(adj) rates and suggest the importance of the structural organization of the Y chromosome and the role of alternative mitotic DNA repair mechanisms.     

Effects of caffeine-pretreatment on SCE and chromosome aberrations induced by monofunctional- and bifunctional-mitomycin C in bloom syndrome lymphocytes

Bloom syndrome (BS) lymphocytes, which are characterized by a high incidence (75.4 per cell) of SCE, were treated with caffeine (CAF) during the first cell cycle and with monofunctional-(M-MC) and bifunctional-(MC)mitomycin C during the second cycle. The effect on the SCE level was synergistic. The CAF-pretreated cells in combination with M-MC and MC post-treatments, had significantly higher (SCE values 152.5 and 167.9 SCE per cell, resp.) than those treated with M-MC or MC alone during the second cycle (101.1 and 116.4 SCE per cell, resp.). M-MC and MC in the presence of BrdU (without CAF) for 2 cell cycles increased SCE to 157.6 and 169.4 per cell (about twice the control level). M-MC + CAF and MC + CAF treatments for 2 cell cycles did not produce a synergistic effect on the SCE frequency in BS cells; the SCE level was not significantly greater than that with M-MC or MC alone. Normal cells treated with MC and CAF for 2 cycles had a maximum SCE frequency of 156 per cell. This suggests that cells with SCE frequencies above this level may not be able to survive, i.e., this is the “saturation” level of SCE. However, CAF alone had almost no effect on SCE in either BS or normal cells and did not produce multiple chromosome aberrations. The lack of CAF effect on BS cells suggests that the lesions in DNA strands of BS cells which lead to SCE are double-strand lesions. In normal cells CAF is known to significantly slow down DNA-chain growth; the reduced rate of DNA-chain growth in BS is an inherent defect of the cells. Therefore, though CAF enhanced SCE and chromosome aberrations (shattered chromosomes) in combination with alkylating agents, CAF alone did not significantly increase the SCE rate in either BS cells or in normal cells. Thus, processes which may induce SCE are not only related to retarded rate of DNA-chain growth, but also to breaks in the template strand permitting double-strand exchanges to occur.     

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.     

Chromosomal aberrations and sister-chromatid exchanges induced by N-nitroso-2-acetylaminofluorene and their modifications by arsenite and selenite in Chinese hamster ovary cells.

The frequencies of chromosomal aberrations (CA) and sister-chromatid exchanges (SCE) in Chinese hamster cells were significantly increased by the direct-acting mutagen N-nitroso-2-acetylaminofluorene (N-NO-AAF) at the concentration of 0.1 mM. N-NO-AAF was prepared by nitrosation of the protohepatocarcinogen 2-acetylaminofluorene. The induced CA, which included chromatid breaks, chromatid exchanges, chromosome breaks, and chromosome ring formation were significantly potentiated by the presence of sodium arsenite (10 microM), but not by hydroxyurea (20 mM) or cytosine arabinoside (25 microM). On the other hand, the clastogenic effect of N-NO-AAF was effectively inhibited by sodium selenite (100 microM). Arsenite (10 microM) was shown to be moderately active in CA induction which was partially blocked by the presence of selenite (10 nM). N-Nitroso compounds such as N-nitroso-N-methylurea, N-nitroso-N-ethylurea and N-methyl-N'-nitro-N-nitrosoguanidine were equally or more active in the induction of CA and SCE in CHO cells when compared with N-NO-AAF. The cell cycle was significantly delayed by the intervention of N-NO-AAF.     

SCE variability in lymphocytes and fibroblasts

Summary To determine whether the sister chromatid exchange (SCE) distributions obtained in lymphocytes and fibroblasts from different individuals are comparable, a controlled study was set up. Peripheral blood and skin biopsies were taken on the same day from five individuals living for years under the same environmental conditions. All samples were treated in the same fashion, and the SCEs were scored in 50 metaphases of peripheral blood lymphocytes and of skin fibroblasts in an early and in a late passage. A repeat blood sample was taken from the same five indivuduals 1 year later. Based on the results obtained in this first part of the study, five randomly chosen healthy blood donors were sampled at different times and studied in the same fashion. Each chromosome was identified, and the SCE scores were tabulated per chromosome over 50 metaphases. The statistical analysis consisted of fitting log linear models to these scores and examining the best fit by determining the exceedance probabilities (observed significance level). For lymphocytes, the results indicated that the SCE distributions depended only on the chromosome examined, and not on BrdU-exposure time, individuals, or time of sampling. Treatment with ethyl methane sulfonate (EMS) increased the number of SCEs proportionally on all chromosomes. Analysis of the SCE scores on lymphocytes and fibroblasts of the five individuals and on their low and high passage fibroblast cultures revealed the necessity of including higher order interactions in order to fit a suitable model to the data. Therefore comparison of the SCE scores of lymphocytes with those of fibroblasts or comparison of scores on fibroblasts from different individuals could not be done. In practice, to compare samples or individuals, it suffices to score the SCE on a limited number of chromosomes (e.G., the A group) of 50 metaphases.