Higher Sister Chromatid Exchange Frequency Research Articles

Trivalent chromium is neither cytotoxic nor mutagenic in permeabilized hamster fibroblasts

BHK cells became reversibly permeable by a 30-min incubation in hypertonic medium. During permeabilization they were exposed to water-soluble Cr(VI) (K 2Cr 2O 7) and Cr(III) (CrCl 3). Thymidine uptake in the intracellular nucleotide pool, DNA replication, DNA damage and repair and sister-chromatid exchanges (SCE) were examined to detect the cytotoxic and genetic effects of Cr compounds. Cr(III) remained inactive also in permeabilized cells. An apparent induction of DNA damage by Cr(lII), suggested by the Painter's test, was considered unreliable. Cr(VI) cytotoxic and genetic activity was enhanced in permeabilized cells, as demonstrated by increased inhibition of DNA replication and higher frequency of SCE.

Sister chromatid exchange in malignant lymphomas

Sister chromatid exchange (SCE) was evaluated in peripheral lymphocytes from 20 untreated patients with malignant lymphomas: 6 with Hodgkin's disease (HD), 14 with non-Hodgkin lymphoma (NHL), and 5 with lymphadenitis. The mean SCE frequency (± SE) was: 11.2 ± 0.6, 11.0 ± 0.6, and 7.2 ± 0.3 for HD, NHL, and lymphadenitis patients, respectively, and 8.7 ± 0.2 for the control group. No differences in SCE score were observed in HD and NHL. These results allowed us to consider both groups (HD and NHL) as a single neoplastic population (mean ± SE, 11.0 ± 0.4). No significant differences were found between the lymphadenitis and control groups. On the other hand, significantly higher SCE scores were seen in neoplastic populations than in the control and lymphadenitis groups (p < 0.001 and p < 0.01, respectively). When SCE was compared by chromosome number and group between neoplastic patients and controls, a higher SCE frequency was observed in chromosomes #1, #2, #3, and B, C + X, E, F chromosome groups than in controls. SCE levels were significantly higher in lymphoma patients in all chromosome numbers and groups mentioned than in patients with lymphadenitis. It is suggested that the high SCE rate in the malignant lymphoma population is possibly related to an increased chromosomal instability.

Sister chromatid exchanges in betel and tobacco chewers

The incidence of sister-chromatid exchange (SCE) was investigated in the lymphocyte chromosomes of betel and tobacco chewers. Betel chewers and betel-with-tobacco chewers showed higher yields of SCE than normal controls. Higher frequencies of SCE were also observed in individuals who chewed more than 10 betel leaves, or betel leaves-with-tobacco, per day, compared to people who chewed less than 10 betel leaves, or betel leaves-with-tobacco, per day, respectively. Subjects who had chewed betel leaves and betel leaves + tobacco for more than 10 years showed an elevated frequency of SCE.

Correlative genotoxicity studies of airborne particles in Salmonella typhimurium and cultured human lymphocytes.

The acetone extracts of ambient air particulates collected locally were tested for their capacity to induce sister chromatid exchanges (SCEs) and chromosomal aberrations (CAs) in human lymphocytes, and to induce gene mutations (GMs) in Salmonella typhimurium. The extracts caused dose-related clastogenic/mutagenic responses in all three assay systems. With the same concentration, it seems that the Ames Salmonella/microsomal assay with TA98 gave the highest, and the chromosomal aberration assay with human lymphocytes the lowest, mutagenic/clastogenic responses, respectively. Because high frequencies of SCEs were induced by solvent extracts of airborne particles, this study further indicated the usefulness of SCE assay in human lymphocytes for genotoxicity studies of airborne particles.

Statistical analysis of high SCE frequency cells in human lymphocytes.

The existence of a subpopulation of lymphocytes in which genetic damage persists was identified in experiments on rabbits injected with low levels of mitomycin C. This led to the suggestion that an important parameter to quantify after exposure to a possibly harmful agent may be the proportion of cells with high sister chromatid exchange (SCE) frequency, i.e., high frequency cells (HFCs). In the research reported here we describe the statistical properties of HFCs, particularly their utility in detecting low levels of genetic damage which may be undetected by a simple t-test comparing the means of two samples of SCEs.

DNA interstrand cross-linking, repair, and SCE mechanism in human cells in special reference to Fanconi anemia.

The relation of DNA cross-linking and repair to sister chromatid exchange (SCE) formation was studied in normal human, Fanconi anemia (FA), and xeroderma pigmentosum (XP) cells. Despite a hypersensitive lethality response in FA cells, the SCE induction rates by mitomycin C (MMC), trimethylpsoralen (TMP)-light, cisplatin, and diepoxybutane were twice as high as in normal cells. For MMC, the induced SCE frequency in normal cells was reduced in a biphasic fashion with a repair incubation time (the first decline t1/2 = 2 hr; the second t1/2 = 14-18 hr) which corresponds exactly to the molecular kinetics of cross-link and monoadduct removal. However, FA cells lack the first half-excision process and exhibit a lack of the first rapid decline SCE component. The slow decline component is present, and a higher SCE frequency is observed 24 to 48 hr after treatment. By contrast, XP cells capable of the half-excision process reveal the first rapid decline component, followed by an extremely slow second-reduction component (t1/2 = 48 hr) due to defective monoadduct repair. The endoreduplication-SCE method revealed that rates of both twin (first cycle) and single (second cycle) SCE formations by MMC and TMP-light were higher in FA cells than in normal cells. These results indicate that cross-links remaining unrepaired induce SCEs as do monoadducts. The probabilistic SCE induction occurs at a rate of 1 SCE per 35 MMC cross-links in FA cells. Further, a non-SCE-forming tolerance mechanism also operates in hypersensitive FA cells. These molecular and cytogenetic results allow us to construct a new probabilistic model for cross-link-induced SCE into which the replication-fork model, random cross-link transfer to both chromatids, and chromatid breakage-reunion are incorporated.

The relationship between sister-chromatid exchange induction and the formation of specific methylated DNA adducts in Chinese hamster ovary cells

The ability of simple alkylating carcinogens to induce sister-chromatid exchange (SCE) has been well established (Perry and Evans, 1975; Carrano et al., 1978), although the alkyl lesion(s) leading to SCE formation have not been delineated. Several lines of evidence have led to the suggestion that persistent DNA damage, particularly O6-alkylguanine, is important in inducing SCE (Wolff, 1978, 1982; Goth-Goldstein, 1977). After treatment with simple alkylating agents, human cells which are defective in the removal of O6-alkylguanine have higher SCE frequencies than cells with normal levels of O6-methylguanine removal (Wolff et al., 1977; Day et al., 1980). The results of liquid-holding studies, however, argue that repairable DNA damage is involved in SCE formation. In these studies, the mutation rate (Jostes, 1981) or the level of O6-methylguanine (Connell and Medcarf, 1982) remained constant while SCE frequencies decreased after holding the cells using conditions that prohibited replication. These results suggested that SCE were the result of repairable DNA damage such as 3-alkyladenine or 7-alkylguanine (Jostes, 1981; Connell and Medcalf, 1982), although no single DNA adduct was identified which was repaired at the rate that SCE-causing damage was removed. The results of time-course experiments are consistent with the liquid-holding studies. In these experiments, the initial SCE frequency induced by exposure to methyl methanesulfonate (MMS) decreased in subsequent generations after treatment (Ockey, 1981; Muscarella and Bloom, 1982). SCE frequency returned to near control levels approximately 10 generations after treatment (Muscarella and Bloom, 1982). Studies in which levels of specific DNA adducts induced by different alkylating agents were quantified and compared with SCE frequencies have not yet yielded conclusive results. The results of Swenson et al. (1980) were supportive of

Proliferative kinetics and mitomycin C-induced chromosome damage in Fanconi's anemia lymphocytes.

Lymphocytes from two sisters with Fanconi's anemia (FA) were studied for cell cycle kinetics, sister chromatid exchanges (SCEs), and chromosomal aberrations when they had undergone one, two, or three or more divisions in mitomycin C (MMC)-treated cultures. Lymphocytes from the parents, another sister of the probands, and a healthy unrelated adult were examined as controls. Analyses of cell cycle kinetics by the sister chromatid differential staining method revealed that the relative frequency of metaphase cells at their third or subsequent divisions was much smaller in untreated FA cultures than in normal cultures fixed at 96 h after phytohemagglutinin stimulation. These data indicate that FA cells proliferate much more slowly than normal cells. MMC treatments of FA and normal cells led to a clearly dose-related delay in cell turnover times, the duration of delay being much longer in FA than in normal cells. FA cells had about 1.4 times higher frequencies of SCEs than normal cells in both MMC-treated and untreated cultures. FA cells also showed several times higher frequencies of chromosomal aberrations than normal cells, and the frequency of chromosomal aberrations decreased through subsequent mitoses by approximately 60% in both FA and normal cells.

Developmental and cytogenetic effects of potassium dichromate on mouse embryos in vitro.

AbstractMouse blastocysts and egg cylinders were treated in vitro with potassium dichromate (K2Cr2O7, 5 × 10−7 to 2 × 10−6 M), and the effects on postimplantation development and induction of sister chromatid exchanges (SCEs) were determined. When blastocysts were treated for six days with 1 × 10−6 M K2Cr2O7, the number of embryos with growing inner cell masses (ICMs) and ICMs that were differentiated into primary endoderm and ectoderm were significantly reduced. The number of embryos forming trophoblast outgrowths was not reduced, but outgrowth formed in the presence of 2 × 10−6 M K2Cr2O7 were small and began to degenerate by the end of the treatment. Hatching and attachment were not affected at any concentration. The frequency of SCEs was significantly increased at ≥ 1 × 10−6 M. When egg cyclinders were treated with K2Cr2O7 for 24 hours, the number of embryos with blood islands, fusion of the allantois with the chorion, and beating heart was significantly reduced at all concentrations tested, and the crown‐rump lengths of embryos were significantly reduced at 1 to 2 × 10−6 M. The frequency of SCEs was increased in embryonic tissues of egg cylinders cultured in ≥ 1 × 10−6 M K2Cr2O7. Increased SCE frequencies were observed at lower concentrations in extraembryonic tissues, which had higher SCE frequencies than embryonic tissues at all concentrations tested. The correlation between developmental effects and SCE frequency suggests that cytogenetic damage may be largely responsible for the detrimental effects of dichromate on mouse embryos.

Differential features of sister-chromatid exchange responses to ultraviolet radiation and caffeine in xeroderma pigmentosum lymphoblastoid cell lines

Sister-chromatid exchange (SCE) induced by ultraviolet (UV) irradiation and viability after UV irradiation were studied in lymphoblastoid cell lines derived from 7 patients with xeroderma pigmentosum (XP) and 6 normal donors. UV irradiation caused significant increases of SCEs in both XP and normal cells. In 3 XP cell lines, which were deficient in unscheduled DNA synthesis (UDS) and sensitive to the killing effect of UV, very high SCE frequencies were observed after UV irradiation. Cells from a patient with the De Sanctis-Cacchione syndrome were the most sensitive to UV in terms of both SCE induction and cell killing. In 2 of 4 UDS-proficient XP cell lines tested, the incidences of UV-induced SCEs were similar to those in normal cell lines, but in 2 other UDS-proficient lines from 2 XP patients with skin cancer, the frequencies of UV-induced SCEs were significantly higher than in normal cells. Continuous post-UV treatment with 1 mM caffeine markedly enhanced UV-induced SCEs in 3 of 4 UDS-proficient XP cell lines but had only slight effects on cells from the 4th UDS-proficient XP patient and from normal individuals.

A CHO-cell strain having hypersensitivity to mutagens, a defect in DNA strand-break repair, and an extraordinary baseline frequency of sister-chromatid exchange

A mutant of CHO cells (strain EM9) previously isolated on the basis of hypersensitivity to killing by ethyl methanesulfonate (EMS) is approx. 10-fold more sensitive than the parental line, AA8, to killing by both EMS and MMS. It is also hypersensitive to killing by other alkylating agents (ethyl nitrosourea and N-methyl- N′-nitro- N′-nitrosoguanidine), X-rays, and ultraviolet radiation. The production and repair of DNA single-strand breaks (SSB) were studied using the technique of alkaline elution of DNA from filters. After exposure to 4 Gy of X-rays at 0°C and subsequent incubation at 25°C, SSb were repaired within 12 min in AA8, but little repair occurred in EM9. Similarly, with doses of EMS or MMS that produced comparable numbers of SSB in AA8 and EM9 at the end of a 10-min exposure, repair of SSB occurred more rapidly in AA8 than in EM9, suggesting that individual SSB are longer lived in EM9. EM9 was found to be hypersensitive also to the induction of mutations and sister-chromatid exchanges (SCE) by EMS; per unit dose the mutant had twice as many mutations to thioguanine resistance, 3 times as many mutations to azaadenine resistance, and a 7-fold enhancement in SCE, compared to AA8. Moreover, the baseline frequency of SCE in the mutant was extraordinarily high, i.e., 8.6 ± 0.6 vs. 107 ± 5 SCE/cell for AA8 and EM9, respectively, with 10 μM BrdUrd in the medium. The high frequency in EM9 did not vary significantly with BrdUrd concentration over the range examined from 2.5 to 20 μM, and the percentage of 5-bromouracil substitution in the DNA was the same in EM9 and AA8 under these conditions. These data, however, do not rule out the possibility that the high SCE frequency in EM9 is a consequence of an altered sensitivity to incorporated BrdUrd. Thus, EM9 may carry a pleiotropic mutation affecting some function in DNA replication and/or DNA repair and ausing the variety of phenotypic properties described in this study.

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 G 0 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 G 0 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 G 0 exposure time. To compare SCE induction between cultures, we calculated the mean frequencies of SCEs induced per metaphase/unit dose MMC/unit G 0 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 G 0 exposure to a 10-fold range in concentration of MMC for time intervals of 30 min to 24 h.

The effect of gamma irradiation of human cells and somatic cell hybridization on sister chromatid exchange frequencies in the hamster chromosomes of hamster-human hybrid lines.

Sister chromatid exchange (SCE) frequency was studied in human/hamster hybrid lines in which the human cells had been exposed to gamma irradiation at doses of 0, 10, 20, and 40 grays. A higher SCE frequency was found in the hamster chromosomes in the hybrid lines than was found in the hamster parental cell line CHW1103. There was also a dose-dependent increase of SCE frequency in the unirradiated hamster chromosomes in the hybrid lines in which the human parental cells had been irradiated.

Increased sister chromatid exchange frequencies in lymphocytes of nurses handling cytostatic drugs.

In oncology units, personnel handling chemotherapeutic drugs may occasionally be exposed to small amounts of genotoxic agents. This exposure was obviously the cause of the increased frequencies of sister chromatid exchange (SCE) observed in nurses in daily contact with cytostatics (N = 20, mean SCEs/cell +/- SE 9.4 +/- 0.3) as compared to a group of office workers (N = 10, mean SCEs/cell 8.1 +/- 0.3). The oncology nurses also had a higher SCE frequency than other hospital nurses (N = 10, mean SCEs/cell 8.7 +/- 0.2), but this difference was not statistically significant. The SCEs of patients under chemotherapy were about five times higher (mean SCEs/cell 36.8 +/- 0.6) than those of healthy subjects.

Sister chromatid exchanges in children treated with azathioprine and cyclophosphamide, and effect of protein-bound polysaccharide (PS-K).

Sister chromatid exchange (SCE) was examined in cultured lymphocytes of nephrotic children and some other juvenile patients treated with immunosuppressive agents, azathioprine (AZA) and cyclophosphamide (CYC), and CYC in combination with a protein-bound polysaccharide (PS-K). The frequency of SCEs in children treated with CYC showed a highly significant increase as compared with the non-treated control. AZA treatment also resulted in an increased frequency of SCEs, though the mean value was lower than that obtained for CYC. An extremely high frequency of SCEs resulted from a combined treatment with AZA and CYC. After 3–4 months of cessation of CYC medication, the frequency of SCEs returned to the normal level. The SCE frequency decreased significantly when CYC was used in combination with PS-K.

Cell-cycle duration and sister-chromatid exchange frequency in cultured human lymphocytes.

Whole heparinized blood samples from normal human donors were grown in culture media containing 10 μg/ml of bromodeoxyuridine. Lymphocytes were harvested after 58, 70, 72 and 80 h and scored for sister-chromatid exchanges (SCEs) under a fluorescence microscope. SCEs which occured during the first and second cell cycles were counted in second or third generation cells selected on the basis of their chromosome fluorescence patterns. The results of a preliminary study showed the mean SCE frequency per cell at 72 h to be 9.0 for second generation cells and 7.8 in third generation cells ( P < 0.01). A second study, using culture medium with heat-inactivated fetal-calf serum, gave similar results (9.4 vs. 7.8, P 0.001) at 70 h. Therefore, the difference in SCE frequency between second and third generation cells at 70 or 72 h cannot be attributed to heat-labile substances of serum origin. An additional finding in the second study was that SCE frequencies in third division cells at 70 and80 h were the samee as those of second division cells at 58 h but significantly less ( P < 0.001) than the frequency in second division cells at 70 h. These data were interpreted as arising from at least two different lymphocyte populations; one group of cells that is either slower growing or slower in phytohemagglutinin stimulation, with a higher SCE frequency which does not reach second division until 70 or 80 h, and a more rapidly dividing (or more quickly stimulated by phytohemagglutinin) population with a lower SCE frequency which reaches second division at 58 h and third division by 70–80 h. Whether or not this hypothesis is correct, the data show that SCE frequency varies significantly with cell-cycle duration. Since some carcinogens have been shown to alter cell kinetics (Craig-Holmes and Shaw, Mutation Res. 46 (1977) 375), changes in SCE frequency which are caused by a change in cell kinetics must be considered a factor in determining the mutagenicity of an agent by its ability to increase SCE frequency.

Sister chromatid exchange and cell cycle in fibroblasts of Bloom's syndrome

Sister chromatid exchange (SCE) has been studied in the fibroblasts of five Bloom's syndrome patients, one heterozygote, and two normal individuals. The high frequency of SCE already known in the lymphocytes of Bloom's syndrome was also found in the fibroblasts of all five patients. However, populations with low and high frequency of SCE were not found. In addition, chromosome aberrations appeared with a lower frequency. The cell cycle duration in the Bloom's fibroblasts appeared to be similar to that in the normal cell line, and the difference in the growth pattern appeared to be due to the variation in the mitotic index. The cell cycle lasted about 24 h in at least four of the Bloom's lines studied during the present experiments.

Sister chromatid exchange studies for monitoring DNA damage and repair capacity after cytostatics in vitro and in lymphocytes of leukaemic patients under cytostatic therapy

The effect of various cytostatic drugs was studied on the frequency of sister chromatid exchanges (SCEs) in vitro and in PHA-stimulated lymphocytes of leukaemic patients under cytostatic therapy. The lymphocyte system is a sensitive one for the detection of DNA damage after administration of cytostatic drugs in vitro. Mitomycin C, busulphan, vincristine, chlorambucil, cytosine arabinoside, cyclophosphamide and lycurim were tested. All except cyclophosphamide induced high frequencies of SCEs in the first mitosis after their administration. The experiments with PHA-stimulated lymphocytes in vivo from patients treated with cytostatics showed that cytosine arabinoside, in combination with thioguanine, did not induce higher frequencies of SCEs, whereas in patients who were treated with cyclophosphamide alone or in combination with other cytostatic drugs, there was a higher incidence of SCEs during treatment. About 10 days after the termination of the treatment the elevated frequencies of SCEs returned to the initial level. After administration of some mutagens, especially alkylating agents in vivo, the lymphocyte system can be used to assess induced DNA repair by continuously monitoring for SCEs.

Sister chromatid exchanges in Drosophila melanogaster cell lines in vitro

The frequency of sister chromatid exchanges (SCEs) in two cell lines of Drosophila melanogaster with different karyotypes (XX and XY) was determined, considering (1) the distribution of SCEs within each chromosome, with reference to eu- and heterochromatin and (2) the distribution of SCEs in different chromosomes. A comparison was made between chromosome pairs within each karyotype and between the two different karyotypes. The following results were obtained. The SCEs are not randomly distributed along chromosomes, since exchanges were never observed in heterochromatin. SCEs are more frequent in XY than in XX cells; moreover, in both cell types there exists a significantly higher frequency of SCEs in the X chromosome than in the autosomes. These findings are discussed in relation to chromosome aberrations and mitotic recombination.

Replication bypass model of sister chromatid exchanges and implications for Bloom's syndrome and Fanconi's anemia.

A model of the sister chromatid exchange (SCE) process is outlined as a replication mechanism to bypass DNA crosslinks. The model suggests that when normal bidirectional replication advances from both sides towards a crosslink along the two opposite parental strands, the complementary parental strand segments can be temporarily displaced at each contralateral 5' side from the crosslink. The free ends produced in this first step will be terminally aligned but will have opposite polarity. The second step of the bypass can, however, be completed by either of two rejoining processes--terminal ligation of the free ends via nascent Okazaki pieces or aberrant complementation by overlapping the free ends. This bypass mechanism (1) allows replication to continue past a crosslink leaving it intact but (2) results in the switching of parental strands and their attached incomplete nascent strands above and below the crosslink site producing an exchange between sister chromatids. This model is compatible with the findings of current SCE studies using the new BUDR/stain techniques as well as with previous autoradiographic studies. It also suggests that the chromatid breaks and deletions in Fanconi's Anemia represent a defect in step two of the replication bypass mechanism and that the high frequency of SCE's and quadriradials in Bloom's Syndrome represent the SCE overload effects of a defect in crosslink repair.