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.     

Suppression of spontaneous and mitomycin C-induced chromosome aberrations in Fanconi's anemia by cell fusion with normal human fibroblasts

Summary The high rate of spontaneous and mitomycin C-induced chromosome aberrations in Fanconi's anemia fibroblasts is fully corrected after euploid somatic cell hybridization with normal human fibroblasts.     

Frequency and types of induced and spontaneous chromosome aberrations in relation to cell kinetics

Summary Induced and spontaneous structural chromosome aberrations (SCA) were studied in a child accidentally radiated with a high dose of ¹⁹²Ir, and in three sibs with Fanconi's anemia, analyzing by separate first division metaphases (FDM) and second division metaphases (SDM). The results showed that the number of SCA, number of cells with aberrations, and SCA per cell were markedly higher in FDM in all patients.Furthermore, for some type of structural changes like dicentric chromosomes and chromatid interchanges, the differences were particularly striking. The importance of ascertaining FDM identified with proper techniques, for the study of the clastogenic effect of environmental agents and some aspects related to the differences in cytogenetic features found in diverse tissues in Fanconi's anemia are discussed.     

Effects of various cyclophosphamide concentrations in vivo on sister chromatid exchanges (SCE) and chromosome aberrations of Chinese hamster bone marrow cells

Summary The in vivo SCE formation and the induction of chromosome aberrations in the bone marrow of Chinese hamsters (Cricetulus griseus) were studied after various concentrations of cyclophosphamide, and the sensitivity of the two test methods was compared. The administration of 1.0, 5.0, 13.3, 25.0, and 40.0 mg/kg body weight induced a dose-dependent increase in SCE. The frequency of chromosome aberration, however, was not increased significantly with doses of 1.0 and 5.0 mg/kg body weight. Only with doses of more than 13.3 mg is a significant induction of chromosome aberrations seen. Therefore the SCE test system seems to be 10 times more sensitive than the induction of chromosome aberrations in the same cell type.This work is a part of the M.D. thesis of G. Roszinsky-Köcher, to whom offprint requests should be sent     

The effect of aphidicolin on Fanconi's anemia lymphocyte chromosomes

The cytogenetic effect of the DNA polymerase alpha inhibitor aphidicolin (APC) at a dose which did not affect cell cycle progression was determined in normal and Fanconi's anemia (FA) lymphocytes. APC enhanced sister-chromatid exchange (SCE) levels by about twice both in control and FA cells, while the yields of chromosome breakage increased up to 20 times in normal lymphocytes and 4 times in FA cells. APC did not act synergistically with the bifunctional alkylating diepoxybutane in terms of SCE either in normal or in FA lymphocytes. However, chromosome aberrations in cultures from normal subjects were much more than expected by an additive mode of action.     

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.     

The rate of sister chromatid exchanges parallel to spontaneous chromosome breakage in Fanconi's anemia and to trenimon-induced aberrations in human lymphocytes and fibroblasts.

The incidence of structural chromosome aberrations and the rate of sister chromatid exchanges (SCE) was investigated in lymphocyte cultures from a patient with typical Fanconi's anemia and his parents. The rate of SCEs was found to be normal. In experiments with the alkylating agent Trenimon the SCE rates proved to be a sensitive indicator for the induction of structural aberrations: in presence of an induced aberration rate half as high as the spontaneous rate in the Fanconi's anemia case, the rate of SCEs was found to be quintupled. Dose-effect relationships for the induction of SCE rates by Trenimon were studied over a wide dose range in lymphocyte and fibroblast cultures. The results reflect the same difference in sensitivity earlier observed in the induction of structural chromosome aberrations, fibroblasts being far more sensitive.     

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.     

Effects of Ultraviolet and X-Ray Radiation on in vitro Cultivated Cells of Haplopappus gracilis

A cell strain of Haplopappus gracills was used for investigations of the effects of UV (2537 A) and X-ray irradiation. Mitotic inhibition and killing after UV exposure were studied. A survival curve of UV treated and then plated cells is presented. The LD₅₀ seems to be about 2000 erg. mm^?2 under the experimental conditions used. All types of chromosome aberrations are induced by UV irradiation, but the frequency is relatively low at doses which do not completely inhibit cell division. A mutant strain of chromosome type is isolated after UV treatment and then plating. Mitotic inhibition and killing after X-ray treatment were studied. A survival curve is presented and the LD₅₀ under the culture conditions used seems to be about 2000 R. The frequency of chromosome aberrations induced by X-rays is highly increased by aeration during X-ray treatment which indicates that some degree of cell anoxia exists in a cell suspension. There arr indications that chromosome aherrations may not cause growth inhibition to such an extent as is usually believed.     

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.     

Effect of 5-bromodeoxyuridine substitution on sister chromatid exchange induction by chemicals

The fluorescence-plus-Giemsa (FPG) technique for analysis of sister chromatid exchange (SCE) is widely used as an assay for mutagenic carcinogens. There is very little information, however, on whether incorporation of the bromodeoxyuridine (BrdU) necessary for visualization of SCEs affects the sensitivity of the SCE test system to different chemical agents. We have investigated the effect of BrdU incorporation on SCE induction by labeling cells with BrdU for either the first cell cycle or the first and second cell cycles. The cells were then treated with bleomycin, which produces DNA strand breakage; proflavine, which intercalates into DNA; mitomycin C, which produces monoadducts and DNA crosslinks; or aphidicolin, which inhibits DNA polymerase . Chemicals were added before BrdU exposure or during the first, second, or both cell cycles. Only mitomycin C, which induces long-lived lesions, elevated the SCE frequency when cells were treated before BrdU labeling. When bleomycin, proflavine, or mitomycin C was present concurrently with BrdU, the frequency of SCEs was increased independently of the BrdU labeling protocol. Aphidicolin, on the other hand, induced more SCEs when present for the second cell cycle, when DNA replicates on a template DNA strand containing BrdU. We also examined the induction of SCEs in the first cell cycle (twins) and in the second cell cycle (singles) after continuous treatment of cells with BrdU and the test chemicals. Only aphidicolin increased SCE frequency in the second cell cycle. These results indicate that aphidicolin, but not bleomycin, proflavine, or mitomycin C, affects BrdU-substituted DNA and unsubstituted DNA differently. This type of interaction should be taken into consideration when the SCE test is used as an assay system.     

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.     

Higher inductions of twin and single sister chromatid exchanges by cross-linking agents in Fanconi's anemia cells

Summary Twin and single sister chromatid exchanges (SCEs) induced by short treatments with mitomycin C (MC) and 4,5,8-trimethylpsoralen (TMP)-plus-near ultraviolet light (NUV) were analyzed in colcemid-induced endoreduplicated normal human and typical Fanconi's anemia (FA) fibroblasts with diplochromosomes. The induction rate of twin SCEs that had occurred in the first cycle (S1) after the treatment was 1.7–2.4 times higher in FA cells than in normal cells. The induction rate of single SCEs that had arisen during the second cycle (S2) long after the treatment was also much higher, though less than the twin SCE rate, in FA cells than the almost neglible rate after repair of cross-links and monoadducts in normal cells. These results in FA cells, which specifically lack the first half-excision step of the two-step cross-link repair but retain the normal monoadduct repair, indicate that MC or TMP cross-links remaining unrepaired are indeed responsible for higher inductions of twin (S1 exchange) and single SCEs (S2 exchange). Thus, these findings indicate that Shafer's model of replication bypass for cross-link-induced SCE, which predicts greatly reduced twin SCE formation in FA cells due to half cancellation, is apparently inadequate as such. We present three plausible models, incorporating the ordinary replication model, random unilateral cross-link transfer, and chromatid breakage/reunion, that can account for the probabilistic inductions of single and twin SCEs and even for no SCE formation.This work was supported in part by a grant-in-aid for cancer research from the Ministry of Education, Science and Culture, Japan     

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.     

Paradoxical changes in SCE frequencies persistently elevated in vivo, on exposure to a mutagen in vitro

Lymphocyte cultures from 4 individuals with persistently significantly elevated frequencies of sister-chromatid exchange (SCE) were examined with no treatment, and with 2 concentrations of mitomycin C. In each of the 4 cases, the mean level of SCEs in the untreated lymphocytes exhibited a paradoxical reduction in SCE frequency when exposed to the lower (0.005 microgram/ml) of the two doses of mitomycin C. At the second higher dose of mitomycin C (0.025 microgram/ml) the mean level of SCE/cell exceeded the untreated mean. When the distributions of SCE/cell were examined it appeared that the untreated cultures had two or more populations of cells; one was in the normal SCE frequency range, while the second population was in an elevated SCE frequency range. The paradoxical reduction in SCE frequency was apparently due to elimination of, or mitotic inhibition of cells in the highest range of SCE frequency, while a small elevation in SCEs was initiated in the cells with a normal SCE frequency. Thus, mean levels of SCE/cell can be misleading. This data suggests that new exposure to the same or a different genotoxic agent might possibly result in a misleading lowering of the mean SCE frequency.     

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.     

Cytogenetic toxicity of antitumor platinum compounds in Fanconi's anemia

Summary Peripheral blood lymphocytes of patients with Fanconi's anemia (FA) were tested for their susceptibility to chromosome breakage by cis-platinum(II)-diamminedichloride [cis-Pt(II)], cis-platinum(IV)diamminetetrachloride [cis-Pt(IV)], and trans-platinum(IV)diamminetetrachloride [trans-Pt(IV)]. Low doses (0.1 g/ml) of the DNA-DNA cross-linking agents cis-Pt(II) and cis-Pt(IV) dramatically increased the chromosome breakage level in FA cultures without affecting the controls. The predominantly DNA-protein cross-linking compound trans-Pt(IV), however, was much less effective in producing chromosomal damage in FA. The differential response of FA cells to cis-Pt(IV) and trans-Pt(IV) suggests that the high susceptibility of FA to bifunctional cross-linking agents is due to an impairment of the cells to tolerate DNA-DNA cross-links, rather than DNA-protein cross-links.     

Numerical relationship between cells with radiation-induced chromosome aberrations and cells lethally injured by radiation

Summary The numerical relationship between radiation-induced chromosome aberrations in humanG ₀ lymphocytes and reproductive lethality of human bone-marrow lymphocytes is analysed within a large LET interval. The comparison is based upon the evaluation of the coefficient of the linear component of the corresponding dose-effect-relation for the frequency of cells without aberrations and for the frequency of surviving cells respectively. The good correlation between these coefficients over a large LET interval supports the hypothesis that structural chromosome aberrations and reproductive cell death both result from the same gross chromatin damage.This work was supported by the Bundesministerium des Innern, Bonn     

Chromosome aberrations, sister chromatid exchanges (SCE) and cell division kinetics in human lymphocytes exposed in vitro to purified trypsin inhibitor from Ascaris

The purified trypsin inhibitor (TI) isolated from nematode Ascaris suum was tested in vitro for chromosome aberrations and sister chromatid exchanges (SCE). TI was obtained from the musculocutaneous sac homogenate of adult Ascaris by the modified method of Rola and Pudles. The inhibitor was isolated and purified from the SF5 fraction of proteins by gel filtration on Sephadex G-50 and electrophoresis SDS-PAGE of the obtained fraction after molecular filtration. TI showed a high inhibitory activity against crystalline trypsin (18.8 Kassell's units/mg of protein). Genotoxicity assessment of TI 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), without exogenous metabolic activation. TI was tested in doses: 25, 50 and 100 microg per mL of culture. Kinetics of cell divisions was determined by the replication index (RI). We found that TI in vitro did not induce chromosome aberrations. It induced a higher number of SCE per cell but less than double frequency as compared to the control. The difference was significant only for the dose 50 microg/mL. For all doses, replication index (RI) values were significantly higher and mitotic index (MI) values were significantly lower than in the control. Thus the Ascaris trypsin inhibitor did not show any genotoxic properties but exhibited a mitostatic activity.     

Bimodal induction of sister-chromatid exchanges by luminol,an inhibitor of poly(ADP-ribose) synthetase,during the S-phase of the cell cycle

The cell cycle dependence of sister chromatid exchanges (SCEs) induced by luminol, a new potent inhibitor of poly(ADP-ribose) synthetase, was studied in Chinese hamster V79 cells. Continuous treatment with luminol during two whole cell cycles in the presence of 5-bromo-2-deoxyuridine (BrdUrd), or in the first or second cycle induced SCEs very efficiently in a linear dosedependent manner. However, no enhancement of SCE levels was observed after luminol treatment in a cycle preceding BrdUrd treatment, in contrast to results found with other strong SCE inducers such ascis-diammine-dichloroplatinum (II) (CDDP) and mitomycin C (MMC). Luminol was about ten times as effective in inducing SCEs as 3-aminobenzamide (3AB), an inhibitor of the NAD⁺ site of poly(ADP-ribose) synthetase. The induction of SCEs by luminol was restricted to the Sphase of the cell cycle with peaks at an early and a late stage, corresponding to the biphasic replication of DNA. The mechanism of SCE appears to be the same at the early and late stages of S-phase for luminol-induced SCE formation.