Synergistic induction of sister-chromatid exchanges in lymphocytes from normal subjects and from patients under cytostatic therapy by inhibitors of poly(ADP-ribose)polymerase and antitumour agents

The effects of nicotinamide on SCE rates induced in vitro by chlorambucil (CBC or melphalan (MELPH) or mitomycin C (MMC) was studied. The combined treatments with either CBC or MELPH or MMC and nicotinamide showed the potentiating ability of the latter drug. Theophylline and MELPH were also found to act synergistically on the induction of SCEs. In a combined in vivo and in vitro study, lymphocytes taken from 7 cancer patients who had been given cytoxan by injection 3 h before, were treated with nicotinamide or diphylline (DP) in vitro, and found to have synergistically increased exchange rates. This has implications for interpreting the repair processes involved, for monitoring drug combinations that synergistically damage DNA in vivo and in vitro and for identifying interindividual variation in the response to the treatment.     

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.     

Characterization of the enhancing effect of caffeine on sister-chromatid exchanges induced by ultraviolet radiation in excision-proficient xeroderma pigmentosum lymphoblastoid cells

Cells of some excision-proficient xeroderma pigmentosum (XP) cell lines are highly sensitive to post-UV caffeine treatment in terms of sister-chromatid exchange (SCE) induction as well as cell lethality. In the present study, we conducted a detailed investigation of the enhancing effect of caffeine on SCE frequency induced by UV in excision-proficient XP cells, and obtained the following results. (1) Continuous post-UV treatment with 1 mM caffeine markedly enhances UV-induced SCEs and such enhanced SCEs occur with similar frequency during either the 1st or the 2nd cell cycle in the presence of caffeine and 5-bromodeoxyuridine (BrdUrd). (2) The high sensitivity of the cells to post-UV caffeine treatment persists for at least 2 days after UV when irradiated cells are held in either the proliferating or the nonproliferating state prior to the addition of BrdUrd. (3) Caffeine exerts its effect on cells in S phase. (4) Neither BrdUrd in the medium nor the incorporated 5-bromodeoxyuridine monophosphate (BrdUMP) in DNA plays an appreciable role in the expression of the enhancing effect of caffeine. The most likely explanation for our findings is as follows. In excision-proficient XP cells, the cause of SCE formation such as UV-induced lesions or resulting perturbations of DNA replication persists until the 2nd round or more of post-UV DNA replication. If caffeine is given as post-UV treatment, such abnormalities may be amplified, resulting in a synergistic increase in SCE frequency.     

Developmental and cytogenetic effects of caffeine on mouse blastocysts, alone or in combination with benzo(a)pyrene

Mouse blastocysts were treated with caffeine and/or benzo(a)pyrene (BP), and the effects on development and on induction of sister chromatid exchanges (SCEs) were examined. Caffeine interfered with blastocyst development in a dose-related manner. At 4 mM, the highest concentration tested, caffeine interfered with development of blastocysts to all four endpoints: hatching, trophoblast outgrowth, inner cell mass (ICM) growth, and two-layer (primary endoderm and ectoderm) differentiation of ICMs. At 2 mM, caffeine reduced the incidence of both ICM growth and differentiation but did not affect hatching or formation of trophoblast outgrowths. At 1 mM, caffeine interfered only with ICM differentiation. Cell proliferation was least sensitive to caffeine and was reduced at concentrations of greater than or equal to 2 mM. Induction of SCEs was most sensitive to caffeine exposure; an increase in SCE frequency was observed at 0.1 and 0.5 mM. When caffeine was added to cultures with BP (1 microM, a concentration that was not embryotoxic and did not induce SCEs), both embryotoxic effects and SCE frequency were increased. The enhancing effect on SCE induction was particularly marked; as little as 0.1 mM caffeine was sufficient to cause doubling of induced SCE frequencies when added to cultures with BP.     

Molecular dosimetry of the chemical mutagen ethyl methanesulfonate in Escherichia coli and in V-79 chinese hamster cells

Caffeine increases the number of sister-chromatid exchanges (SCE) induced by mitomycin C (MMC) in human peripheral lymphocytes in culture. This enhancement decreases when the treated cells are held in medium before phytohemagglutinin (PHA) stimulation, or when caffeine is added to cultures some time after PHA stimulation but prior to DNA synthesis. There thus appears to be a caffeine-sensitive prereplication repair system, presumably an excision mechanism, capable of repairing a fraction of the MMC-induced DNA lesions.     

Enhancement of cytogenetic damage by chlorpromazine in human lymphocytes treated with alkylating antineoplastics and caffeine

In cultured human lymphocytes chlorpromazine (CPZ) was found to induce cell division delays and to have no effect on sister-chromatid exchanges (SCEs) or on mitotic indices (MIs). CPZ induces cytotoxic effects in combination with caffeine (CAF) and alkylating agents. In combination with CAF it induced cell division delays and suppression of MIs. In combination with melphalan (MEL) and CAF, CPZ synergistically induced SCEs, caused cell division delay and suppressed MIs. In combination with chlorambucil (CBC) and CAF, CPZ produced synergism on induction of SCEs, enhanced cell division delays and reduced MIs.     

Cytogenetic damage by melphalan and hyperthermia in patients with an initial epileptic attack.

Sister-chromatid exchanges (SCEs) and cell kinetics in cultured lymphocytes of patients with an initial epileptic attack, and prior to any anticonvulsant treatment, were studied. Spontaneous melphalan (MEL) and MEL-hyperthermia (MEL-HYP) induced SCE frequencies have been studied in 18 adults with an initial epileptic seizure. Fifteen age and sex matched healthy subjects were used as the control group. The incidence of spontaneous SCEs in lymphocytes from epileptics was not significantly greater than in those from the control subjects. However, when exposed to MEL in vitro, cells from both groups showed an increase in SCE frequency. When exposed to MEL and HYP (41 degrees C for 3 h) in vitro, cells from both groups showed a further increase in SCE frequency with yields from epileptics higher (P less than 0.05) than from controls. HYP in combination with MEL enhanced synergistically SCEs and cell division delays in both groups with synergistic effects in cells from epileptics (P less than 0.01 and P less than 0.01 respectively) higher than from controls (P less than 0.05 and P less than 0.05 respectively.     

Antagonizing effect of 3-aminoharman on induction of sister-chromatid exchanges by mutagens

3-Aminoharman (3AH, 3-amino-1-methyl-9H-pyrido[3,4-b]indole), which has been reported as a novel substance with an antagonistic effect on induction of sister-chromatid exchange (SCE) by polycyclic mutagens in the presence of the metabolic activation system, was examined with a cultured human lymphoblastoid cell line, NL3, for its effect on SCE induction by direct-acting mutagens such as mitomycin C (MMC), nitrogen mustard N-oxide (NMO), methyl methanesulfonate (MMS), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 4-nitroquinoline 1-oxide (4NQO) and 3-hydroxyamino-1-methyl-5H-pyrido[4,3-b]indole (OH-Trp-P-2), and also by ultraviolet light (UV) irradiation. The results obtained on simultaneous treatment with 3AH and mutagens were as follows: (1) 3AH suppressed more than 50% of SCEs induced by MMC, NMO and OH-Trp-P-2; (2) 4NQO- and MNNG-induced SCEs were also suppressed by 3AH but to a lesser degree; (3) MMS-induced SCEs were not, however, altered by 3AH; and (4) the suppression of SCE by 3AH was dose-dependent. Treatment of cells with 3AH for 2 h immediately before MMC exposure suppressed SCE induction to a significant degree similar to the simultaneous treatment, but post-treatment with 3AH was much less effective. 3AH inhibited SCE induction by NMO when 3AH treatment was carried out either before or after NMO treatment, to an extent similar to the simultaneous treatment. Treatments with 3AH either before or after UV exposure did not change the UV-induced SCEs. Results with these direct-acting mutagens ruled out the relevance of metabolic activation as a necessary step for the antagonizing effect of 3AH.     

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.     

黄鳝染色体体内SCE检测系统的建立

应用IdU-毛玉米油体内SCE技术,以不同剂量的典型诱变剂MMC和CP对70尾黄鳝的脾、肾、血淋巴细胞进行了体内诱发SCE敏感性测试。结果:三种细胞的染色体SCE自发频率均较低,不同剂量MMC和CP诱发黄鳝三种细胞SCE频率均较对照组显著增加。诱变剂剂量与诱发SCE频率呈线性关系。三种细胞染色体SCE对MMC和CP的敏感性次序为肾>脾>血淋巴细胞。与几种鱼和其它动物比较,黄鳝三种细胞的SCE自发频率均较低,对MMC和CP诱发SCE的敏感性均较高,因此认为黄鳝可作为较理想的体内SCE检测系统。     

Dependency of the yield of sister-chromatid exchanges induced by alkylating agents on fixation time. Possible involvement of secondary lesions in sister-chromatid exchange induction

Chinese hamster V79 cells were pulse-treated (for 60 min) with various mutagens three, two or one cell cycles before fixation (treatment variants A, B and C, respectively) and the frequencies of induced SCEs were analysed and compared. The degree of increase in frequency of SCEs with dose in the treatment variants depended on the mutagen used. For the methylating agents MNU, MNNG and DMPNU, high yields of SCEs were obtained in the treatment variants A and B, and there was no difference in the efficiency with which these agents induced SCEs in these treatment variants. In the treatment variant C, however, no SCEs were induced with mutagen doses yielding a linear increase in SCE frequency in treatment variants A and B. A slight increase in SCE frequency in treatment variant C was observed only when relatively high doses of MNU or MNNG were applied. Like the above agents, EMS, ENU and MMS induced more SCEs in treatment variants A and B than in C, but for these agents treatment variant B was most effective and SCEs were induced over the entire dose range, also in treatment variant C. As opposed to the methylating and ethylating agents, MMC induced SCEs with high efficiency when treatment occurred one or two generations prior to fixation. There was no difference in SCE frequency between these treatment variants. MMC was completely ineffective for the induction of SCEs when treatment occurred three generations before fixation. The unexpectedly low SCE frequencies induced by the methylating and ethylating agents when treatment occurred one generation before fixation were not due to the exposure of cells to BrdU prior to mutagen treatment. From the results obtained, it is concluded that DNA methylation and ethylation lesions give rise to SCEs only with very low probability during the replication cycle after the lesion's induction, and that subsequent lesions produced during or after replication of the methylated or ethylated template (secondary lesions) are of prime importance for SCE formation after alkylation. For MMC, however, primary lesions seem to be most important for SCE induction.     

Psoralen/UVA treatment and chromosomes

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

Sister-chromatid exchanges and cell-cycle kinetics in human lymphocyte cultures exposed to alkylating mutagens: apparent deformity in dose-response relationships

Experiments have been carried out using human whole-blood cultures to determine the effects of sampling times and of the duration of 5-bromodeoxyuridine (BrdUrd) treatment before fixation on sister-chromatid exchange (SCE) frequencies following exposure to mitomycin C (MMC). Cells were pulse treated for 1 h with 3 X 10(-6) M MMC at G1, and then sampled at 4-h intervals up to 88 h after stimulation of cultures with phytohemagglutinin (PHA). Results showed that this MMC treatment induced a 5-6 h proliferation delay per cell cycle, and that SCE frequencies first increased with time of fixation, peaking at 68 h, and then decreased. When cells were similarly treated with MMC, but subsequently exposed to BrdUrd for various times before fixation of cultures at 72 h, the SCE frequencies markedly increased with increasing durations of BrdUrd incubation times. These data indicate that, in mutagen-treated cultures, lymphocytes having relatively longer cell-cycle times show a higher mean frequency of SCEs. In a subsequent experiment, cells were treated for 1 h with increasing doses of MMC or 4-nitroquinoline 1-oxide (4NQO) at 0, 24, or 48 h, and then fixed at 72 h after PHA stimulation. Results showed that the optimal treatment times at which the agents could most efficiently produce SCEs were different for MMC and 4NQO, and that the dose-response curves tended to 'bend down' at very high doses; that is, treatments with very high doses induced smaller than expected numbers of SCEs. However, cells similarly treated with very high doses showed a higher, expected frequency of SCEs when sampled at 84 h, but again had a lower than expected SCE frequency when fixed at 96 h. The results indicate that there is an optimal time for sampling at which one can observe the maximum increase in SCE frequencies following mutagen exposure, and strongly suggest that the higher the dose, the later the optimal sampling time. Because of the apparent deformity of dose-response curves obtained after various treatments and sampling times, it seems necessary that extra fixation-time points be included in test protocols so as to avoid false negatives or confirm possible positives.     

Sister chromatid exchanges induced by sarcolysine in human lymphocytes in vitro

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

DNA crosslinking, sister-chromatid exchange and specific-locus mutations

Chinese hamster ovary cells were treated with the DNA-crosslinking chemicals, mitomycin C (MMC) and porfiromycin (POR), and their monofunctional derivative decarbamoyl mitomycin C (DCMMC). After exposure, the cells were studied for the induction of sister-chromatid exchanges (SCEs) and mutations at the hypoxanthine phosphoribosyltransferase and adenine phosphoribosyltransferase loci. The frequency of SCEs varied significantly in successive sampling intervals, requiring the weighting of each interval by the percentage of second-division mitosis in that interval to obtain the mean SCE frequency for each dose. All 3 compounds were potent inducers of SCEs but weakly mutagenic. All 3 chemicals by concentration were approximately equally effective in inducing SCEs or mutations. When the induced SCEs and mutations were compared at equal levels of survival, DCMMC was slightly more effective than MMC or POR in inducing SCEs and somewhat less mutagenic. These results indicate that the DNA interstrand crosslink is not the major lesion responsible for the induction of SCE or mutation by these compounds.     

Mitomycin C-induced damage and repair in human and pig lymphocytes

Human and pig lymphocytes were used to compare the chromosomal sensitivity to MMC and the efficiency of repair of MMC-induced DNA adducts. No significant interspecies differences were found. The results obtained show that SCE frequencies are linearly correlated with MMC doses. During the G0 period there are indications that lymphocytes may half-repair the DNA-interstrand crosslinks transforming bi- into mono-adducts. SCEs induced by MMC decrease to near control levels in the second cell cycle. Therefore, most MMC lesions responsible for SCEs should be repaired between the moment in the first S phase in which they induce the exchanges and the onset of the second S period.     

SCE evaluations in human lymphocytes after G0 exposure to mitomycin C Lack of expression of MMC-induced SCEs in cells that have undergone greater than two in vitro divisions

We conducted a series of experiments designed to determine whether DNA damage induced in G₀ lymphocytes by mitomycin C (MMC) would be expressed as sister-chromatid exchanges during the second and third post-treatment cell cycles. Lymphocytes from normal donors were exposed to MMC for 2 h prior to culture in the presence of phytohemagglutinin. MMC-treated and control cells were subsequently exposed to bromodeoxyuridine (BrdUrd) for the entire culture period (i.e. 48 h or 72 h) or for the terminal 24 h of 72-h cultures. We observed a 3–4-fold increase in SCEs in MII metaphases from lymphocytes treated with MMC and cultured in the presence of BrdUrd for the entire culture period. In contrast, in replicate cultures of MMC-treated lymphocytes that were exposed to BrdUrd for the terminal 24 h only, the SCE frequency in uniformly harlequinized metaphases was not significantly different from that observed in control cultures. We interpret these data as providing evidence that MMC-induced lesions (or alterations) in the DNA of G₀ lymphocytes are probably expressed as SCEs during the first period of mitogeninduced DNA synthesis, and that these lesions do not persist and give rise to SCEs in subsequent cell divisions.     

A schedule to demonstrate radiation-induced sister chromatid exchanges in human lymphocytes

Summary The reciprocal interchange between the chromatids of a chromosome, termed sister chromatid exchange (SCE), is considered to be one of the most sensitive and accurate cytogenetic parameters and respond to toxic chemicals at very low doses. But the response of SCE to ionizing radiation is very poor. Human lymphocytes fail to give SCE response when irradiated at G₀. Probably the primary lesions induced at G₀ do not remain available long enough to find expression as SCEs. Based on this assumption a schedule was developed using caffeine to demonstrate radiation induced SCEs. Following this schedule a dose-dependent increase in the frequency of radiation induced SCEs has been observed.     

Repeated measurement of spontaneous and clastogen-induced sister-chromatid exchange

Sister-chromatid exchanges (SCE), both spontaneous and chemically-induced [blomycin (BLM), mitomycin-C (MMC), streptonigrin (SN), and 4-nitroquinoline-1-oxide (4NQO)], were studied in the lymphocytes of 24 normal individuals on 2 or 3 different occasions, separated by periods of up to 2 years. For all BLM-induced SCEs, the variation in SCE frequency among the samples from a single individual was significantly greater than the variation between replicate cultures on a given day. These results raise questions concerning the validity of conclusions based on a single observation of chemically-induced SCEs.     

Repeated measurement of spontaneous and clastogen-induced sister-chromatid exchange.

Sister-chromatid exchanges (SCE), both spontaneous and chemically-induced [bleomycin (BLM), mitomycin-C (MMC), streptonigrin (SN), and 4-nitroquinoline-1-oxide (4NQO)], were studied in the lymphocytes of 24 normal individuals on 2 or 3 different occasions, separated by periods of up to 2 years. For all BLM-induced SCEs, the variation in SCE frequency among the samples from a single individual was significantly greater than the variation between replicate cultures on a given day. These results raise questions concerning the validity of conclusions based on a single observation of chemically-induced SCEs.