Effect of caffeine in G2 on X-ray-induced chromosomal aberrations and mitotic inhibition in ataxia telangiectasia fibroblast and lymphoblastoid cells

Summary The effect of post-treatments with caffeine in G₂ on the frequency of X-ray-induced chromatid aberrations was studied in normal and ataxia telangiectasia (A-T) fibroblast and lymphoblastoid cells. Caffeine was found to potentiate the X-ray-induced aberration yield in both normal fibroblast and lymphoblastoid cells. An enhancement was also observed in A-T lymphoblastoid cells, whereas the X-ray-induced aberration frequency in A-T fibroblasts was unaffected by the presence of caffeine. The influence of caffeine on the radiationinduced mitotic inhibition was investigated in normal and A-T fibroblasts; in both types of cell less inhibition was obtained in the presence of caffeine.     

Post-treatments with sodium arsenite during G2 enhance the frequency of chromosomal aberrations induced by S-dependent clastogens

Treatment with sodium arsenite during the G2 phase potentiated the chromatid breaks and chromatid exchanges induced by ultraviolet light or 4-nitroquinoline 1-oxide but not those induced by methyl methanesulfonate, ethyl methanesulfonate, mitomycin C or cisplatin in Chinese hamster ovary cells. A comparison was made between the effects of treatment during G2 with sodium arsenite, cytosine-β- -arabinofuranoside, aphidicolin, hydroxyurea, caffeine, 3-aminobenzamide and novobiocin on the frequency of chromosomal aberrations induced by the above-mentioned S-dependent clastogens. It was found that the effects varied considerably, both quantitatively and qlalitatively. However, potentiation was more often observed in the chromosomal aberrations induced by ultraviolet light and 4-nitroquinoline 1-oxide than by other S-dependent clastogens, and the frequency of chromatid exchanges was potentiated only in cells pretreated with ultraviolet light or 4-nitroquinoline 1-oxide. Furthermore, for all of the S-dependent clastogens studied, treatment with cytosine-β- -arabinofuranoside during the G2 phase potentiated the frequency of chromatid breaks but not the frequency of chromatid exchanges.     

G2 effects of DNA-repair inhibitors on chromatid-type aberrations in root-tip cells treated with maleic hydrazide and mitomycin C

In recent years the existence of a DNA-repair process in G2 has been proposed to explain the potentiating effects of DNA-repair inhibitors given in G2 on chromatid aberrations (CA) induced by S-dependent as well as S-independent DNA-damaging agents. In the present report, root-tip cells of Allium cepa were exposed to maleic hydrazide (MH) or mitomycin C (MMC) and post-treated in G2 with caffeine (Caff) and various inhibitors of DNA synthesis. No enhancement of chromosome damage was observed when Caff was present in G2, but hydroxyurea (HU) or 5-fluorodeoxyuridine (FdUrd) potentiated the frequencies of CA. A slight additional increase of CA frequencies was observed following treatment with Ara C and excess thymidine in G2. When MH-damaged cells were pulse-treated with Caff earlier during recovery, the yield of CA was enhanced. The earlier Caff was present following MH treatment, the stronger was the potentiation.     

Cytological characterization of Chinese hamster ovary X-ray-sensitive mutant cells xrs 5 and xrs 6. I. Induction of chromosomal aberrations by X-irradiation and its modulation with 3-aminobenzamide and caffeine

We have studied two X-ray-sensitive mutants xrs 5 and xrs 6 (derived from the CHO-K1 cell line), known to be defective in repair of double-strand breaks, for cell killing and frequency of the chromosomal aberrations induced by X-irradiation. The survival experiments showed that mutants are very sensitive to X-rays, the D0, for the wild-type CHO-K1 was 6-fold higher than D0 value for the mutants. The modal number of chromosomes (2 n = 23) and the frequency of spontaneously occurring chromosomal aberrations were similar in all 3 cell lines. X-Irradiation of synchronized mutant cells in G1-phase significantly induced both chromosome- and chromatid-type of aberrations. The frequency of aberrations in xrs mutants was 12-fold more than in the wild-type CHO-K1 cells. X-Irradiation of G2-phase cells also yielded higher frequency of aberrations in the mutants, namely 7-8-fold in xrs 5 and about 3.5-fold in xrs 6 compared to the wild-type CHO-K1 cells. There was a good correlation between relative inability to repair of DNA double-strand breaks and induction of aberrations. The effect of 3-aminobenzamide (3AB), an inhibitor of poly(ADP-ribose) synthetase on the frequency of X-ray-induced chromosomal aberrations in these 3 cell lines was also studied. 3AB potentiated the frequency of aberrations in G1 and G2 in all the cell types. In the mutants, 3AB had a potentiating effect on the frequency of X-ray-induced chromosomal aberrations only at low doses. X-Ray-induced G2 arrest and its release by caffeine was studied by cytofluorometric methods. The relative speed with which irradiated S-G2 cells progressed into mitosis in the presence of caffeine was CHO-K1 greater than xrs 5 greater than xrs 6. Caffeine could counteract G2 delay induced by X-rays in CHO-K1 and xrs 5 but not in xrs 6. Large differences in potentiation by caffeine were observed among these cells subjected to X-rays and caffeine post-treatment for different durations. These responses and possible reasons for the increased radiosensitivity of xrs mutants are discussed and compared to ataxia telangiectasia (A-T) cells and a radiosensitive mutant mouse lymphoma cell line.     

Effects of G2 treatments with inhibitors of DNA synthesis and repair on chromosome damage induced by X-rays and chemical clastogens in root tips of Vicia faba. Comparison with corresponding effects in cultured human lymphocytes

The frequencies of chromatid aberrations produced in roots of Vicia faba by clastogenic (chromosome-damaging) agents were strongly enhanced by exposing the root-tip cells to inhibitors of DNA synthesis during the G2 phase. Chromosome damage produced by both S-dependent (maleic hydrazide, methyl methanesulfonate, thio-TEPA) and S-independent (X-rays, streptonigrin) mechanisms was enhanced by the inhibitor treatments. The types of aberrations affected by the inhibitors were mainly chromatid gaps and breaks and isochromatid breaks of the non-union type. Most effective among the inhibitors tested were hydroxyurea (HU) and 5-fluorodeoxyuridine (FdUrd). Post-treatments with caffeine were effective in enhancing clastogen-induced chromosome damage when given during the S phase. All types of aberrations, exchanges as well as breaks, were enhanced by the post-treatments. When given during the G2 phase, caffeine enhanced only the frequency of chromatid aberrations produced by X-rays. The enhancement was slight and obtained only when the cells were irradiated in the G2 phase and immediately post-treated with caffeine. Clastogen-treated cultures of human lymphocytes responded to post-treatments with inhibitors of DNA synthesis in very much the same way as clastogen-treated root-tip cells of Vicia faba. Thus, the frequencies of chromatid gaps and breaks and isochromatid breaks of the non-union type were strongly enhanced by exposing clastogen-treated lymphocytes to inhibitors of DNA synthesis during the G2 phase. The efficiency of the inhibitors, however, varied considerably in the two materials. On the whole, the number of inhibitors capable of enhancing induced chromosome damage was much larger in lymphocytes than in bean root tips. Only HU was equally effective in both materials. The most striking difference between the two materials was found when caffeine was given as a post-treatment. Thus, in human lymphocytes the frequencies of chromatid aberrations induced by most clastogenic agents were strongly enhanced when caffeine was given during the G2 phase, but little affected by post-treatments with caffeine during the S phase.     

The effect of caffeine posttreatment of X-ray-induced chromosomal aberrations in human blood lymphocytes in vitro

Summary The potentiating effect of caffeine on X-ray-induced chromosomal aberrations in human blood lymphocytes has been investigated, with special reference to cell cycle stages (G0 and G2). Both quantitative and qualitative differences in the yield of chromosomal aberrations were detected in caffeine-posttreated cells, depending on the cell stage irradiated. The studies on caffeine potentiating effects on X-irradiated G0 lymphocytes from normal adults, newborns, Down syndrome patients, and an ataxia telangiectasia patient pointed to interindividual variations in the response to caffeine potentiation among normal probands and a very profound effect in ataxia cells.     

X-ray- and mitomycin C (MMC)-induced chromosome aberrations in spermiogenic germ cells and the repair capacity of mouse eggs for the X-ray and MMC damage

Chromosome aberrations induced at the first-cleavage metaphase of eggs fertilized with sperm recovered from spermiogenic cells which had been X-irradiated and treated with mitomycin C (MMC) at various stages were observed using in vitro fertilization and embryo culture technique. Furthermore, the repair capacity of the fertilized eggs for X-ray- and MMC-induced DNA damage which was induced in the spermiogenic cells and retained in the sperm until fertilization was investigated by analysis of the potentiation effects of 2 repair inhibitors, 3-aminobenzamide (3AB) and caffeine on the yield of chromosome aberrations. The frequency of chromosome aberrations observed in the eggs fertilized with sperm recovered from the early spermatid to late spermatocyte stage with X-irradiation of 4 Gy (16-20 days after X-irradiation) was markedly higher than that in the eggs fertilized with sperm recovered from spermatozoa to late spermatid stage (0-8 days after X-irradiation). The induced chromosome aberrations predominantly consisted of chromosome-type aberrations, the main type being chromosome fragment followed by chromosome exchange through all the spermiogenic stages. On the other hand, a high frequency of chromosome aberrations was not induced through all the stages with MMC treatment of 5 mg/kg. The remarkable potentiation effects of 3AB and caffeine were found in the eggs fertilized with sperm recovered from almost all the spermiogenic stages after X-irradiation. In the MMC treatment, a remarkable caffeine effect was observed occasionally in mid-early spermatids to late spermatocytes where a large amount of MMC damage could be induced. These results suggest that the large amount of DNA lesions induced in spermiogenic cells by X-rays and MMC persist as reparable damage until sperm maturation and are effectively repaired in the cytoplasm of the fertilized eggs.     

A comparative study of the potentiating effect of caffeine and poly-D-lysine on chromosome damage induced by X-rays in plant cells.

X-Ray-induced chromosomal aberrations (CA) were potentiated by post-treatments in G2 with either caffeine (caff) or poly-D-lysine (PDL) in root-tip cells of Allium cepa. The enhancement of the yield of CA was concomitant with an increase in the frequency of mitosis. Our results seem to support the idea of a direct relationship between radiation-induced G2 delay and repair of chromosome damage. Here we report on similarities between caffeine and PDL in both decreasing G2 delay and enhancing chromatid aberration yield. The possible molecular mechanism(s) of action responsible for the cytogenetic effects observed are discussed.     

Induction of sister chromatid exchanges by UV light and its inhibition by caffeine

Sister chromatid exchanges in Chinese hamster chromosomes were studied after pulse-labeling cells with ³H-thymidine at various concentrations. Whereas the frequency of chromatid aberrations varied widely, depending upon tritium dose, there was no significant change in the sister chromatid exchange frequency, even with a 40-fold range of variation in the tritium concentration in the medium. When cells were exposed immediately after labeling to UV light at 40 erg/mm² and examined at the second mitosis, the frequency of sister chromatid exchanges was found to be 4 times higher than that of the unirradiated controls. A synchronization treatment utilizing 2 mM thymidine also caused a two-fold rise in the exchange frequency above the control level. Furthermore, when synchronized cells were irradiated with UV light at a dose of 40 erg/mm², the exchange frequency exceeded 5 times that of the untreated controls. However, this effect was detectable only when cells were irradiated at the earlier part of the S phase, while no change was detected when irradiated at the late S or G2 phase. A post-treatment of irradiated cells with caffeine caused a remarkable decrease in the frequency of sister chromatid exchanges. On the other hand, the frequency of chromatid aberrations of the deletion type increased strikingly after the same treatment. The results appear to suggest a certain correlation between the mechanism involved in the induction of sister chromatid exchanges and a post-replication repair of DNA damage.     

Effect of caffeine and adenosine on G2 repair: mitotic delay and chromosome damage

Proliferating plant cells treated during the late S period with 5-aminouracil (AU), give the typical response that DNA-damaging agents induce, characterized by: an important mitotic delay, and a potentiation of the chromosome damage by caffeine post-treatment. The study of labelled prophases, after a tritiated thymidine pulse, allowed evaluation of the mitotic delay induced by AU as well as its reversion by caffeine, while chromosome damage was estimated by the percentage of anaphases and telophases showing chromosomal aberrations. Post-treatment with adenosine alone has shown no effect on mitotic delay or chromosomal damage. However, when cells after AU were incubated in caffeine plus adenosine, the chromosome damage potentiation was abolished without affecting the caffeine action on mitotic delay. As a consequence, we postulate that caffeine could have two effects on G2 cells with damaged DNA: the first, to cancel their mitotic delay and the second to inhibit some DNA-repair pathway(s). Only this last effect could be reversed by adenosine.     

Effects of caffeine and inhibitors of DNA synthesis on chromatid-type aberrations induced by acetaldehyde in root-tip cells

Root-tip cells of Allium cepa were exposed to acetaldehyde (AA) and post-treated with caffeine and 3 inhibitors of DNA synthesis, namely hydroxyurea (HU), 5-fluorodeoxyuridine (FdUrd), and arabinofuranosylcytosine (araC). Caffeine strongly potentiated the frequency of chromatid-type aberrations when given immediately after the AA treatment or as a 5-h treatment starting 10 h before the addition of colchicine. In contrast, no enhancement was observed when caffeine was present for the last 2.5 h, simultaneously with colchicine. The inhibitors of DNA synthesis were given following this last schedule. Both HU and FdUrd clearly enhanced the yield of AA-induced chromatid aberrations, while no enhancement fo chromosone damage was observed after exposure in araC.     

Protective effect of caffeine on chromosomal structures of mammalian cells irradiated with UV-rays]

The effect of caffeine (2mM) on the frequency of structural mutations induced by UV light (lambda = 265 nm at an incident dose of 40 erg/mm2) in the primary culture of mouse embryonic fibroblasts is studied. A half-hour treatment with caffeine of cells at the time of the first mitosis metaphase decreased approximately by 2 times the frequency of chromosome aberrations induced by UV light at the S stage and observed at the metaphase of this or the next C-mitosis. The frequency of both breaks and exchanges decreased as the result of caffeine treatment. The persistence of the protective effect of caffeine at the time of the second C-mitosis suggests that the observed decrease of the aberration rate is accompanied by the true reparation of pre-mutational lesions in chromosomes; the nature of the reparative process and the time when it takes place is as yet not clear. Caffeine did not decrease the frequency of spontaneous structural mutations.     

Clastogenic action of ellipticine over the cell cycle of human lymphocytes and influence of posttreatments with caffeine and ara-C at G2

The clastogenic potential of the intercalating compound ellipticine, an antitumor alkaloid, has been demonstrated in mammalian cells. To characterize the mechanism of action of this drug over the cell cycle, human lymphocyte cultures from 2 healthy donors were treated with 3 micrograms/ml ellipticine in 30-min pulses during different phases of the cell cycle and analyzed for chromosomal aberrations and sister-chromatid exchanges. The G2 phase was most sensitive in terms of induction of aberrations, followed by S and G1. Chromatid-type aberrations were the most common type of chromosomal damage. Induction of SCEs was significantly high only after treatment at G1, when the frequencies of SCEs doubled. The post-treatment effect of lymphocytes with inhibitors of DNA repair, 10(-3) M caffeine and 5 x 10(-6) M 1-beta-D-arabinofuranosylcytosine, was also tested by adding 3 micrograms/ml ellipticine at G2 in 30-min pulses and immediately followed by caffeine and/or ara-C during the last 3 h before harvesting. Three experiments performed on blood from 3 donors showed a moderate potentiation effect on the frequency of chromatid-type aberrations (about 2-3 times) by both inhibitors. Likewise, a 3-fold increase was observed in the frequencies of chromosomal aberrations when caffeine and ara-C were combined. The present data demonstrate that posttreatment with caffeine and ara-C at G2 can modify the response of human lymphocytes treated with ellipticine by increasing the clastogenic action of this compound or by changing the cell-cycle progression.     

G2 repair and chromosomal damage in lymphocytes from workers occupationally exposed to low-level ionizing radiation

The effect of the G2 repair of chromosomal damage in lymphocytes from workers exposed to low levels of X- or gamma-rays was evaluated. Samples of peripheral blood were collected from 15 radiation workers, 20 subjects working in radiodiagnostics, and 30 healthy control donors. Chromosomal aberrations (CA) were evaluated by scoring the presence of chromatid and isochromatid breaks, dicentric and ring chromosomes in lymphocytes with/without 5 mM caffeine plus 3 mM-aminobenzamide (3-AB) treatment during G2. Our results showed that the mean value of basal aberrations in lymphocytes from exposed workers was higher than in control cells (p < 0.001). The chromosomal damage in G2, detected with caffeine plus 3-AB treatment was higher than the basal damage (untreated conditions), both in control and exposed populations (p < 0.05). In the exposed workers group, the mean value of chromosomal abnormalities in G2 was higher than in the control (p < 0.0001). No correlation was found between the frequency of chromosome type of aberrations (basal or in G2), and the absorbed dose. Nevertheless, significant correlation coefficients (p < 0.05) between absorbed dose and basal aberrations yield (r = 0.430) or in G2 (r = 0.448) were detected when chromatid breaks were included in the total aberrations yield. Under this latter condition no significant effect of age, years of employment or smoking habit on the chromosomal aberrations yield was detected. However, analysis of the relationship between basal aberrations yield and the efficiency of G2 repair mechanisms, defined as the percentage of chromosomal lesions repaired in G2, showed a significant correlation coefficient (r = -0.802; p < 0.001). These results suggest that in addition to the absorbed dose, the individual G2 repair efficiency may be another important factor affecting the chromosomal aberrations yield detected in workers exposed to low-level ionizing radiation.     

Cytogenetical characterization of Chinese hamster ovary X-ray-sensitive mutant cells xrs 5 and xrs 6. VII. Complementation analysis of X-irradiated wild-type CHO-K1 and xrs mutant cells using the premature chromosome condensation technique

The complementation effect of wild-type CHO-K1 and xrs mutants after fusion, as judged by the frequencies of X-ray-induced G1 and G2 premature chromosome condensation (PCC), was studied. For induction of PCC, X-irradiated interphase cells (G1 and G2) were fused immediately with untreated mitotic cells of the same cell line or with mitotic cells of another line. The frequencies of breaks in G1-PCC, or breaks and chromatid exchanges in G2-PCC were determined and the latter parameter was compared with the frequency of chromosomal aberrations in mitotic cells following G2 irradiation. CHO-K1 cells were capable of complementing the X-ray sensitivity of both xrs 5 and xrs 6 cells. However, full restoration of the repair defect in xrs cells could never be accomplished. The mutants failed to complement each other. In CHO-K1 cells, the incidence of chromosomal aberrations was significantly higher in G2-PCC (2.5-fold) than that observed in mitotic cells at 2.5 h after irradiation. The ratio of the induced frequency of aberrations in G2-PCC to that in mitotic cells was correlated with the degree of repair of DNA double-strand breaks (dsb) and reached almost 1 in xrs 5 cells indicating no repair. In addition the data indicated that, during the period of recovery of CHO-K1 cells, X-ray-induced breaks decreased but exchanges remained at the same level. In contrast, due to a deficiency in rejoining of dsb in xrs mutants, breaks remained open for a long period of time, allowing the formation of additional chromatid exchanges during recovery time.     

Cell cycle-dependent potentiation of X-ray-induced chromosomal aberrations by 3-aminobenzamide

The poly(ADP-ribose) polymerase inhibitor 3-aminobenzamide had dramatically different effects on X-ray-induced cytogenetic damage in human lymphocytes depending on the stage of the cell cycle in which cells were irradiated. 3-Aminobenzamide (0.08-3.00 mM) potentiated the frequency of chromosomal aberrations when lymphocytes were irradiated in G1, S, or late G2. No effect was observed, however, when lymphocytes were irradiated in G0 or at the S/G2 boundary 6 h before termination of culture. These results indicate that poly(ADP-ribose) polymerase may be involved in chromosomal repair of radiation damage only during specific stages of the cell cycle.     

The Production of Chromosome Aberrations in Various Mammalian Cells by Triethylenemelamine

The cytogenetic effects of triethylenemelamine (TEM) were studied using five different mammalian tissues. Treatments of 0.1 and 0.2 mg/kg TEM on differentiating mouse spermatogonia and bone marrow cells showed no significant differences in the frequency of chromosomal aberrations produced in these two tissues. At higher doses, however, the sensitivites of the two tissues appear to be different. The frequency of aberrations varies with time after treatment, with the greatest amount occurring at the latter fixation times. Results of an experiment on primary spermatocytes indicated a correlation between the frequency of chromosome aberrations and DNA replication. Human peripheral leukocytes were utilized in an attempt to clarify the cell-stage specificity of TEM-induced chromosome aberrations. Cultures were treated with TEM prior to PHA stimulation (G0), as well as various time intervals after stimulation (late G,1 S, and G2). The most sensitive stages of the cell cycle to aberration induction were later G1 and S, with chromatid aberrations the predominant type. A very low yield of chromosome damage was observed with the G0 and G1 treated stages. The experiments described tend to support the view that TEM is most effective at inducing aberrations when an intervening round of DNA replication has occurred.     

3-Aminobenzamide does not affect X-ray-induced cytogenetic damage in G0 human lymphocytes

The inhibition of poly(ADP-ribose) polymerase by 3-aminobenzamide (3AB) has been reported to have very different effects on X-ray-induced chromosome aberrations in G0 human lymphocytes. One group of investigators observed a 2-3-fold increase in the yield of rings, dicentrics and chromosome breaks after X-irradiation and 3AB treatment, whereas another group found that 3AB had no effect on X-ray-induced chromosome aberrations. To resolve this discrepancy, we repeated the experiments as described by both groups and found no effect of 3 mM or 5 mM 3AB on the frequency of chromosome aberrations induced by either 1 Gy or 2 Gy of X-rays. Furthermore, we found no effect of 3AB on X-ray-induced aberration yields in C-banded prematurely condensed chromosome preparations from unstimulated human lymphocytes. These results indicate that poly(ADP-ribose) polymerase is not involved in the repair of cytogenetic damage in G0 human lymphocytes.     

Suppressing effect of antimutagenic flavorings on chromosome aberrations induced by UV-light or X-rays in cultured Chinese hamster cells

Chromosome aberrations induced by UV-light or X-rays were suppressed by the post-treatment with antimutagenic flavorings, such as anisaldehyde, cinnamaldehyde, coumarin, and vanillin. UV- or X-ray-irradiated surviving cells increased in the presence of each flavoring. X-ray-induced breakage-type and exchange-type chromosome aberrations were suppressed by the vanillin treatment in the G1 phase of the cell cycle and a greater decrease in the number of X-ray-induced chromosome aberrations during G1 holding was observed in the presence of vanillin. Furthermore, a greater decrease in the number of X-ray-induced DNA single-strand breaks was observed in the presence of vanillin. Treatment with vanillin in the G2 phase suppressed UV- and X-ray-induced breakage-type but not exchange-type chromosome aberrations. The suppression of breakage-type aberrations was assumed to be due to a modification of the capability of the post-replicational repair of DNA double-strand breaks. These G1- and G2-dependent anticlastogenic effects were not observed in the presence of 2',3'-dideoxythymidine, an inhibitor of DNA polymerase beta. Based on these results, the anticlastogenic effect of vanillin was considered to be due to the promotion of the DNA rejoining process in which DNA polymerase beta acts.     

Effects of caffeine and cycloheximide during G2 prophase in control and X-ray-irradiated human lymphocytes

The effect of caffeine and cycloheximide during the G2 phase on frequency of chromosomal aberrations and G2 duration was studied in control and X-ray-irradiated human lymphocytes in vitro. Caffeine treatments alone increase the frequencies of chromatid breakage and decrease the average G2 duration in control and X-ray-irradiated lymphocytes (40 R). Both caffeine effects are reversed by 0.5 micrograms/ml cycloheximide in combination treatments. Cycloheximide treatments alone prolong G2 duration in control as well as in X-ray-irradiated lymphocytes although no improvement in chromosome repairing by this inhibitor of protein synthesis was observed under the conditions of our experiments. We propose that the cycloheximide effect is associated with a low level of mitotic factors, required for the entrance into mitosis, which is maintained at a higher level in caffeine treatment alone. Finally, G2 delay has generally been associated with certain genome damage. The fact that the caffeine and cycloheximide effects on X-irradiated lymphocytes are also present in control lymphocytes (without X-rays) suggests that control of the G2 duration constitutes one of the mechanisms involved in DNA repair operating during the G2 phase.