Effect of caffeine on in vivo processing of alkylated bases in proliferating plant cells.

DNA damage was induced by either 2 mM ethylmethanesulfonate or 1 Gy of gamma-irradiation in Allium cepa L. root meristems. The percentage of DNA that migrated towards the anode during microelectrophoresis after alkali denaturation (pH approximately 13.5) of the isolated nuclei (comet assay) reflects the amount of single strand breaks present in them. There was some DNA migration (12.8+/-2.4%) in untreated roots. This percentage doubled at the end of 1.5 h treatment with the mono-functional alkylating agent 2 mM ethylmethanesulfonate, and trebled after a single exposure to 1 Gy of gamma-rays. A proportion of the DNA migration caused by these two treatments was reversed (repaired) by a 2 h long period of in vivo recovery. However, when 5 mM caffeine was applied after removal of the alkylating agent, the amount of DNA migrating to the comet tail over the same 2 h period was almost double that at the onset of recovery. In both control and irradiated nuclei, caffeine also increased the initial level of DNA migration in the comet assay, but to a lesser extent. These results indicate that caffeine increases the DNA damage that accumulates during the processing of alkylated bases and, to a lesser extent, of the DNA bases damaged by gamma-irradiation. Thus, the potentiation effect of caffeine on induced chromosomal damage may not just be due to caffeine-induced cancellation of the G2 checkpoint, but also to a direct effect this methylxantine has on the processing of DNA damage.

Ataxia telangiectasia: G2 checkpoint and chromosomal damage in proliferating lymphocytes.

There is a checkpoint pathway in eukaryotic cells that depends on ATM (ataxia telangiectasia mutated) kinase which activates the processes leading to the repair of DNA damage and also lengthens the G(2) stage of the cell cycle. In cells from ataxia telangiectasia patients, due to their lack of active ATM kinase, an increase in chromosomal aberrations and a failure to induce G(2) lengthening could be expected. However, the basal G(2) timing in ataxia telangiectasia cells was longer than in controls and was further extended after X-ray irradiation (0.4 Gy), although to a lesser extent than in controls. Moreover, in control cells caffeine shortened G(2) and increased chromosomal damage 7-fold, while in ataxia telangiectasia cells caffeine only trebled aberration yield without shortening G(2). As caffeine is an inhibitor of ATM kinase, these results suggest the existence of some redundant ATM-independent checkpoint in G(2) of ataxia telangiectasia cells. The differential response to caffeine of ataxia telangiectasia and control lymphocytes may be explained by the presence of two different subpathways in the G(2) checkpoint: one regulating the processing and repair of damaged DNA and the other controlling G(2) timing. While in controls both subpathways may be mediated by ATM kinase, in ataxia telangiectasia cells caffeine-sensitive ATR kinase and the caffeine-insensitive DNA-PK kinases might be responsible for DNA repair and the G(2) delay subpathways, respectively. Confirmation of this model in ataxia telangiectasia cells with another cell type in which both subpathways are mediated by DNA-PK should define whether a metylxanthine such as caffeine may also have an additional direct inhibitory effect on DNA repair.

Fanconi anemia lymphocytes: effect of DL-alpha-tocopherol (Vitamin E) on chromatid breaks and on G2 repair efficiency.

The high frequency of chromosomal breaks in Fanconi anemia (FA) lymphocytes has been related to the increased oxidative damage shown by these cells. The effect of 100 microM DL-alpha-tocopherol (Vitamin E) on the level of chromosomal damage in mitosis was studied in lymphocytes from five FA patients and from age matched controls, both under basal conditions and when G2 repair was prevented by 2.5 mM caffeine (G2 unrepaired damage). In addition, the effect of this antioxidant on G2 duration and the efficiency of G2 repair was also evaluated in the sample. alpha-Tocopherol (AT) decreased the frequency of chromosomal damage (under basal and inhibited G2 repair conditions) and the duration of G2 in FA cells. This antioxidant protective effect, expressed as the decrease in chromatid breaks, was greater in FA cells (50.8%) than in controls (25%). The efficiency of the G2 repair process (G2 R rate) defined as the ratio between the percentage of chromatid breaks repaired in G2 and the duration of this cell cycle phase was lesser in FA cells (10.6) than in controls (22.6). AT treatment slightly increased this G2 R rate, both in FA cells and controls. These results suggest that an increased oxidative damage and a lower G2 repair rate may be simultaneously involved in the high frequency of chromatid damage detected in FA cells.

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.

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 g-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 5mM caffeine plus 3mM-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