Modification of p53 protein profile by gamma irradiation followed by methyl donor starvation.

The possible beneficial radio-protective effects of one-carbon transfer agents namely folate, choline and methionine have been the subject of extensive investigation. Ionizing radiation is known to extensively damage the DNA. One-carbon transfer agents have been proposed to have important role in context of DNA repair via their role in purine and thymidylate synthesis and in DNA methylation. Sufficient dietary availability of one-carbon transfer agents therefore, might have ability to modify radiation effects. In present study modifications in level of tumor suppressor protein p53 by gamma irradiation followed by methyl donor starvation was observed. Experiments showed an increase in nuclear and cytoplasmic p53 protein concentration in liver, spleen and thymus. The overall rise in the level of p53 protein in liver was found to be less than that in spleen and thymus. Moreover significant heterogeneity in the basal level of expression of the p53 protein in liver, spleen and thymus was observed as the level of p53 protein in spleen and thymus was found to be 7-8 fold more than that in liver. Results indicated that radiation stress followed by methyl donor starvation could significantly induce p53 protein in spleen and thymus where there was a dramatic accumulation of p53 following irradiation, while in other tissues, particularly the liver, no such dramatic response was seen. Folate contribution of intestinal bacteria was found to influence p53 protein levels. Our observations indicated a prominent role played by the methyl donors in protecting the cell against harmful effects of ionizing radiation.

Modulation of enzymes involved in folate dependent one-carbon metabolism by gamma-radiation stress in mice.

The role of various enzymes in folate dependent one-carbon metabolism, which are involved in mobilizing the folate pool for DNA synthesis and the DNA methylation reaction, was investigated. Male Swiss mice (6 weeks old) were subjected to 2, 5 and 7 Gy total body gamma-irradiation. The animals were killed at intervals of 24, 48, 72, 96, 120 and 192 h and the livers were removed. Using a 12000 x g supernatant of 10% tissue homogenate, the activities of dihydrofolate reductase, thymidylate synthase and methylenetetrahydrofolate reductase were determined. The profiles of these folate enzymes were correlated to DNA damage by monitoring p53 protein profile and by comet tail moment analysis. A significant increase in activity of dihydrofolate reductase and thymidylate synthase was observed up to 96 h post-irradiation and the activity subsided thereafter, reaching control value after 192 h. A sharp decline in methylenetetrahydrofolate reductase activity was observed until 192 h after irradiation. Total folates declined by 54% after 96 h following irradiation, and p53 protein concentration in nuclei increased after irradiation, proportionate to radiation dose, and subsided slowly. Thus results indicate a significant drop in total folate levels and rise in p53 protein concentration in the liver after total body gamma-irradiation. It may appear that, under radiation stress conditions, levels of enzymes involved in one-carbon metabolism for DNA repair, are modulated up to a certain time interval, in a dose specific manner. It may also appear that the requirements of folate for nucleotide base synthesis seem to be met at the expense of other one-carbon transfer reactions.

Increased folate catabolism following total body gamma-irradiation in mice.

Concentrations of total folates and their oxidative degradate, para-aminobenzoyl glutamic acid, were determined in mouse after 2-7 Gy total body gamma-irradiation (TBI). Total liver folate levels were drastically reduced by almost 47% over a period of 120 h after TBI with 7 Gy. Oxidative damage, splitting the folate molecule into pterin and p-aminobenzoylglutamic acid (p-ABG), was observed after TBI. p-ABG levels, which 24 h after irradiation were raised by 15%, were further elevated to more than three-fold over the control after 120 h. A dose-dependent increase in the oxidative degradation of folate was observed. The oxidative cleavage of folate may be one factor contributing to folate deficiency in radiation stress.