Dietary L-methionine supplementation mitigates gamma-radiation induced global DNA hypomethylation: enhanced metabolic flux towards S-adenosyl-L-methionine (SAM) biosynthesis increases genomic methylation potential.

The objective of this study was to examine the effect of (60)Co-gamma (γ) radiation on modulation of genomic DNA methylation, if any, of mice maintained (6 weeks) on normal control diet (NCD) and L-methionine supplemented diet (MSD). To elucidate the possible underlying mechanism(s), we exposed the animals to γ-radiation (2, 3 and 4 Gy) and investigated the profile of downstream metabolites and enzymes involved in S-adenosyl-L-methionine (SAM) generation. Liver samples were also subjected to histopathological examinations. Compared to NCD fed and irradiated animals, hepatic folate, choline and L-methionine levels decreased moderately, while hepatic SAM levels increased in MSD fed and irradiated animals. Under these conditions, a marked modulation of methionine adenosyltransferase (MAT) and L-methionine synthase (MSase) activities was observed. Concomitant with increase in liver SAM pool, increased DNA methyltransferase (dnmt) activity in MSD fed mice indicated enhanced metabolic flux towards DNA methylation. Further results showed that genomic DNA methylation and 5-methyl-2'-deoxy cytidine residues were maintained at normal levels in MSD fed and irradiated mice compared to NCD fed and irradiated animals. In conclusion, our results suggest that increasing supply of preformed methyl groups, via dietary L-methionine supplementation might significantly increase methylation potential of radiation stress compromised DNA methylation cycle.

Mitigation of gamma-radiation induced abasic sites in genomic DNA by dietary nicotinamide supplementation: metabolic up-regulation of NAD(+) biosynthesis.

The search for non-toxic radio-protective drugs has yielded many potential agents but most of these compounds have certain amount of toxicity. The objective of the present study was to investigate dietary nicotinamide enrichment dependent adaptive response to potential cytotoxic effect of (60)Co γ-radiation. To elucidate the possible underlying mechanism(s), male Swiss mice were maintained on control diet (CD) and nicotinamide supplemented diet (NSD). After 6 weeks of CD and NSD dietary regimen, we exposed the animals to γ-radiation (2, 4 and 6Gy) and investigated the profile of downstream metabolites and activities of enzymes involved in NAD(+) biosynthesis. Increased activities of nicotinamide phosphoribosyltransferase (NAMPT) and nicotinamide mononucleotide adenylyltransferase (NMNAT) were observed up to 48h post-irradiation in NSD fed irradiated mice. Concomitant with increase in liver NAMPT and NMNAT activities, NAD(+) levels were replenished in NSD fed and irradiated animals. However, NAMPT and NMNAT-mediated NAD(+) biosynthesis and ATP levels were severely compromised in liver of CD fed irradiated mice. Another major finding of these studies revealed that under γ-radiation stress, dietary nicotinamide supplementation might induce higher and long-lasting poly(ADP)-ribose polymerase 1 (PARP1) and poly(ADP-ribose) glycohydrolase (PARG) activities in NSD fed animals compared to CD fed animals. To investigate liver DNA damage, number of apurinic/apyrimidinic sites (AP sites) and level of 8-hydroxy-2'-deoxyguanosine (8-oxo-dG) residues were quantified. A significant increase in liver DNA AP sites and 8-oxo-dG levels with concomitant increase in caspase-3 was observed in CD fed and irradiated animals compared to NSD fed and irradiated mice. In conclusion present studies show that under γ-radiation stress conditions, dietary nicotinamide supplementation restores DNA excision repair activity via prolonged activation of PARP1 and PARG activities. Present results clearly indicated that hepatic NAD(+) replenishment might be a novel and potent approach to reduce radiation injury.

Interaction between γ-radiation and dietary folate starvation metabolically reprograms global hepatic histone H3 methylation at lysine 4 and lysine 27 residues.

The objective of the present study was to investigate the regulatory control of histone H3 methylation at lysine 4 (H3K4) and lysine 27 (H3K27) residues in response to the effect of folate deficiency and gamma (γ)-radiation. Male Swiss mice maintained on folate sufficient diet (FSD) and folate free diet (FFD) based on AIN-93M formula, were subjected to 2-4 Gy total body γ-irradiation. There was a significant decrease in liver folate levels with concomitant depletion of S-adenosylmethionine (SAM) reserves. Folate deficiency and γ-radiation together induced H3K4 histone methyltransferase (H3K4HMTase) and suppressed H3K27 histone methyltransferase (H3K27HMTase) activities in a dose and time dependent manner. Our studies suggested radiation induced metabolic reprogramming of H3K4/H3K27 methylation patterns in FFD animals. We showed that radiation toxicity diverted one-carbon (C1) flux in FFD fed animals towards H3K4 methylation. Present work on methylation pattern of histone lysine residues gains particular importance as methylation of H3K4 residues is associated with euchromatin while methylated H3K27 residues promote gene silencing. In conclusion, our study suggests that maintenance of genomic histone methylation under γ-radiation stress might be a very dynamic, progressive process that could be modulated by dietary folate deficiency leading to formation of epigenetically reprogrammed cells.

Interaction between total body gamma-irradiation and choline deficiency triggers immediate modulation of choline and choline-containing moieties.

PURPOSE: The objective of this study was to examine the effect of 60Co-gamma (γ) radiation on acute phase modulation, if any, of choline and choline-containing moieties in choline-deficient subjects. Corresponding results could provide information that might be useful in the management of adverse effects of γ-radiation. MATERIALS AND METHODS: Male Swiss mice maintained on a choline-sufficient diet (CSD) and choline-free diet (CFD) based on AIN-93M formula, were subjected to whole body γ-irradiation (2-6 Gy). Liver, serum and brain samples from each group were then tested for: (i) Alterations in choline and choline-containing moieties such as phosphatidylcholine (PC) and sphingomyeline (SM); and (ii) modulation of choline profile modulating enzymes such as phospholipase D (PLD) and total sphingomyelinase (t-SMase). Liver and brain samples were also subjected to histo-pathological examinations. RESULTS: No significant changes were observed in folate, choline, choline-containing moieties and choline-modulating enzymes in choline-sufficient mice. In contrast, interaction between cytotoxic effects of γ-radiation and choline deficiency modulated choline and choline-containing moieties. Feeding CFD reduced hepatic concentrations of choline, PC and SM whereas PLD and t-SMase activities were significantly raised. The decrease in liver choline and choline-containing moieties was accompanied by an increase in blood choline concentration. Despite choline deficiency, the level of choline and acetylcholine synthesizing enzyme choline acetyltransfease (ChAT) significantly increased in the brain. CONCLUSIONS: We propose that choline deprivation and γ-radiation interact to modulate choline reserves of hepatic tissue, which might release choline to blood. Our studies also clearly showed that interaction between choline deficiency and γ-radiation might substantially enhance liver adipogenesis.

Enhanced one-carbon flux towards DNA methylation: Effect of dietary methyl supplements against gamma-radiation-induced epigenetic modifications.

Radiation exposure poses a major risk for workers in the nuclear power plants and other radiation related industry. In this context, we demonstrate that gamma-radiation is an efficient DNA demethylating agent and its injurious effect can be minimized by dietary methyl supplements (folate, choline and vitamin B12). To elucidate the possible underlying mechanism(s), male Swiss mice were maintained on normal control diet (NCD) and methyl-supplemented diet (MSD). After 2 weeks of NCD and MSD dietary regimen, we exposed the animals to gamma-radiation (2, 4 and 6Gy) and investigated the profile of downstream metabolites and activity levels of one-carbon (C(1)) flux generating enzymes. In MSD fed and irradiated animals, hepatic folate levels increased (P<0.01), while hepatic homocysteine levels decreased (P<0.01) compared to NCD fed and irradiated animals. Although hepatic folate level increased significantly in MSD fed animals (P<0.01), it showed a decrease in response to high doses of gamma-irradiation. Under these conditions, a marked suppression of S-adenosylmethionine (SAM) levels occurred in NCD fed and irradiated animals, suggesting reduced conversion of homocysteine to SAM. Concomitant with decline in liver SAM Pool, activities of DNA methyltransferase (Dnmt, that methylates DNA) and methionine synthase (MSase, that regenerates methionine from homocysteine) were both decreased in NCD fed and irradiated mice. However, in MSD fed and irradiated mice, they were increased. These results strongly indicated that increased levels of dnmt and MSase may enhance C(1) flux towards DNA methylation reactions in MSD fed animals. These results were confirmed and further substantiated by measuring genomic DNA methylation levels, which were maintained at normal levels in MSD fed and irradiated mice compared to NCD fed and irradiated animals (P<0.01). In conclusion, our results suggest that maintenance of genomic DNA methylation under gamma-radiation stress might be a very dynamic, progressive diet dependent process that could involve increased one-carbon flux through various C(1) metabolites.

Interaction between cytotoxic effects of gamma-radiation and folate deficiency in relation to choline reserves.

The search for non-toxic radio-protective drugs has yielded many potential agents but most of these compounds have certain amount of toxicity. Recent studies have indicated that bio-molecules such as folate and choline might be of radio-protective value as they are, within broad dose ranges, non-toxic to humans and experimental animals. The objective of the present study was to investigate choline dependent adaptive response to potential synergistic cytotoxic effect of folate deficiency and gamma-radiation. Male Swiss mice maintained on folate sufficient diet (FSD) and folate free diet (FFD) based on AIN-93M formula, were subjected to 1-4Gy total body gamma-irradiation. To investigate liver DNA damage, apurinic/apyrimidinic sites (AP sites) were quantified. A significant increase in liver DNA AP sites with concomitant depletion of liver choline reserves was observed when gamma-radiation was combined with folate deficiency. Further work in this direction suggested that cytotoxic interaction between folate deficiency and gamma radiation might induce utilization of choline and choline containing moieties by modifying levels of key regulatory enzymes dihydrofolate reductase (DHFR) and choline oxidase (ChoOx). Another major finding of these studies is that significant liver damage at higher doses of radiation (3-4Gy), might release considerable amounts of choline reserves to serum. In conclusion, a plausible interpretation of the present studies is that folate deprivation and gamma-radiation interact to mobilize additional choline reserves of hepatic tissue, for redistribution to other organs, which could not be utilized by folate deficiency alone. Present results clearly indicated a distinct choline pool in liver and kidney tissues that could be utilized by folate deficient animals only under radiation stress conditions.

Folate deficiency followed by ionizing radiation perturbs hepatic dihydrofolate reducatse activity.

There is lot of interest in the folate metabolism because of the essential role of folate coenzymes in nucleic acid synthesis. Gamma (gamma) radiation is well known for inducing damage in the DNA. To counteract these damage, a variety of DNA repair pathways have evolved that require regular supply of DNA bases whose biosynthesis in turn depends on sufficient pools of folate dependent enzymes like dihydrofolate reductase (DHFR). In the present study, we examined the ionizing radiation mediated perturbation of DHFR activity in folate deficient and folate sufficient conditions. In folate deficient animals a potent inhibition of liver DHFR activity was observed. Our results showed that combination of folate starvation and ionizing radiation might adversely affect the DHFR activity, compared to their individual treatments. Measurement of apurinic/apyrimidinic sites (AP sites), a major type of DNA damage generated by radiation induced loss of purine and/or pyrimidine base, indicated a dose dependent DNA damage in folate deficient animals. In conclusion our data suggest an interactive role of folate deficiency and radiation injury in inhibiting DHFR activity.

Modulation of DNA methyltransferase profile by methyl donor starvation followed by gamma irradiation.

DNA methylation is an important epigenetic mechanism of transcriptional control, which plays an essential role in maintaining cellular function. Role of one-carbon transfer agents/methyl donors namely folate, choline and methionine in DNA methylation has been the subject of extensive investigation. The methylation pattern of DNA is established during embryogenesis by DNA methyltransferase 3 (dnmt3) and is subsequently maintained by maintenance methylation activity of the enzyme DNA methyltransferase 1 (dnmt1). Ionizing radiation is known to extensively damage the DNA. Sufficient dietary availability of methyl donors is known to contribute towards one-carbon transfer mediated repair of damaged DNA where folate is involved in nucleotide base synthesis. In the present study, modification in activities of dnmt1 and dnmt3 by methyl donor starvation followed by gamma-irradiation was observed. Assays were based on the catalytic transfer of (3)H-methyl groups from S-adenosyl-L: -methionine to a DNA substrate. Experiments showed a dose and methyl donors starvation dependent attenuation in dnmt1 activity. Attenuation of dnmt1 activity was most significant for diet deprived of all the three-methyl donors. No significant change in nuclear or cytoplasmic dnmt3 activity was observed when either or all the three possible source of dietary methyl group supply were removed. Ionizing radiation and methyl donor deficiency were observed to act synergistically towards inhibiting dnmt1 activity. Present results suggested possibility of interaction among folate, methionine and choline deficiency to potentiate symptoms of ionizing radiation stress. These enzymatic modifications might contribute to altered DNA methylation after chronic feeding of methyl donor free diets followed by gamma irradiation. These results suggested that dietary availability of methyl donors and gamma-radiation stress might significantly alter the dnmt1 profile.

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.