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 Vipen Batra ⇑ , Poonam Verma Radiation Biology and Health Sciences Division, Bhabha Atomic Research Centre, Mumbai 400 085, India article info Article history: Received 7 January 2014 Accepted 28 March 2014 Available online 8 April 2014 Keywords: Diet Metabolism DNA methylation L-methionine Radiation toxicity abstract The objective of this study was to examine the effect of 60 Co-gamma (c) radiation on modulation of geno- mic DNA methylation, if any, of mice maintained (6 weeks) on normal control diet (NCD) and L-methio- nine supplemented diet (MSD). To elucidate the possible underlying mechanism(s), we exposed the animals to c-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 irra- diated 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 0 - 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. Ó 2014 Elsevier Ltd. All rights reserved. 1. Introduction Dietary countermeasures against radiation toxicity require therapeutic agents that are safe and easy to administer (Singh et al., 2012). Earlier studies have indicated that supplementation of diet with nutrients might alleviate harmful effects of radiation such as DNA damage and DNA hypometylation (Koturbash et al., 2005; Brown et al., 2010). In this context, dietary one-carbon (C 1 ) transfers agents namely folate, choline and L-methionine are quite important (Batra and Devasagayam, 2009). Liver, seat of C 1 metab- olism, plays a central role in L-methionine metabolism (Yun et al., 2013). Currently, it is believed that C 1 transfer agents affect DNA through two potential pathways. The first is through epigenetic regulation of gene expression by covalent modification of cytosine residues on DNA helix (Anderson et al., 2012). The second pathway involves role of folate coenzymes in purine and pyrimidine biosyn- thesis (Champier et al., 2012). Under radiation stress conditions both of these metabolic pathways for DNA methylation and nucle- otide synthesis appear to compete (Mason and Choi, 2000). Increased utilization of cellular folate pool for purine/pyrimidine biosynthesis may decrease the methionine synthase (MSase) cata- lyzed folate dependent C 1 flux towards S-adenosyl-L-methionine (SAM) generation (Hoffbrand and Jackson, 1993). Reduced SAM synthesis might eventually lead to unwarranted hypomethylation of genomic DNA (Koturbash et al., 2005). Such undesirable DNA hypomethylation might facilitate manifestation of late (months to years) effects (cancer) of ionizing radiation (Ehrlich, 2009). Sim- ilarly, radiation exposure adversely affects choline reserves as it prompts choline utilization for repair of oxidized phosphatidylcho- line (PC) molecules in bio-membranes (Batra et al., 2011). Effect of L-methionine on availability of SAM for DNA methylation reactions has obtained less attention; in the studies (Batra et al., 2010, 2011) dealing with supplementation of dietary C 1 transfer agents under http://dx.doi.org/10.1016/j.fct.2014.03.040 0278-6915/Ó 2014 Elsevier Ltd. All rights reserved. ⇑ Corresponding author. Address: Radiation Biology and Health Sciences Division, Modular Laboratories, Bhabha Atomic Research Centre, Room No. 3-47-S, Mumbai 400 085, India. Tel.: +91 22 25590411; fax: +91 22 25560750/25505151. E-mail address: [email protected](V. Batra). Food and Chemical Toxicology 69 (2014) 46–54 Contents lists available at ScienceDirect Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox
The objective of this study was to examine the effect of 60Co-gamma (c) radiation on modulation of geno-mic DNA methylation, if any, of mice maintained (6 weeks) on normal control diet (NCD) and L-methio-nine supplemented diet (MSD). To elucidate the possible underlying mechanism(s), we exposed theanimals to c-radiation (2, 3 and 4 Gy) and investigated the profile of downstream metabolites andenzymes involved in S-adenosyl-L-methionine (SAM) generation. Liver samples were also subjected tohistopathological examinations. Compared to NCD fed and irradiated animals, hepatic folate, cholineand L-methionine levels decreased moderately, while hepatic SAM levels increased in MSD fed and irra-diated 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 fluxtowards DNA methylation. Further results showed that genomic DNA methylation and 5-methyl-20-deoxy cytidine residues were maintained at normal levels in MSD fed and irradiated mice compared toNCD fed and irradiated animals. In conclusion, our results suggest that increasing supply of preformedmethyl groups, via dietary L-methionine supplementation might significantly increase methylationpotential of radiation stress compromised DNA methylation cycle.
� 2014 Elsevier Ltd. All rights reserved.
1. Introduction
Dietary countermeasures against radiation toxicity requiretherapeutic agents that are safe and easy to administer (Singhet al., 2012). Earlier studies have indicated that supplementationof diet with nutrients might alleviate harmful effects of radiationsuch as DNA damage and DNA hypometylation (Koturbash et al.,2005; Brown et al., 2010). In this context, dietary one-carbon (C1)transfers agents namely folate, choline and L-methionine are quiteimportant (Batra and Devasagayam, 2009). Liver, seat of C1 metab-olism, plays a central role in L-methionine metabolism (Yun et al.,2013). Currently, it is believed that C1 transfer agents affect DNAthrough two potential pathways. The first is through epigeneticregulation of gene expression by covalent modification of cytosine
residues on DNA helix (Anderson et al., 2012). The second pathwayinvolves role of folate coenzymes in purine and pyrimidine biosyn-thesis (Champier et al., 2012). Under radiation stress conditionsboth of these metabolic pathways for DNA methylation and nucle-otide synthesis appear to compete (Mason and Choi, 2000).Increased utilization of cellular folate pool for purine/pyrimidinebiosynthesis may decrease the methionine synthase (MSase) cata-lyzed folate dependent C1 flux towards S-adenosyl-L-methionine(SAM) generation (Hoffbrand and Jackson, 1993). Reduced SAMsynthesis might eventually lead to unwarranted hypomethylationof genomic DNA (Koturbash et al., 2005). Such undesirable DNAhypomethylation might facilitate manifestation of late (monthsto years) effects (cancer) of ionizing radiation (Ehrlich, 2009). Sim-ilarly, radiation exposure adversely affects choline reserves as itprompts choline utilization for repair of oxidized phosphatidylcho-line (PC) molecules in bio-membranes (Batra et al., 2011). Effect ofL-methionine on availability of SAM for DNA methylation reactionshas obtained less attention; in the studies (Batra et al., 2010, 2011)dealing with supplementation of dietary C1 transfer agents under
Casein supplied by central Drug House (P) Ltd. (New Delhi, India), cornstarch anddextrinized cornstarch (feed grade) by Vijaya Enterprises (Mumbai, India), sucroseby Sisco research laboratory, (Mumbai, India), soyabean oil by Bharat Foods Co-operative Ltd. (Gandhidham, India), cellulose fiber by Maple Biotech (P) Ltd., (Pune,India), Mineral mix by MP biomedicals, USA, L-cysteine, choline bitartarate and tert-butylhydroquinone by Sigma Aldrich Company, (St. Louis, USA).
V. Batra, P. Verma / Food and Chemical Toxicology 69 (2014) 46–54 47
radiation stress. Therefore, in present study we hypothesized thatreduced availability of folate and choline under radiation stresswould necessitate preferential utilization of L-methionine forDNA methylation. To test our hypothesis, we supplementedAIN93M rodent diet with L-methionine, which provides preformedmethyl groups for DNA methylation reactions.
Metabolism of the L-methionine methyl group involves its acti-vation to SAM followed by the transfer of the methyl group to DNA(Chen et al., 2010). Methionine adenosyltransferase (MAT) cata-lyzes the synthesis of SAM from L-methionine and ATP (Elremalyet al., 2012). SAM acts as the ultimate methyl donor in the DNAmethyltransferase (dnmt) mediated DNA methylation reaction,resulting in the formation of S-adenosyl-L-homocysteine (SAH)after donating its methyl group to the DNA substrate (Smithet al., 2012). Because the dnmt reaction is dependent on the supplyof SAM and removal of SAH, the SAM: SAH ratio is used as a ‘geno-mic methylation potential’ to indicate the likelihood of hyper-methylation or hypomethylation of DNA (Dominguez-Salas et al.,2013). A decrease in the SAM: SAH ratio is known to inhibit DNAmethylation (Sibani et al., 2002 Evidence suggests that reducedavailability of methyl group(s) for genomic cytosine(s) methylationmay facilitate transcriptional initiation and expression of other-wise silenced oncogenes (Jones et al., 1998; Ono et al., 2012). Inpresent work, we attempted comparative evaluation of the effectsof normal control diet (NCD) that contained normal levels ofL-methionine and experimental L-methionine-supplemented diet(MSD) that contained therapeutic levels of L-methionine, on globalDNA methylation under radiation induced stress conditions. Theexperiments reported here represent initial attempts to examineour hypothesis by comparing the metabolism of L-methionine ina mice liver homogenate system.
2. Materials and methods
2.1. Chemicals and reagents
ATP, alkaline phosphatase, bovine serum albumin (BSA), casein, choline bitar-trate, deoxycholic acid, L-dithiothreitol (DTT), dowex 50X4 cation exchange column,EDTA, 5-formyltetrahydrofolate, D, L-homocysteine, igepal CA-630, lauryl sulfate, L-methionine, NaCl, nuclear extraction kit, peroxidase (type I), nuclease P1 and pro-tease inhibitor cocktail were purchased from Sigma–Aldrich Chemical Co (St. Louis,MO, USA). DNA methylation quantification kit was from Epigentek Inc. (Brooklyn,NY). L-methionine and SAM assay kit was purchased from Mediomics Inc. (St. Louis,MO, USA). De-ionized water was purified with a Milli-Q water purification system(Millipore, Bedford, MA). All other chemicals/reagents were purchased fromreputed manufacturers and were of analytical grade.
2.2. Animal maintenance and feeding
Male Swiss mice obtained from the departmental animal house facility ofBhabha Atomic Research Centre (BARC) were maintained (for 6 weeks) on normalcontrol diet (NCD) and L-methionine supplemented diet (MSD) based on AIN-93Mformula (Reeves et al., 1993), which recommends a protein content of 14% (w/w)for rodents (Table 1). Due to availability of limited literature on radioprotectiveeffect of L-methionine, preliminary experiments (data not shown) were conductedto select optimum L-methionine dose. Dietary L-methionine dose (2 g/kg diet) facil-itated maintenance of liver L-methionine levels under radiation stress conditionswithout corresponding enhancement in serum alanine transferase (ALT) levels.Therefore, L-methionine (2 g/kg diet) equivalent to 340 mg/kg body weight/daywas supplemented to produce MSD (Table 1). NCD and MSD diets were supple-mented with folate and vitamin B12 to compensate for adverse effect of irradiationon stability and bioavailability of these nutrients. The mice were given free accessto food and water throughout the study. Mice were housed four per cage in a roomwith a constant temperature of 23 ± 1 �C and a 12 h light–dark cycle.
2.3. Experimental design
The mice were randomly distributed into fourteen different groups of four ani-mals each under identical conditions and were grouped as follows:
Group 1
Served as NCD fed control (NCD-C) and was not irradiated Group 2 Served as MSD fed control (MSD-C) and was not irradiated
Group 3, 4and 5
NCD fed animals received 2, 3, and 4 Gy of 60Co-c-radiation,respectively and liver and blood were removed after 24 h ofirradiation
Group 6, 7and 8
NCD fed animals received 2, 3, and 4 Gy of 60Co-c-radiation,respectively and liver and blood were removed after 48 h ofirradiation
Group 9, 10and 11
MSD fed animals received 2, 3, and 4 Gy of 60Co-c-radiation,respectively and liver and blood were removed after 24 h ofirradiation
Group 12,13and 14
MSD fed animals received 2, 3, and 4 Gy of 60Co-c-radiation,respectively and liver and blood were removed after 48 h ofirradiation
Animals were killed by CO2 inhalation and liver and blood wereremoved after different time intervals, as indicated above. The han-dling and sacrifice of the mice were done as per the guidelinesissued by BARC (Bhabha Atomic Research Center) animal ethicscommittee. Animals (6 weeks old) were subjected to total bodyc-radiation at a rate of 50 cGy/min by using a 60Co Bhabhatronradiotherapy unit (Department of Atomic Energy, Mumbai, India).Area of exposure was kept constant.
2.4. Folate, choline and L-methionine assay
Folate activity was determined by microbiological assay, using Lactobacilluscasei ATCC 7469 with 5-formyltetrahydrofolate (Sigma Chemical Co., Missouri,USA), as standard (after correction for the inactive isomer), as described earlier(Pote et al., 1998). The total folate was assayed after prior digestion of the samplewith chicken liver folyl conjugase.
Choline was assayed using fluorometric method (Woollard and Indyk, 2000). Inthe assay free choline was oxidized to betaine, via the intermediate betaine alde-hyde. The reaction generates H2O2 that reacts with 4-aminoantipyrine to producecolor (570 nm) and fluorescence (excitation/emission 535/587).
The L-methionine fluorescence assay was based on a novel assay platformdesign that utilized increased affinity of the DNA sequence-specific L-methioninerepressor protein for its unique DNA binding site in the presence of its ligand,SAM. The L-methionine from test sample was converted to SAM by SAM synthetasein the presence of ATP and Mg2+. L-methionine concentrations in test samples aredetermined using an L-methionine standard curve.
48 V. Batra, P. Verma / Food and Chemical Toxicology 69 (2014) 46–54
2.5. Determination of DNA methylation potential
Determination of DNA methylation potential involved assay of liver SAM andSAH levels. SAM fluorescence assay was based on a novel assay platform design thatutilized increased affinity of the DNA sequence-specific L-methionine repressor pro-tein for its unique DNA binding site in the presence of its ligand, SAM. For this assay,the MetJ consensus sequence was split into two approximately equal DNA ‘‘half-sites’’ with one half fragment labeled with fluorescein and the other half fragmentlabeled with Oyster� 645 fluorophore. Relative amount of SAM present in a testsample influenced the amount of DNA-MetJ protein complex formation in the assay.Complex formation brought the fluorescence labeled-DNA half-sites into closeproximity and thus caused a measurable change (increase) in fluorescence signalemission that could be readily measured using a microplate reader (excitation/emission: 485 nm/665 nm).
SAH levels were estimated (Capdevila et al., 2007) by competitive immunoassaysuitably modified in our laboratory. The assay was based on competition betweenSAH in sample and immobilized SAH bound to the walls of microtitre plate for bind-ing sites on a monoclonal anti-SAH antibody. In brief, 25 ll of the samples or cali-brators were transferred to SAH coated micro-titer wells. 200 ll of anti-SAHantibody (67 lg/L in assay buffer 2) was added to each well, and the samples wereincubated at ambient temperature (18–25 �C) for 30 min. The micro-titer plate waswashed 3 times, each time with 400 ll of washing solution per well, after which200 ll of HRP-conjugated antibody (1.3 mg/L in stabilization buffer) was added toeach sample. This plate was then incubated at ambient temperature for 30 minand then washed 3 times with 400 ll of washing solution per well. After additionof 200 ll of the HRP substrate to each well, the plate was incubated at ambient tem-perature for 10 min. The HRP reaction was stopped by adding 100 ll of 0.8 mol/Lsulfuric acid per well, and the yellow color produced was measured at 450 nm.
2.6. Methinine adenosyltransferase (MAT) and methionine synthase (MSase) assay
Liver tissue was rinsed at least twice with phosphate buffered saline. To 100 mgof incised tissue, 1 ml of cell lysis buffer (50 mM Tris–HCl; pH 7.5, 5 mM EDTA;150 mM NaCl; 0.1% lauryl sulfate, sodium salt in deionized water (DW); 0.5%deoxycholic acid in DW; 1% Igepal CA-630 in DW; Protease inhibitor cocktail wasadded followed by incubation for 15 min. It was followed by transfer of samplealong with cell lysis buffer to pre-chilled micro-homogenizer where the tissuewas homogenized. The lysed sample were centrifuged (12,000g, 10 min) and pro-tein-containing supernatant was removed to a chilled test and kept on ice till usedfor MAT and MSase assay.
The MAT assay used was a modification of method described earlier (Bensonet al., 1993) based on the ability of tissue MAT to catalyze the reaction of ATPand L-methionine to produce SAM. MAT assay system contained, in a total volumeof 1 ml, 100 mM L-methionine, 50 mM ATP, 23 mM MgCl2 and 55 mM KCl and 5 lgenzyme protein in 50 mM Tris–HCl buffer (pH 7.4). SAM was isolated using Dowex50X4 cation exchange column and quantified by a novel SAM assay platform designdescribed earlier. MSase assay was carried out by the method described earlier(Drummond et al., 1995). This assay involved methyl group transfer from 5-meth-yltetrahydrofolate to homocysteine to give L-methionine and tetrahydrofolate. Thelater was derivatized with a formylating agent under acidic conditions to producemethenyltetrahydrofolate, which absorbed at 350 nm.
2.7. DNA methyltransferase (dnmt) assay
Total DNA methyltransferase activity was measured by using liver tissuenuclear extracts as described earlier (Suzuki et al., 2008). Nuclear extracts were iso-lated using the nuclear extraction kit. Nuclear extracts were incubated with meth-ylation substrate for 1.5 h at 37 �C, and then exposed to capture antibody for 60 minand detection antibody for 30 min, at room temperature. Absorbance was deter-mined using a microplate spectrophotometer at 450 nm, and dnmt activity was cal-culated according to the following formula: (Sample OD – blank OD)/(nuclearprotein in lg � 1.5 h) � 1000), as per manufacturer’s instructions. Results wereexpressed as O.D./h/mg nuclear protein.
2.8. Global DNA methylation assay
Genomic DNA was isolated from liver tissue using DNAeasy Blood and tissue kit(Qiagen, Chatsworth, CA, USA). To quantify the global DNA methylation levels, aDNA methylation quantification kit (Epigentek Inc., Brooklyn, NY) was used. In thisassay, methylated fraction of the DNA immobilized on a strip, was recognized by 5-methylcytosine antibody and quantified through ELISA. Results were expressed asabsolute percentage of 5-methylcytosine. In brief, 28 ll of DNA binding solutionand 2 ll (200 ng) of sample DNA were incubated at 37 �C for 2 h followed by a sec-ond incubation (to evaporate the solution) at 60 �C for 20–30 min. For blank, 30 llof DNA binding solution was used instead of DNA. To each dried well, 150 ll ofblocking solution was added followed by incubation at 37 �C for 30–45 min. Wellswere aspirated with 150 ll of wash buffer and diluted, capture antibody (1 lg/ml)was then added followed by incubation at room temperature for 60 min. Wellswere again aspirated and washed with wash buffer. To each well, detecting anti-
body was added and incubated at room temperature for 30 min. Wells were aspi-rated and washed with 150 ll of wash buffer (5 times). 100 ll of developingsolution was added to each well and plate was incubated at room temperaturefor 2–10 min in the dark. Color development was monitored in the sample andthe control well. Finally, the reaction was stopped using stop solution (50 ll) andthe absorbance was read on a microplate reader at 450 nm. Results were calculatedas percent of methylated DNA.
2.9. 5-methyl-20-deoxy cytidine quantification
Radiation induced modulation of DNA methylation was evaluated by competi-tive enzyme immunoassay kit (Cayman Chemical Company, Ann Arbor, MI, USA)that measured 5-methyl-20-deoxy cytidine, a marker of DNA methylation changes,in the liver. Briefly, genomic DNA was extracted (DNAeasy Blood and tissue kit, Qia-gen, Chatsworth, CA, USA) and suspended in 135 ll of 20 mM sodium acetate (pH4.8). DNA was digested to nucleotide with nuclease P1 (40 U/ml) at 37 �C for 1 h.Then, 15 ll of 1 M Tris/HCl (pH 7.4) was added to the samples and they were thentreated with alkaline phosphatase (25 U/ml) at 37 �C for 1 h. The 5-methyl-20-deoxycytidine assay in the digested DNA solution was based on the competition between5-methyl-20-deoxy cytidine and 5-methyl-20-deoxy cytidine-acetylcholinesterase(AChE) conjugate (5-methyl-20-deoxy cytidine tracer) for a limited amount of5-methyl-20-deoxy cytidine monoclonal antibody. The absorbance of product ofreaction was read at 412 nm using a microplate reader. The intensity of the colorwas proportional to amount of 5-methyl-20-deoxy cytidine tracer bound to the well,which is inversely proportional to amount of 5-methyl-20-deoxy cytidine presentin the sample. The 5-methyl-20-deoxy cytidine levels were expressed as5-methyl-20-deoxy cytidine (ng/ml)/DNA (lg/ll).
2.10. Histopathology examination for toxicity assessment
Liver were removed and weighed. Part of the liver was frozen in liquid nitrogenfor metabolite determinations. The rest of the liver were fixed in buffered formalinand embedded in paraffin. The slides for histologic study were prepared with meth-ods that included hematoxylin–eosin staining for routine histologic analysis of fatcontent, apoptotic foci, necrotic areas, vascular damages and for detection of fibroustissue.
2.11. Statistical analysis
All values were expressed as mean ± standard error of means of samples with nand p indicated in the Results section. Statistical analyses were performed usingStudent’s t-test (GraphPad QuickCalcs, San Diego, CA, USA). Values with P < 0.05were considered statistically significant.
3. Results
3.1. Food consumption and animal weight
Average food consumed (g/mouse/day) for each group was asfollows: NCD control, 4.4 ± 0.25; MSD control, 4.3 ± 0.25; 2 Gy irra-diated: 4.3 ± 0.21; 3 Gy irradiated: 4.3 ± 0.22 and 4 Gy irradiated4.2 ± 0.21. The difference in food consumption between differentdiet groups, however, was found to be statistically insignificant(P < 0.01). Average initial body weight of the mice was22.1 ± 1.6 g. There was no significant change in body weight inthe different groups.
Folate, choline and L-methionine metabolism intersect at theformation of L-methionine from homocysteine and thereforeperturbing the metabolism of one of these nutrient results in com-pensatory changes in the others. Our results demonstrated thathepatic folate concentrations in MSD fed control animals showedno change compared to NCD fed control mice (P > 0.05). After com-pletion of the dietary intervention, the liver folate concentrationmeasured in the NCD control group maintained on unfortified dietwas 702 ± 66 ng/g liver (Table 2). After 6 weeks of consumption ofMSD, the liver folate concentrations were measured again. In theMSD fed un-irradiated mice, the mean liver folate concentrationwas 709 ± 67 ng/g liver. These results suggested that MSD dietary
Table 2Effect of dietary L-methionine supplementation (6 weeks) followed by various doses of c-radiation on folate, choline and methionine levels in liver (24 h and 48 h post-irradiation).
Radiation (Gy) Normal control diet (NCD) L-Methionine supplemented diet (MSD)
# Liver folate levels are expressed as nanogram per gram (ng/g) liver. Liver choline and methionine values are given as nmol/g liver. Liver choline values are multiple of 100.All values are mean ± standard error of samples (in triplicate) from 4 animals in each group.
a P < 0.05: indicate significance of difference within the same diet group between unirradiated control and irradiated mice.b P < 0.01: indicate significance of difference within the same diet group between unirradiated control and irradiated mice.c P < 0.05: indicate significance of difference between combined stimuli of L-methionine supplementation and radiation exposure compared to radiation exposure alone.d P < 0.01: indicate significance of difference between combined stimuli of L-methionine supplementation and radiation exposure compared to radiation exposure alone.
V. Batra, P. Verma / Food and Chemical Toxicology 69 (2014) 46–54 49
regimen had no effect on hepatic folate reserves. However, dietaryL-methionine supplementation could compensate for radiation-induced depletion of folate. As shown in Table 2, liver folate levelsin 4 Gy irradiated MSD fed animals were higher compared to 4 Gyirradiated NCD fed mice (P < 0.05). These findings clearly demon-strated the radio-protective ability, if any, of MSD dietary regimensmight be partly attributed to folate sparing effect in liver.
Liver choline levels were measured to verify the choline statusof NCD and MSD fed and irradiated animals (Table 2). Choline con-centration of the liver was significantly depleted (31.6 ± 2.5 nmol/gliver) after 24 h of 4 Gy exposure in mice fed on NCD as comparedto animals maintained on MSD (39.4 ± 3.2; (P < 0.01) (Table 2).After 48 h of irradiation, hepatic choline levels decreased furtherto 33.0 ± 2.7 (3 Gy) and 26.5 ± 2.4 (4 Gy). Our results showed thatalthough at 2 Gy, liver choline level was not significantly affectedin NCD or MSD fed animals, it decreased progressively withincrease in dose and maximum fall was seen after 4 Gy in NCDfed mice.
No significant change in L-methionine levels was observed after24 and 48 h of 2 Gy irradiation indicating absence of L-methioninemobilization for systemic usage. However, L-methionine levelsdecreased sharply in NCD fed mice after 48 h of 3 Gy (41.3 ± 3.5)and 4 Gy (32.3 ± 3.0) exposure. In contrast, L-methionine levelsdecreased only moderately when MSD fed animals were exposedto 3 and 4 Gy radiation (Table 2).
3.3. Interaction between 60Co c-radiation and dietary L-methioninesupplementation increased hepatic SAM levels and maintained higherand long-lasting DNA methylation potential
Data on liver SAM profile are presented in Table 3. Liver SAMlevel was 43.5 ± 3.8 nmol/g in the un-irradiated NCD fed mice.
Table 3Effect of various doses of c-irradiation on liver S-adenosylhomocysteine (SAH) and S-supplemented diet (MSD) fed animals after 24 and 48 h post-irradiation.
# Liver SAH and SAM levels are expressed as nmole/g liver. All values are mean ± standa P < 0.05: indicate significance of difference within the same diet group between unirra
b P < 0.01: indicate significance of difference within the same diet group between unic P < 0.05: indicate significance of difference between combined stimuli of L-methionid P < 0.01: indicate significance of difference between combined stimuli of L-methioni
MSD feeding significantly (P < 0.05) increased SAM in unirradiatedmice to 52.3 ± 4.9 nmol/g. Compared to MSD fed and irradiatedanimals SAM levels declined sharply in NCD fed mice after 3 and4 Gy irradiation (at 24 h). NCD maintained mice showed maximumdecline in SAM concentration (19.5 ± 2.3; P < 0.01) after 48 h of4 Gy irradiation; showing a decline of approximately 54% com-pared to MSD fed and 4 Gy irradiated mice. Lesser decline inSAM levels in MSD fed mice after radiation exposure suggestedconservation of SAM pool by enhanced biosynthesis from L-methi-onine precursor.
Table 3 shows the liver SAH profile in NCD and MSD fed animalsafter 24 and 48 h post-irradiation. Our studies showed that L-methionine supplementation moderately increased (P < 0.05)baseline hepatic SAH in un-irradiated mice. After 24 and 48 h ofirradiation at 2 Gy, SAH levels were lower in NCD fed animals(P < 0.05) compared to MSD fed mice. Liver SAH level was7.6 ± 0.6 nmol/g in the un-irradiated NCD fed mice and 9.3 ± 0.7in un-irradiated MSD fed mice. However, 48 h after exposure to4 Gy the differences were statistically significant. In NCD as wellas MSD fed mice, SAH levels increased progressively in dose-dependent manner with a maximum level of 17.4 ± 1.7 and18.8 ± 1.9 (P < 0.01) after 48 h of 4 Gy, respectively (Table 3). Thesedata indicated that MSD feeding could not alter effect of c-radia-tion on SAH generation. However, MSD regimens significantly min-imized decreases in liver SAM levels, as compared to NCD fedanimals.
In present studies, we used SAM: SAH ratio to evaluate the‘‘genomic DNA methylation potential’’. The SAM: SAH ratio inMSD fed and irradiated mice were significantly higher than NCDfed and irradiated animals (Table 4). The mean intracellular SAM:SAH ratio in MSD fed mice after 48 h of 4 Gy (2.3 ± 0.19) was signif-icantly higher than the ratio (1.1 ± 0.08) of NCD fed mice (P < 0.01).
adenosylmethionine (SAM) levels of normal control diet (NCD) and L-methionine
ard error of samples (in triplicate) from 4 animals in each group.diated control and irradiated mice.rradiated control and irradiated mice.ne supplementation and radiation exposure compared to radiation exposure alone.ne supplementation and radiation exposure compared to radiation exposure alone.
Table 4Effect of various doses of c-irradiation on liver S-adenosylmethionine (SAM) to S-adenosylhomocysteine (SAH) ratio (genomic methylation potential) in normal control diet (NCD)and L-methionine supplemented diet (MSD) fed animals after 24 and 48 h post-irradiation.
Radiation (Gy) Normal control diet (NCD) Methionine supplemented diet (MSD)
All values are mean ± standard error of samples (in triplicate) from 4 animals in each group.a P < 0.05: indicate significance of difference within the same diet group between unirradiated control and irradiated mice.b P < 0.01: indicate significance of difference within the same diet group between unirradiated control and irradiated mice.c P < 0.05: indicate significance of difference between combined stimuli of L-methionine supplementation and radiation exposure compared to radiation exposure alone.d P < 0.01: indicate significance of difference between combined stimuli of L-methionine supplementation and radiation exposure compared to radiation exposure alone.
Fig. 1. Effect of dietary L-methionine supplementation followed by c-radiation (24and 48 h post-irradiation) on liver methionine adenosinetransferase (MAT) andmethionine synthase (MSase) activities. NCD-C and MSD-C indicate normal diet andL-methionine supplemented diet fed controls. Liver MAT activity is expressed asinternational units (IU) and MSase activity is expressed as mU/mg protein. Allvalues are mean ± standard error of samples (in triplicate) from 4 animals in eachgroup. aP < 0.05, bP < 0.01: indicate significance of difference within the same dietgroup between unirradiated control and irradiated mice. cP < 0.05, dP < 0.01:indicate significance of difference between combined stimuli of L-methioninesupplementation and radiation exposure compared to radiation exposure alone.
50 V. Batra, P. Verma / Food and Chemical Toxicology 69 (2014) 46–54
The effects of L-methionine supplementation on the DNA methyla-tion potential suggested that high dietary intake of L-methioninemay mitigate radiation induced DNA hypomethylation. Theincrease in DNA methylation potential was due to less effect ofradiation on SAM levels of MSD fed mice in all cases (Table 4).
3.4. 60Co c-Radiation exposure differentially modified methionineadenosytransferase (MAT) and methionine synthase (MSase) activitiesin NCD and MSD fed mice
To determine possible metabolic mechanisms leading to altera-tions in DNA methylation, we analyzed the protein expression ofMAT, an enzyme catalyzing SAM synthesis. Significant increase inMAT activity was seen in animals maintained on MSD (Fig. 1Aand B). The enzyme activity started increasing steeply in MSDfed mice exposed to 3 Gy (Fig. 1A) where enzyme levels were33.5 ± 2.5 IU (NCD-3 Gy: 19.7 ± 1.4). As shown in Fig. 1B MSD ani-mals maintained up to 48 h post-irradiation after 4 Gy, showedmost significant increase (P < 0.01) in the enzyme activity (approx-imately 3.7-fold increase) compared to NCD animals exposed tosimilar dose. These results showed that interaction between L-methionine supplementation and ionizing radiation mightpromptly induce liver MAT enzyme (Fig. 1A and B), which in turncould alter methylation of genomic DNA.
To understand effect of L-methionine supplementation on folateand choline dependent C1 flux towards DNA methylation, the influ-ence of c-radiation on the levels of MSase was studied in NCD andMSD fed mice (NCD control: 6.9 ± 0.5 mU/mg protein, MSD control:6.6 ± 0.5). No statistically significant difference in liver MSaseactivity was noticed when animals maintained on MSD wereexposed to c-radiation (Fig. 1C and D). However, in NCD fed mice24 h after irradiation, the MSase activity increased significantlyafter 3 Gy (13.2 ± 1.1, P < 0.01) and 4 Gy (17.3 ± 1.4, P < 0.01). After48 h of 4 Gy exposure, further increase (21.8 ± 1.6, P < 0.01) inMSase activity was noticed (Fig. 1D).
The data in Fig. 2 shows 24 (Fig. 2A) and 48 h (Fig. 2B) post-irra-diation profile (expressed as O.D./h/mg nuclear protein) of dnmt inliver tissues of NCD and MSD fed animals. Significant increase indnmt levels occurred in MSD group after 24 and 48 h of variousdoses of radiation (P < 0.01). Compared to dnmt profile of NCDfed mice, the dnmt levels of MSD fed mice (MSD control:22.1 ± 1.5) significantly increased after 24 h of 3–4 Gy exposures.The dnmt levels (Fig. 2A) increased to 51.5 ± 3.2 after 3 Gy(P < 0.01) and 63.8 ± 3.9 after 4 Gy (P < 0.01). A significant changein dnmt level of MSD fed mice was noticed 48 h after irradiation
in L-methionine supplemented mice. At 2 Gy dnmt level was31.4 ± 2.5 (Fig. 2B). At 3 Gy, dnmt levels rose up to 54.9 ± 3.1(P < 0.01) followed by further increase to 69.5 ± 3.7 (P < 0.01) afterexposure to 4 Gy (Fig. 2B). These results demonstrate that in MSDfed mice, increase in dnmt is an early event meant to protect
Fig. 2. Effect of various doses of c-radiation on liver total DNA methyltransfease(dnmt) activity# of normal control diet (NCD) and L-methionine supplemented diet(MSD) fed animals after 24 (A) and 48 h (B) post-irradiation. Liver dnmt activityis expressed as O.D./h/mg nuclear protein. All values are mean ± standard error ofsamples (in triplicate) from 4 animals in each group. aP < 0.05, bP < 0.01: indicatesignificance of difference within the same diet group between unirradiated controland irradiated mice. cP < 0.05, dP < 0.01: indicate significance of difference betweencombined stimuli of L-methionine supplementation and radiation exposurecompared to radiation exposure alone.
Fig. 3. Effect of c-irradiation (4 Gy) on global DNA methylation. Layer A showspercentage of methylated DNA (genomic) of normal control diet (NCD) fed controland irradiated (24 and 48 h post-irradiation profile) mice. Layer B shows percentageof methylated DNA (genomic) of L-methionine supplemented diet (MSD) fedcontrol and irradiated (24 and 48 h post-irradiation profile) mice. All values aremean ± standard error of samples (in triplicate) from 4 animals in each group.aP < 0.05, bP < 0.01: indicate significance of difference within the same diet groupbetween unirradiated control and irradiated mice. cP < 0.05, dP < 0.01: indicatesignificance of difference between combined stimuli of L-methionine supplemen-tation and radiation exposure compared to radiation exposure alone.
Fig. 4. Effect of dietary L-methionine supplementation (6 weeks) followed byvarious doses of c-irradiation on 5-methyl-20-deoxy cytidine levels in liver DNA. (A)24 h post-irradiation profile and (B) 48 h post-irradiation profile. NCD and NSDindicate two different controls. The 5-methyl-20-deoxy cytidine levels are expressedas 5-methyl-20-deoxy cytidine (ng/lg DNA). All values represent mean ± SEM for 4mice in each group. aP < 0.05, bP < 0.01: indicate significance of difference withinthe same diet group between unirradiated control and irradiated mice. cP < 0.05,dP < 0.01: indicate significance of difference between combined stimuli of L-methionine supplementation and radiation exposure compared to radiation expo-sure alone.
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against c-radiation induced epigenetic modification in genomicDNA.
Alteration in global DNA methylation is likely to be animportant epigenetic modification, which may contribute to thesusceptibility to cellular disorders caused by gene expression andsilencing. Preliminary studies suggested no significant change inglobal DNA methylation level at 2 Gy in liver tissue of animalsmaintained on NCD or MSD diet. Therefore, only 24 and 48 hpost-irradiation (4 Gy) DNA methylation profiles of animals (NCDand MSD) were studied. Significant decrease in DNA methylationlevels in NCD fed animals was seen after 4 Gy of radiation(P < 0.01). There was approximately 26% decline in DNA methyla-tion level after 24 h and the decrease became prominent after48 h (ffi39%) (Fig. 3). In MSD fed animals, there was no significantchange in global DNA methylation levels of animals after 24 hand 48 h post-irradiation (P > 0.05).
3.7. Quantification of 5-methyl-20-deoxy cytidine residuessubstantiated role of dietary L-methionine supplementation inincreasing DNA methylation potential
Data on 5-methyl-20-deoxy cytidine levels in genomic DNA ispresented in Fig. 4A and B. 5-methyl-20-deoxy cytidine level was74.2 ± 6.9 (ng/lg DNA) in the un-irradiated NCD fed mice. Mea-surement of 5-methyl-20-deoxy cytidine residues in NCD fed mice,in general showed significant c-radiation-induced change. After24 h of irradiation at 2 Gy, no significant change in 5-methyl-20-deoxy cytidine levels was observed in NCD animals compared toMSD fed mice (Fig. 4A). 5-Methyl-20-deoxy cytidine levelsdecreased progressively in NCD fed mice with a maximumdecrease to 44.5 ± 3.8 (P < 0.01) after 24 h of 4 Gy (Fig. 4A). Maxi-mum decrease in 5-methyl-20-deoxy cytidine levels in NCD fedmice was 41.9 ± 3.7 at 48 h after exposure to 4 Gy (P < 0.01)(Fig. 4B). However no significant perturbation in 5-methyl-20-deoxy cytidine levels was seen in MSD fed mice after 24 and48 h post-irradiation (P > 0.05).
3.8. Dietary L-methionine supplementation (2 g/kg diet; 6 weeks)showed no toxic effect on liver
The effect of c-irradiation on liver of mice maintained on NCDand MSD is shown in Fig. 5. Histological examination revealed nopathological changes in liver due to L-methionine supplementationfor 6 weeks duration. No pathological change in liver was observedwhen mice maintained on NCD and MSD were exposed to 2 and3 Gy dose (data not shown). However, NCD as well as MSD fed
Fig. 5. Histological evaluation (scale bar = 50 lM; hematoxylin–eosin staining) ofthe liver of mice maintained on normal control diet (NCD) and L-methionine-supplemented diet (MSD) for six weeks followed by c-irradiation. (A) Liver: NCD-control (0 Gy) animals; (B) liver: MSD-control (0 Gy); (C) liver: NCD followed by4 Gy; (D) liver: MSD followed by 4 Gy; (E) ALT profile of NCD and MSD animals.
52 V. Batra, P. Verma / Food and Chemical Toxicology 69 (2014) 46–54
animals showed slightly blurred hepatocyte lining after 4 Gy expo-sures (Fig. 5C and D).
Since the liver is a major site of L-methionine metabolism andtherefore a potential target of L-methionine toxicity, animals weretested for serum ALT levels. Supplementation with L-methionine inabsence of radiation stress had no significant effect (P > 0.05) onALT levels in NCD (22.5 ± 1.3) and MSD (23.3 ± 1.4) fed mice(Fig. 5E). However, significant increase in serum ALT activity wasobserved upon irradiation of NCD and MSD fed mice. Notably,serum ALT activity is an established marker of hepatic injury (Ram-adan et al., 2002). Mice on NCD as well as MSD showed theexpected increase of ALT levels to 47.2 ± 2.9 (units/liter) and46.7 ± 2.8 after 48 h of 4 Gy, respectively (Fig. 5E). Takentogether these data indicate that dietary L-methionine supplemen-tation did not exacerbated radiation-induced liver damage andhad a mitigating effect on radiation-induced genomic DNAhypomethylation.
4. Discussion
The objective of this study was to understand the cytotoxiceffect of 60Co c-irradiation on DNA methylation in the absenceand presence of dietary L-methionine supplementation in micein vivo. The acute phase metabolic mechanisms translating radia-tion exposure into long-term pathologies such as cancer manifes-tation involve hypomethylation of genomic DNA (Watanabe and
Maekawa, 2010). Because DNA hypomethylation occurs quite earlyin the tumorigenic process, it has been suggested that the loss ofcytosine methylation destabilizes the genome by promoting a vari-ety of mutational effects (Aypar et al., 2011). Earlier studies havealso shown that radiation impaired intestinal nutrient absorptioncould progressively deplete hepatic L-methionine levels (Rocheet al., 2011). Therefore, Based on existing knowledge that SAM isindispensable for dnmt dependent DNA methylation reaction, wehypothesized that dietary L-methioine supplementation mightmitigate c-radiation induced DNA demethylation. To test ourhypothesis, we comprehensively examined the metabolic machin-ery that mobilizes dietary L-methionine towards SAM biosynthesis,the ultimate C1 donor for DNA methylation. Results showed thatdietary L-methionine supplementation might alleviate radiationinduced substantial loss of genomic DNA methylation in murineliver tissue.
Metabolic pathways are coordinated sequence of chemical reac-tions catalyzed by enzymes and connected via their substrates andproducts. In present studies we explored implications of dietaryL-methionine supplementation on intermediates of C1 metabolicpathway that might influence DNA methylation under radiationstress. Earlier work has shown that radiation stress mightadversely affect homocysteine–methionine cycle that generatesC1 flux for DNA methylation (Wilson et al., 2012). Present resultsdemonstrated that shortage of L-methionine regeneration viahomocysteine–methionine cycle might decrease SAM levels inNCD fed mice compared to MSD fed animals. Reduced SAM levelsmight adversely affect DNA methylation and thereby facilitatedevelopment of radiation-related late pathologies such as cancer(Koturbash et al., 2005). Our work showed that compared to radi-ation-induced SAM decline in NCD fed animals, higher SAM levelscould be maintained by dietary L-methionine supplementation.Elevated SAH and diminished SAM levels in NCD fed mice, led usto suggest that radiation induced decrease in SAM: SAH ratio(referred as genomic methylation potential) may be responsiblefor decreased DNA methylation capacity (Pogribny et al., 2006).In times of radiation stress, hepatic metabolic demands are extre-mely higher and protective doses of nutrients, prescribed for nor-mal conditions, become inadequate (Savarese et al., 2003).Present results show that radiation-toxicity disrupts cellular C1
metabolism as it deforms and damages regulatory enzymes andintermediary macromolecules. Therefore, dietary supplementationof L-methionine at levels higher than recommended dietary allow-ances might become essential to maintain methylation level ofgenomic DNA.
Recent reports have indicated that activities of several enzymesin liver might be markedly altered by dietary conditions (Pooyaet al., 2006). Therefore we investigated levels of certain keyenzymes, which may regulate C1 flux from folate, choline and L-methionine towards DNA methylation. To this end, our resultsshowed dose dependent induction of MAT and dnmt activities inMSD animals. In contrast, MSase activity in NCD animals wasmarkedly increased after c-irradiation. This increased MAT activityin MSD animals may account in a large part for substrate depen-dent enzyme expression while increased Msase activity in NCDfed mice mobilized folate pool towards DNA methylation. Resultsof these experiments suggested that L-methionine supplementa-tion altered normal balance of enzymes to mobilize preformedmethyl groups of L-methionine towards DNA methylation.Moreover, correlation between SAM and DNA methylation levelsconfirmed that SAM might be a limiting factor for the dnmt activityunder radiation stress conditions (Wang et al., 2011). Based onpresent data, we propose strong rationale behind dietaryL-methionine supplementation mediated mitigation of radiationtoxicity, as it prevents DNA hypomethylation on genomic scale,by depriving dnmt of its metabolic substrate.
V. Batra, P. Verma / Food and Chemical Toxicology 69 (2014) 46–54 53
Underlying question in all these studies was to determine dif-ferential advantage of dietary L-methionine supplementation, ifany, with respect to maintenance of genomic DNA methylationunder radiation stress conditions. Our findings indicated thatMSD feeding mitigated radiation induced DNA hypomethylation.Radiation induced cell killing is well known to result in detectabletissue reactions. Some of these reactions such as modified meta-bolic perturbations and molecular damages occur early (days)while other effects such as tumor formation occur late (monthsto years) after irradiation. In this context, several studies have sug-gested that radiation induced genomic DNA hypmethylation mightbe a significant step towards tumorigenesis (Koturbash et al.,2005). The observed alterations in global DNA methylation profileof NCD fed animals was influenced by potential factors such asradiation dose, time after irradiation and concentration of immedi-ate substrate i.e. SAM. To further confirm the effect of L-methioninesupplementation on DNA methylation, we then quantified thelevels of 5-methyl-20-deoxy cytidine residues in NCD and MSDfed animals. Significant decreases in 5-methyl-20-deoxy cytidineresidues in NCD irradiated mice and no significant decrease inMSD irradiated animals further confirmed role of dietaryL-methionine enrichment in mitigation of epigenetic modifications.In brief, our data indicated that dietary L-methionine supplementa-tion could be construed to be a ‘‘radio-protective strategy.’’Existing recommendations for adequate dietary supply ofL-methionine are designed to provide levels that are sufficient toprevent overt clinical symptoms of deficiencies. However, theserecommendations may not be appropriate for mammalian cell sys-tems subjected to radiation toxicity. Thus there is a need for stud-ies to reevaluate dietary recommendations for such nutrientsunder radiation induced stress conditions. L-methionine dosesused for this study were slightly higher than those used typicallyfor the correction of nutritional deficiencies. However, we did not
Fig. 6. Schematic diagram showing effect of dietary L-methionine supplementationon gamma-radiation induced genomic DNA hypomethylation via up-regulation ofradiation-compromised metabolic flux towards S-adenosylmethioninebiosynthesis.
notice signs of L-methionine toxicity. Despite no effect of L-methi-onine supplementation on baseline ALT levels in MSD fed mice,radiation inflicted functional injury to hepatocytes of both NCDand MSD fed mice as indicated by increased serum ALT activity.
L-methionine is a naturally existing essential amino acid, thus adiet could be developed for humans, which would be of littleinconvenience to the individual. Here, we have presented evidencethat L-methionine supplementation may up-regulate C1 flux direc-ted towards DNA methylation, presenting an interesting case forincreased dietary L-methionine intake under c-radiation inducedstress conditions. Activation of several genes (enhanced transcrip-tion) has been ascribed to the demethylation of genes, and silenc-ing of many genes (reduced transcription) has been related to theincreased methylation of these genes (Liotto et al., 2009).Inhibition of c-radiation mediated DNA hypomethylation, by die-tary L-methionine supplementation, may be one of the importantcontributing factors to prevent adverse effects of radiation at met-abolic level (Fig. 6). Moreover, while this study was designed todevelop agents for the mitigation of radiation toxicity sustainedduring nuclear accidents or acts of radiological terrorism, it mayalso have implications for cancer patients who sustain normal tis-sue injury as a result of radiation therapy. Taken together, ourstudy reveals new metabolic mechanisms for the regulation ofDNA methylation patterns in radiation stress environments.
Conflict of Interest
The authors declare that there are no conflicts of interest.
Transparency Document
The Transparency document associated with this article can befound in the online version.
Acknowledgements
The authors wish to acknowledge the support of Dr. JayantBandekar, Head, Radiation Biology and Health Sciences Divisionin carrying out this work. The authors wish to thank Dr. B.N. Pan-dey, Head, Radiation Signaling and Cancer Biology Section for crit-ical evaluation of this manuscript. The authors also wish to thankand gratefully acknowledge Mr. Sanjay Shinde, Ms. Pritam Patiland Ms. Deepika Bhange for their expert technical assistance inconducting the experiments.
References
Anderson, O.S., Sant, K.E., Dolinoy, D.C., 2012. Nutrition and epigenetics: aninterplay of dietary methyl donors, one-carbon metabolism and DNAmethylation. J. Nutr. Biochem. 23, 853–859.
Aypar, U., Morgan, W.F., Baulch, J.E., 2011. Radiation-induced epigenetic alterationsafter low and high LET irradiations. Mutat. Res. 707, 24–33.
Batra, V., Kislay, B., Devasagayam, T.P., 2011. Interaction between total bodygamma-irradiation and choline deficiency triggers immediate modulation ofcholine and choline-containing moieties. Int. J. Radiat. Biol. 87, 1196–1207.
Benson, R., Crowell, B., Hill, B., Doonquah, K., Charlton, C., 1993. The effects of L-dopa on the activity of methionine adenosyltransferase: relevance to L-dopatherapy and tolerance. Neurochem. Res. 18, 325–330.
Brown, S.L., Kolozsvary, A., Liu, J., Jenrow, K.A., Ryu, S., Kim, J.H., 2010. Antioxidantdiet supplementation starting 24 hours after exposure reduces radiationlethality. Radiat. Res. 173, 462–468.
Capdevila, A., Burk, R.F., Freedman, J., Frantzen, F., Alfheim, I., Wagner, C., 2007. Asimple rapid immunoassay for S-adenosylhomocysteine in plasma. J. Nutr.Biochem. 18, 827–831.
Ehrlich, M., 2009. DNA hypomethylation in cancer cells. Epigenomics 1, 239–259.Elremaly, W., Rouleau, T., Lavoie, J.C., 2012. Inhibition of hepatic methionine
adenosyltransferase by peroxides contaminating parenteral nutrition leads to alower level of glutathione in newborn Guinea pigs. Free Radic. Biol. Med. 53,2250–2255.
Hoffbrand, A.V., Jackson, B.F.A., 1993. Correction of the DNAsynthesis defect invitamin B12 deficiency by tetrahydrofolate: evidence in favour of the methyl-folate trap hypothesis as the cause of megaloblastic anaemia in vitamin B12deficiency. Br. J. Haematol. 83, 643–647.
Jones, P.L., Veenstra, G.J., Wade, P.A., Vermaak, D., Kass, S.U., Landesberger, N.,Strouboulis, J., Wolffe, A.P., 1998. Methylated DNA and MeCP2 recruit histonedeacetylase to repress transcription. Nat. Genet. 19, 187–191.
Koturbash, I., Pogribny, I., Kovalchuk, O., 2005. Stable loss of global DNAmethylation in the radiation-target tissue – a possible mechanismcontributing to radiation carcinogenesis? Biochem. Biophys. Res. Commun.337, 526–533.
Liotto, N., Miozzo, M., Giannì, M.L., Taroni, F., Morlacchi, L., Piemontese, P., Roggero,P., Mosca, F., 2009. Early nutrition: the role of genetics and epigenetics. Pediatr.Med. Chir. 31, 65–71.
Mason, J.B., Choi, S.W., 2000. Folate and carcinogenesis: developing a unifyinghypothesis. Adv. Enzyme Regul. 40, 127–141.
Ono, H., Iwasaki, M., Kuchiba, A., Kasuga, Y., Yokoyama, S., Onuma, H., Nishimura, H.,Kusama, R., Ohnami, S., Sakamoto, H., Yoshida, T., Tsugane, S., 2012. Associationof dietary and genetic factors related to one-carbon metabolism with globalmethylation level of leukocyte DNA. Cancer Sci. 103, 2159–2164.
Pogribny, I.P., Ross, S.A., Wise, C., Pogribna, M., Jones, E.A., Tryndyak, V.P., James, S.J.,Dragan, Y.P., Poirier, L.A., 2006. Irreversible global DNA hypomethylation as akey step in hepatocarcinogenesis induced by dietary methyl deficiency. Mutat.Res. 593, 80–87.
Pooya, S., Blaise, S., Garcia, M., Giudicelli, J., Alberto, J.M., Guéant-Rodriguez, R.M.,Jeannesson, E., Gueguen, N., Bressenot, A., Nicolas, B., Malthiery, Y., Daval, J.L.,
Peyrin-stipanuk, Dominy, J.E., Lee, J.I., Coloso, R.M., 2006. Mammalian cysteinemetabolism: new insights into regulation of cysteine metabolism. J. Nutr. 136,1652S–1659S.
Reeves, P.G., Nielsen, F.H., Fahey, G.C., 1993. AIN-93 purified diets for laboratoryrodents: final report of the American institute of nutrition ad hoc writingcommittee on the reformulation of the AIN-76A rodent diet. J. Nutr. 123, 1939–1951.
Roche, M., Kemp, F.W., Agrawal, A., Attanasio, A., Neti, P.V., Howell, R.W., Ferraris,R.P., 2011. Marked changes in endogenous antioxidant expression precedevitamin A-, C- and E-protectable, radiation-induced reductions in smallintestinal nutrient transport. Free Radic. Biol. Med. 50, 55–65.
Savarese, D., Savy, G., Vahdat, L., Wischmeyer, P., Corey, B., 2003. Prevention ofchemotherapy and radiation toxicity with glutamine. Cancer Treat. Rev. 29,501–513.
Sibani, S., Melnyk, S., Pogribny, I.P., Wang, W., Hiou-Tim, F., Deng, L., Trasler, J.,James, S.J., Rozen, R., 2002. Studies of methionine cycle intermediates (SAM,SAH), DNA methylation and the impact of folate deficiency on tumor numbersin Min mice. Carcinogenesis 23, 61–65.
Singh, V.K., Ducey, E.J., Brown, D.S., Whitnall, M.H., 2012. A review of radiationcountermeasure work ongoing at the Armed Forces Radiobiology ResearchInstitute. Int. J. Radiat. Biol. 88, 296–310.
Smith, D.E., Smulders, Y.M., Blom, H.J., Popp, J., Jessen, F., Semmler, A., Farkas, M.,Linnebank, M., 2012. Determinants of the essential one-carbon metabolismmetabolites, homocysteine, S-adenosylmethionine, S-adenosylhomocysteineand folate, in cerebrospinal fluid. Clin. Chem. Lab Med. 50, 1641–1647.
Suzuki, M., Shinohara, F., Rikiishi, H., 2008. Zebularine-induced reduction in VEGFsecretion by HIF-1a degradation in oral squamous cell carcinoma. Mol. Med.Rep. 1, 465–471.
Wang, Y.C., Tang, F.Y., Chen, S.Y., Chen, Y.M., Chiang, E.P., 2011. Glycine-Nmethyltransferase expression in HepG2 cells is involved in methyl grouphomeostasis by regulating transmethylation kinetics and DNA methylation. J.Nutr. 141, 777–782.
Watanabe, Y., Maekawa, M., 2010. Methylation of DNA in cancer. Adv. Clin. Chem.52, 145–167.
Wilson, F.A., Holtrop, G., Calder, A.G., Anderson, S.E., Lobley, G.E., Rees, W.D., 2012.Effects of methyl-deficient diets on methionine and homocysteine metabolismin the pregnant rat. Am. J. Physiol. Endocrinol. Metab. 302, E1531–E1540.
Woollard, D.C., Indyk, H.I., 2000. Determination of choline in milk and infantformulas by enzymatic analysis: collaborative study. J. AOAC Int. 83, 131–138.
Yun, K.U., Ryu, C.S., Lee, J.Y., Noh, J.R., Lee, C.H., Lee, H.S., Kang, J.S., Park, S.K., Kim,B.H., Kim, S.K., 2013. Hepatic metabolism of sulfur amino acids in db/db mice.Food Chem. Toxicol. 53, 180–186.
