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

Dietary modulation to combat various forms of stress, includ- ing that caused by gamma ( )-radiation, has been demonstrated as effective strategy [1]. Among all the cellular targets of radiation, DNA is considered to be the most critical molecule. Nicotinamide, the amide form of vitamin B3 (niacin) functions as substrate for metabolic pathways linked to DNA repair reactions [2]. There is a limited amount of data documenting the effect of dietary nicotinamide (pyridine-3-carboxamide) supplementation on pro- tection against -radiation induced DNA damage [3–5]. Mammals predominantly use nicotinamide as a precursor for nicotinamide adenine dinucleotide (NAD+) biosynthesis [6]. Several studies suggest that nicotinamide, by functioning as precursor for NAD+ , might influence repair of radiation generated purine and pyrimidine free sites (AP sites/abasic sites) in DNA [7–9]. The base excision repair (BER) dependent repair of AP sites in mammalian cells introduces DNA single strand breaks (SSBs) as intermediates, which in turn activate the nuclear enzyme, poly(ADP)-ribose polymerase 1 (PARP1) [10]. PARP1 assists DNA repair events by utilizing NAD+ for formation of poly-ADP-ribose (PAR) derivatives of DNA repair proteins [11]. Due to this underlying mechanism, cellular NAD+ status has been increasingly demonstrated to alter the cell susceptibility to  -radiation exposure, highlighting the possible role of dietary nicotinamide in DNA repair [12,13]. Nicotinamide is recycled to NAD+ by two enzymes, nicotinamide phosphoribosyltransferase (NAMPT) and nicotinamide mononucleotide adenylyltransferase 45 (NMNAT), which convert nicotinamide to NMN and NMN to NAD+, respectively [14].

Existing data clearly demonstrates that immediately after radiation exposure, PARP1 binds to the AP site [15,16]. Earlier studies also suggest that PARP1 may be involved in coordination of AP sites  repair process by attracting other BER proteins [17]. When activated by DNA strand breaks, PARP1 uses NAD+ as a substrate to  form ADP-ribose polymers on DNA repair proteins [18]. High levels of DNA strand breaks have been found to induce extensive polymer formation, with a concomitant lowering of cellular NAD+ levels, which adversely affects BER dependent repair of AP sites [19,20]. 
The ensuing depletion of NAD+ might inhibit glycolytic generation of ATP with consequent ATP depletion, eventuating in cell death. However, besides modifying several DNA repair proteins, PARP1 modifies itself by poly (ADP-ribosyl)ation, which results in a general inhibition of PARP1 activity [21,22]. The in vivo half-life of poly-ADP ribose (PAR) is less than a minute [23]. Poly(ADP-ribose) glycohydrolase (PARG) degrades PAR polymers synthesized by PARP1 [24]. PARG therefore maintains the active state of PARP1 by continuously removing inhibitory ADP-ribose residues from PARP1 [25]. The regulation of PARG activity may therefore influence PARP1-mediated AP sites repair [26].

Y-Radiation induced free radicals react with DNA and inflict damage to purine and pyrimidine bases [27,28]. In this study, we focused on 8-hydroxy-2′-deoxyguanosine (8-oxo-dG), a DNA metabolite, as a marker for radiosensitivity. Ogg1 is the primary enzyme responsible for the excision of 8-oxo-dG from DNA, earlier studies [29] have indicated that Ogg1 binding to PARP-1 plays a functional role in the repair of oxidative DNA damage. Guanine is the highly susceptible target for -radiation mediated oxidative reactions because of its low redox potential [29]. Moreover, 8-oxodG, is potentially mutagenic because of its ability to form base pairs with both cytosine and adenine [30]. Thus, tissue 8-oxo-dG levels are one of the most mutagenic lesions, and the most abundant source of AP site in genomic DNA [31]. Delayed repair of AP sites can also result in replication induced double strand breaks (DSB)  [32]. Therefore reducing AP sites in DNA may be an approach to decrease the adverse effects of -radiation. Existing evidence suggests that nicotinamide deficiency may impair poly (ADP-ribosylation) of DNA repair proteins, leading to accumulation of AP sites in DNA [33]. We therefore asked whether dietary nicotinamide supplementation has the potential to mitigate radiation induced AP sites and delay cell death and, if so, whether this result is due to metabolic up-regulation of NAD+ biosynthesis. Development of new therapeutic strategies using nicotinamide as radio-protective agent rests heavily upon the elucidation of metabolic pathways that link this nutrient to DNA repair reactions.

 

ATP, bicin, casein, l-dithiothreitol (DTT), deoxycholic acid, EDTA, (-(-hydroxyethyl)–piperazineethanesulfonic acid (HEPES), Igepal CA-, dimethyl  thiazolyl diphenyl tetrazolium (MTT), nicotinamide, nicotinamide adenine mononucleotide (NMN), nuclear extraction kit, protease inhibitor cocktail, Tris–HCl, triton X-, tween  and yeast alcohol dehydrogenase were purchased from  Sigma–Aldrich Chemical Co (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 from reputed manufacturers and were of analytical grade. Animal maintenance and feeding 
Male Swiss mice obtained from the departmental animal house facility of Bhabha Atomic Research Center (BARC) were maintained (for  weeks) on normal control diet (CD) and nicotinamide supplemented diet (NSD) based on AIN- M formula [], which recommends a protein content of % (w/w) for rodents (Table ). The mice were given free access to food and water throughout the study. Mice were 
housed three per cage in a room with a constant temperature of  ±  ◦ C and a 
h light–dark cycle. No significant change in the total food consumption and body

Casein supplied by central Drug House (P) Ltd. (New Delhi, India), cornstarch and dextrinizrd cornstarch (feed grade) by Vijaya Enterprises (Mumbai, India), sucrose by 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 bitaratarate and ter-butylhydroquinone by Sigma–Aldrich Company, (St. Louis, USA).

a Vitamin Mix AIN-93-VX (g/kg) Nicotinic acid 3.0, ca pantothenate 1.6, Pyridoxin- HCl 0.700, Thiamine-HCl 0.600, Riboflavin 0.600, Folic acid 0.200, d-Biotin, Vitamin B12 (0.1% IN Mannitol) 2.500, -tocopherol powder (500IU/g) 15.00, Vitamin A palmitate (250,000 U/g) 1.6, Vitamin D3 (400,000 U/g) 0.25, Phylloquinone 0.08, Sucrose 959.7. ** Vitamin mix without folate was vitamin Mix AIN-93-VX except that it did not contain folic acid. weight was noticed, in the CD fed animals compared to animals maintained on NSD. The average initial body weight of the mice was 22.7 ± 1.4 g.