Dietary nicotinamide supplementation delayed onset of apoptosis: evaluation of liver apoptosis by caspase-3 activity

Till recently, the use of nutrients in mitigating radiation injury has focused on enhancing recovery from the acute radiation syndrome . However, with the information available at present, it is difficult to predict the types of dietary modifications that can best reduce the risk of radiation exposure to radiation workers, those working in nuclear power reactors, or those enduring diagnostic or therapeutic radiation exposure. It has been observed that some nutrients can give partial protection to tissues from

genotoxic effects of radiation []. However, limited numbers of studies have dealt with nutrients dependent radioprotection. Nicotinamide is one such important nutrient involved in PARP dependent DNA repair mechanisms []. The direct relation of dietary nicotinamide supply with DNA repair pathways has provided a surge of interest in the therapeutic possibilities where NAD+ anabolism could be explored to alleviate radiation injury [,]. To explore the metabolic role of nicotinamide in DNA repair reactions, we quantified the activities of various enzymes that regulate biosynthesis of NAD+, a moiety which functions as co-substrate for PARP. Present studies involved the dimensions of radiation dose and post-irradiation time. With respect to time, h post-irradiation profile showed substantial decline in DNA repair processes in animals maintained on CD. However in CD fed animals the metabolic response h post-irradiation was characterized by moderate decline in NAD+ concentration and PARP activity. Data presented here shows that nicotinamide mediated NAD+ replenishment conferred remarkable radio-protection to

hepatocytes via mitigation of AP sites in genomic DNA. Liver, is seat  of nicotinamide metabolism and possesses significant concentra-  tion of nicotinamide metabolizing enzymes []. Our data indicated  that NAMPT and NMNAT are adaptively important and thus affected  metabolic flux that generates NAD+ reserves of liver. Present stud-  ies revealed that under radiation stress, NAMPT expression was  positively regulated by dietary nicotinamide enrichment. Enzyme  levels in a multi-step metabolic pathway affect efficiency of form-  ing the final product by preventing loss of intermediates through  diffusion, degradation, or conversion by competitive enzymes [].  NAMPT up regulation in response to increased dietary nicotinamide  supply suggested a potential role of flux generated by this enzyme  in the PARP dependent DNA repair reactions []. Therefore,  we hypothesized that NAMPT might be a high-priority target for  increasing NAD+ content in liver of accidental  -radiation exposure  subjects. NSD fed animals showed significant NAMPT and NMNAT  induction and sustenance of higher NAD+ levels in liver tissue for  prolonged duration. These results indicated that exposure of CD fed  animals to  -radiation diminished NAD+ levels while in NSD fed  animals nicotinamide dependent induction of NAMPT and NMNAT  facilitated increased NAD+ biosynthesis. Further work showed that exposure of CD fed animals to  -radiation diminished ATP levels  while in NSD fed animals ATP levels were maintained after  h of  radiation.

Another major finding of our studies showed that synergistic  interaction between  -radiation and nicotinamide supplementa-  tion could modulate PARP and PARG profile. Numerous studies  show an important contribution of PARP in modulation of  enzymes that process radiation-induced abasic sites in DNA [,].  Present study demonstrated that NAD+ replenishment might pos-  itively regulate PARP activity. In situations where level of DNA  damage is high, the resulting decrease in NAD+ levels from PARP  over-activation prohibits efficient DNA repair []. Under such  circumstances flux generated by nicotinamide induced enzymes  NAMPT and NMN might increase NAD+ concentration. Mount-  ing evidence suggests that modulation of NAD+ concentration is

an effective means of mitigating DNA damage processes in mammals []. Increased activity of PARP can promote DNA repair and its interaction with several transcription factors, whereas hyperactivation of PARP can result in poly(ADP-ribose) accumulation and depletion of NAD+ which may lead to apoptotic or necrotic cell death []. Our data suggests that compared to NSD fed animals, radiation induced surge in PARP activity was not sustained in CD fed mice after  h of irradiation due to insufficient NAD+ levels. Similarly, in CD maintained animals increased PARG activity was not sustained after  h of irradiation while higher activity was observed in NSD fed animals even after  h of  -irradiation. Our results demonstrated that radiation induced increased NAD+ biosynthesis was indeed positively correlated to PARP and PARG activity. Earlier studies have shown that DNA-damaging treatments that raise PARP activity without concurrent increase in PARG activity lead to increased cell death. Levels of AP sites in DNA are the consequence of a balance between lesion induction from radiation induced radical processes and repair. Here, we show that nicotinamide supplementation in the presence of  -radiation exposure prevented progressive increase of AP sites. Earlier studies have shown that PARP’s interaction at the AP site could protect the damaged DNA until AP endonuclease becomes available to ini-  tiate strand incision and AP site repair. For a short period  of time an incised AP site is occupied by PARP and then DNA  polymerase   adds one nucleotide into the repair gap and simulta-  neously removes the ′ -sugar phosphate.

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