The objectives of the proposed research are to determine the mechanism through which niacin intake affects tissue levels of NAD and to define the effects of altered NAD levels on the molecular and cellular responses to carcinogen exposure and oxidative stress. The hypothesis to be tested is that sub-optimal niacin nutriture results in reduced cellular levels of NAD, leading to a predisposition to malignancy following environmental insults to the genome.
Specific Aim 1 is to quantify the relationship of nicotinamide, nicotinic acid and tryptophan to intracellular NAD content in human mammary epithelial (HME) cells maintained in primary culture. HME cells will be obtained from a commercial vendor and cultured in a defined medium containing varying amounts of nicotinamide and nicotinic acid. The levels of cellular NAD and NADP will be determined. Similar experiments will be performed to determine to what extent tryptophan is capable of serving as an NAD precursor in HME cells.
Specific Aim 2 is to determine the intracellular NAD content required in order for HME cells to display an optimal response to carcinogen induced DNA damage or oxidative stress. HME cells, cultured under conditions that lead to varying concentrations of intracellular NAD, will be treated with either N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or xanthine/xanthine oxidase. ADP-ribose polymer metabolism, level of p53 transcription, repair of single strand DNA breaks, sister chromatid exchange, chromosomal aberrations, and cell survival will be examined as a function of NAD content. These in vitro studies will provide the information necessary to design future experiments in which the in vivo relationship between niacin intake, levels of circulating precursors, and breast epithelium NAD content determined and optimized to limit carcinogenic events.
