The acute effect of carcinogenic N-alkyl-N-nitroso compounds on nicotinamide adenine dinucleotide (NAD) metabolism was studied in 3T3 cells with N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) as the model compound. MNNG caused a rapid time- and dose-dependent lowering of the intracellular levels of NAD. Addition of 130 µm MNNG to the culture medium resulted in a 50% reduction in the size of the NAD pool in 1 hr but did not affect the size of the nicotinamide adenine dinucleotide phosphate or adenosine triphosphate pools. The biosynthesis of NAD in vivo was studied by measuring the rate of conversion of [14C]nicotinamide to NAD. The presence of 140 µm MNNG in the culture medium did not affect the rate of biosynthesis of NAD, demonstrating that lowering of NAD must be caused by an increased rate of degradation of this nucleotide. The activities of the known degradative enzymes, NAD glycohydrolase and poly(adenosine diphosphate ribose) [poly(ADP-ribose)] polymerase, were measured in cells made permeable to NAD by a brief cold-shock treatment. Treatment of cells for 30 min with 340 µm MNNG prior to permeabilization did not change the activity of NAD glycohydrolase but resulted in a 7-fold increase in the activity of poly(ADP-ribose) polymerase, suggesting that lowering of NAD is caused by an increased synthesis of poly(ADP-ribose). This possibility was also supported by the observation that the rate of lowering of NAD could be greatly reduced by the addition of the poly(ADP-ribose) polymerase inhibitor, theophylline, to the culture medium. The lowering of NAD was coincident with the appearance of alkali-labile sites in DNA as judged by velocity sedimentation on alkaline sucrose density gradients. In total, the results provide evidence that acute lowering of NAD is caused by an increased synthesis of poly(ADP-ribose) which occurs in response to molecular damage to DNA.

The mutagenicity of a non-carcinogenic nitrosamine, N,N-dibenzylnitrosamine (I), and a chemically synthesized alpha-acetoxy derivative, N-(alpha-acetoxy-benzyl)-N-benzylnitrosamine (II), has been examined in Salmonella typhimurium TA100 and TA1535. Compound (I) was non-mutagenic when tested directly or in the presence of a metabolic activation system while (II) was highly mutagenic when tested directly. This is the first report on the conversion of a non-mutagenic N-nitrosamine to a mutagen by the formation of an alpha-acetoxy derivative.

The functional pathways of nicotinamide adenine dinucleotide (NAD) biosynthesis and their regulation were studied in the dimorphic fungus Candida albicans. The presence of a functional endogenous pathway of NAD biosynthesis from tryptophan was demonstrated. In addition, nicotinamide served as an efficient salvage precursor for NAD biosynthesis but nicotinate was not utilized. The pathway for nicotinamide utilization involved nicotinate and nicotinate nucleotides as intermediates, suggesting that the failure to utilize nicotinate involves a transport defect. The mechanisms that regulate NAD levels during exponential growth operated to maintain constant NAD levels when NAD biosynthesis occurred exclusively from endogenous or salvage pathways or from a combination of the two. The regulation also operated such that the salvage pathway was preferentially utilized.

The chromatographic properties on thin layers of poly(ethyleneimine) cellulose of sixteen compounds containing the pyridine and/or adenine ring have been studied. Chromatographic mobilities have been examined as a function of the concentration of lithium chloride or sodium formate buffer in the chromatographic solvent. These data provide a rationale for the development of rapid and simple separation methods that should prove useful in the study of pyridine and adenine nucleotide metabolism.