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Effect of Carcinogenic N-Alkyl-N-nitroso Compounds on Nicotinamide Adenine Dinucleotide Metabolism¹

Departments of Chemistry and Biochemistry, North Texas State University/Texas College of Osteopathic Medicine, Denton, Texas 76203 [M. K. J., V. L., H. J-S., R. A. B.], and Department of Biology, Texas Woman's University, Denton, Texas 76204 [E. L. J.]

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.

¹ Supported in part by Grants B-633 from The Robert A. Welch Foundation and CA23994 from NIH and by North Texas State University and Texas Woman's University Faculty Research Funds.

² To whom requests for reprints should be addressed.

Received 9/21/79. Accepted 2/20/80.

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