The pyridine nucleotide NAD is directly involved in the regulation of cell growth. We have observed that carcinogenic N-nitroso compounds cause a rapid depression of cellular NAD. This depression appears to be related to NAD-dependent nuclear modification reactions associated with carcinogen-induced damage to DNA and/or repair of DNA damage. A major objective of this proposal is to study in detail the ability of N-nitroso compounds of known carcinogenicity to cause acute depression of NAD in 3T3 cells and in mitogen stimulated human lymphocytes. Several classes of direct-acting and indirect-acting carcinogens will be examined to determine whether acute depression of NAD is a general effect of carcinogens. The extent of DNA damage caused by N-nitroso compounds and the rate of DNA repair of carcinogen induced DNA damage will be studied in normal and NAD depleted 3T3 cells. Sensitive, quantitative methods involving gas chromatography-mass spectrometry and high pressure liquid chromatography for measuring NAD-dependent nuclear modifications as mono- and poly-adenosine diphosphoribose (ADPR) will be developed. The extent of these NAD-dependent nuclear modifications will be studied in normal cells and in cells exposed to carcinogenic N-nitroso compounds. Carcinogen-induced DNA damage and the rate of DNA repair will be studied in the presence of the methylated xanthines caffeine and theophylline which are specific inhibitors of NAD-dependent modifications.