ALTERATION OF NAD METABOLISM BY CHEMICAL CARCINOGENS

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The occurrence of DNA damage and cellular responses to DNA damage are involved in the initiation and perhaps the promotion phase of carcinogenesis. The proposed studies will focus on the mechanism and biological significance of the rapid alteration of poly(ADP-ribose) metabolism cause by environmentally induced DNA damage. These studies will utilize intact cultured mouse cells following treatment with N-methyl-N'-nitro-N nitrosoguanidine (MNNG). The complexity of polymers of ADP-ribose will be studied by isolation of polymers using highly selective boronate affinity resins, fractionation by HPLC molecular sieve chromatography and simultaneous determination of linear and branched internal residues and terminal residues. Such experiments will allow the determination of true polymer size and branching frequency per molecule, information necessary for evaluating the importance of non-covalent interactions of the polymer with other components of chromatin. The turnover of polymers and their distribution within chromatin fractions will be studied by combining the above methods with in vivo labeling protocols and inhibitors of poly(ADP-ribose) polymerase. Labeling methods will also be used to assess the quantitative significance of poly(ADP-ribose) metabolism in non-damaged cells. The function of poly(ADP-ribose) metabolism in cellular recovery from the cytotoxic effects of MNNG will be addressed by studies of cell cycle progression in synchronized C3H10T1/2 cells using flow cytofluorimetry, autoradiography and ADP-ribosylation inhibitors. This system will also be used to make chromatin comparisons designed to yield information concerning the stabilization or alterations of chromatin structure involving poly(ADP-ribose) and normal cell cycle progression in MNNG treated cells. The involvement of poly(ADP-ribose) metabolism in other important biological responses to DNA damage in C3H10T1/2 cells will be studied by evaluating the effect of ADP-ribosylation inhibitors and nutritional manipulation of NAD levels on mutagenesis and malignant transformation. Such studies will utilize conditions where co-cytotoxic effects will be eliminated or minimized and effects on growth during expression carefully monitored. The mechanism of poly(ADP-ribose) metabolism by hyperthermia will be studied and an assessment of a possible general role of poly(ADP-ribose) metabolism in a general cellular response to environmental stress will be made.