The primary objective of this proposal is to determine whether the oxidative burst induced in polymorphonuclear leukocytes (PMNs) by tumor promoting agents causes modification of the thymine moiety in DNA through hydroxyl radical (.OH) attack. PMNs will be isolated from the blood of healthy, informed volunteers by venipuncture into tubes containing anticoagulant. The production of superoxide anion radical, H2O2 and .OH resulting from the response of the PMNs to stimulation by tumor promoters will be measured in the presence or absence of human plasma. Tumor promoters will include 12-0-tetradecanoylphorbol-13-acetate (TPA), a complete promoter, and mezerein, a second stage promoter. To determine whether the extent of thymine modification in DNA exposed to activated PMNs is proportional to .OH formation, DNA will be analyzed for the presence of 5-hydroxymethyl-2'-deoxyuridine (HMdU) and thymidine glycol (dTG), products known to be formed through the .OH-mediated action of ionizing radiation. Initial experiments will be carried out with E. coli (3H)thymidine-labeled DNA co-incubated with activated PMNs. This will be followed by co-incubating (3H)thymidine-labeled HeLa cells with activated PMNs to determine whether the oxygen radical species formed can effect modification of cellular DNA. When the extent and nature of PMN-mediated thymine modification has been determined in these systems, then non-radioactive HeLa cells will be incubated with activated PMNs and thymine modification detected by post-labeling with 32P. A second objective is to characterize the DNA-protein crosslink which we have identified in DNA incubated with activated PMNs. First, E. coli (3H)thymidine-labeled DNA and then HeLa cells whose DNA will be prelabeled with (3H)thymidine will be incubated with TPA-activated PMNs in the presence of autologous human plasma. After digestion of proteins with Proteinase K, the DNA will be enzymatically hydrolyzed and analyzed by HPLC for the presence of HMdU-amino acid adducts using 14C-containing marker compounds which will be synthesized in this laboratory.