Prevention of DNA oxidation by dietary antioxidants will be investigated both in vitro using isolated DNA, tumor cells and non- transformed cells grown in culture, and in vivo using human subjects receiving antioxidants in dietary supplements. The antioxidant effects of lycopene, beta-carotene, and alpha-tocopherol will be quantitatively assessed in vitro by measuring levels of oxidized DNA in calf thymus DNA, human prostate tumor cells and preneoplastic fibroblasts grown in culture: and the extent to which dietary lycopene prevents DNA oxidation in blood and the prostate will be investigated in vivo using a group of 120 human subjects. Because these studies will encompass several in vitro systems as well as a carefiilly planned human dietary intervention study, it will be possible to evaluate the chemopreventive predictability of each in vitro assay. Measurement of DNA oxidation in both peripheral blood cells and in prostate tissue (following prostatectomy) of the same human subjects will permit the evaluation of the predictive value of using the more easily obtained blood samples as indicators of oxidative stress. Following mild enzymatic hydrolysis of DNA, oxidized nucleosides will be quantitated using a new electrospray liquid chromatography-mass spectrometry (LC-MS) method developed during this project, which will facilitate the measurement of all major oxidized deoxynucleosides. In addition to the measurement of oxidized nucleosides, intracellular levels of each carotenoid (including cis- and transisomers), alpha-tocopherol and glutathione will be measured. By comparing cellular (and prostate) carotenoid or tocopherol levels to the amounts added in vitro or fed to human subjects, uptake of specific dietary antioxidants by target cells or organs will be measured. Overall, this investigation will l) assess the importance of measuring multiple DNA oxidation products as biomarkers of oxidative stress and its chemoprevention, 2) evaluate the predictive value of in vitro assays of chemoprevention by direct cornparison to in vivo measurements, 3) detertnine the significance of DNA oxidation products in blood as an indicator of oxidative stress in prostate, and 4)measure cellular uptake in vitro and in vitro of the chemoprevention agent lycopene.
