Molecular and Cellular Radiobiology Projecet Title: Mechgnisms of Action of Free Radicals and Hormones RX-03-02-(c) 17. Expected Results in FY 1974: (Cont'd.) Therefore preliminary ENDOR investigations will be carried out when a demonstration ENDOR spectrometer is available for a few weeks at Varian’s New Jersey applications laboratory. Pending those trials, collaborations with other laboratories with ENDOR capability may be set up. The new two-stage mixer for continuous flow work with Q-band EPR is relatively slow, difficult to set up, and not regularly consistent in performance. Further improvement is a goal, but with limited time remaining for Schmidt to complete on-going work and delay in obtaining a replacement the work is only tentatively scheduled, EPR data collection for analysis of the crystals of irradiated deoxyadenosine-H 70 and l-methyl thymine should be completed, and full interpretation of the computer-enhanced powder spectrum of irradiated dADNS is expected. The TiCLII)/H,0, radiomimetic reactions of the hydroxyl radical on purine derivatives, pursued, studied with Q-band flow methods, will be Oxidative chain reactions involving dissolved oxygen and lipoperoxy free radicals as chain carriers may be involved in the damage reactions of several classes of pollutants, as well as of some drugs, other potentially noxious agents. radiation, and Furthermore, "normal" aging mechanisms and membrane changes associated with malignant transformation of cells may be mediated, in part, through such reactions; and the mixed function oxidase enzymes that both detoxify and activate some pollutants and other carcinogens, as well as many drugs, are membrane-bound systems that produce free radicals . capable of damaging both their own and other intracellular membranes. Preparations are being made to extablish methodologies for lipid extraction from membranes of animal cells, for mixed function oxidase (aryl hydrocarbon hydroxylase) assays, for lipoperoxide assay by luminescence and fluorescence spectro- scopy (as well as by nonspecific and indirect means such as thiobarbituric acid reaction with malondialdehyde), for product analysis by thin-layer and gas-liquid chromatography, and for synthesis of spin-labeled lipids, Experi- ments to test some ofthe effect: of mixed function oxidase activity and of carcinogen and pollutant actio: :n nuclear DNA, as mediated through the nuclear membrane will use nuclei isolated from livers of rats pretreated with tritiated thymidine. Following a modified McGrath and Williams centrifugal separation of DNA on an alkaline sucrose gradient, " strand breakage will ined from the tritium distribution as one measure of deleterious the DNA itself, Another assay of nuclear "damage" will be the incorporation of lipid spin labels into the nuclear envelope monitored by EPR, A modification of this approach designed to evaluate enzymic activity of mixed-function and other (See Continuation Sheet) Lt 19304 RX- 244