App!. Magn. Reson. 18, 483-492 (2000) Applied Magnetic Resonance Springer- Verlag 2000 Printed in Austria Fluorescence and ESR Studies on Membrane Oxidative Damage by Gamma Radiation* B. N. Pandey and K. P. Mishra Cellular and Free Radical Radiation Biology Section, Radiation Biology Division, Bhabha Atomic Research Centre, Mumbai, India Received October 31, 1999; revised December 26, 1999 Abstract. Oxidative damage to cellular membranes critically controls the manifestation of cellular response to ionizing radiation. To gain further insight into the damaging mechanisms, we have in- vestigated the effects of y-radiation-generated free-radical-mediated peroxidative damage in egg yolk lecithin unilamellar liposomal membranes by employing 1,6-diphenyl-1,3,5-hexatriene (DPH). Alter- ations in lipid bilayer fluidity and malondialdehyde (MDA) formation were measured in irradiated liposomal membranes as a function of radiation dose (0.1-1 kGy). A relationship seems to exist be- tween the degree of radiation-induced peroxidative damage and the magnitude of DPH fluorescence decay in irradiated membranes. Radiation-induced membrane rigidization and MDA formation were significantly reduced when u-tocopherol, a natural membrane antioxidant, was present in the lipo- somes suggesting an involvement of lipid free radicals in the mechanism of the damage process. The results of the present study have been compared with those obtained by the electron spin resonance (ESR) technique on human erythrocyte ghost membranes with spin-labeled phospholipids having the unique capability to sensitively report on the dynamic state of the lipid environment inside the bilayer membrane. Iodoacetamide and N-ethylmaleimide spin labels were used to investigate alterations in membrane proteins. These results have contributed to our understanding of mechanisms involved in radiation membrane oxidative damage in terms of lipid peroxidation, fluidity changes and involve- ment of -SH groups of membrane proteins. Combined use of fluorescence and ESR spin-label tech- niques is of potential interest in probing the deeper molecular mechanisms of radiation injury in cellular membranes for developing strategies to modify the radiation damage to cells. I Introduction Ionizing-radiation-generated free-radical-induced oxidative damage of plasma membrane has been known to impair cellular functions causing pathogenesis including cancer or cell death [1-2]. Cellular membranes are recognized as im- portant target for radiation-induced oxidative damage, but the mechanism of the damaging process is poorly understood. Artificial and natural membranes such Presented at the 2nd Asia-Pacific Symposium, Hangzhou, China, October 31 to November 4, 1999.