Effects of ionizing radiation on biological membranes include alterations in membrane proteins, peroxidation of unsaturated lipids accompanied by perturbations of the lipid bilayer polarity. We have measured radiation-induced membrane modifications using two fluorescent lipophilic membrane probes (TMA-DPH and DPH) by the technique of fluorescence polarization on two different cell lines (Chinese hamster ovary CHO-K1 and lymphoblastic RPMI 1788 cell lines). gamma-Irradiation was performed using a 60Co source with dose rates of 0.1 and 1 Gy/min for final doses of 4 and 8 Gy. Irradiation induced a decrease of fluorescence intensity and anisotropy of DPH and TMA-DPH in both cell lines, which was dose-dependent but varied inversely with the dose rate. Moreover, the fluorescence anisotropy measured in lymphoblastic cells using TMA-DPH was found to decrease as early as 1 h after irradiation, and remained significantly lower 24 h after irradiation. This study indicates that some alterations of membrane fluidity are observed after low irradiation doses and for some time thereafter. The changes in membrane fluidity might reflect oxidative damage, thus confirming a radiation-induced fluidization of biological membranes. The use of membrane fluidity changes as a potential biological indicator of radiation injury is discussed.
BACKGROUND Modifications of intracellular transfer, resulting from a loss of membrane integrity may contribute toward setting the cell onto the pathway of apoptosis. METHODS We have developed an original technique of measuring simultaneously, with flow cytometry, changes in membrane fluidity and cell death status. Our aim was to assess the extent to which radio‐induced cell death and membrane alterations are linked. Investigations were performed on lymphocytes 24 h after whole human blood γ‐irradiation. RESULTS Our results confirmed the expected increase in the percentage of apoptotic cells as a function of dose, but revealed that the percentage of necrotic cells appeared stable after irradiation. At the same time, the fluorescence anisotropy of the living lymphocyte subpopulation decreased significantly and dose dependently as measured 24 h post‐irradiation. With TMA‐DPH, the anisotropy index of apoptotic lymphocytes was always lower than that of the viable lymphocyte subpopulation. On the other hand, 1,6‐diphenyl‐1,3,5‐hexatriene (DPH) anisotropy was similar in apoptotic and viable cells after irradiation. These findings suggest that apoptotic lymphocytes are characterised by a membrane fluidisation that mainly occurs on the cell membrane surface. CONCLUSION Our study made technical advances in using cytometric fluorescence anisotropy measurement as an early biological indicator of apoptosis after cellular exposure to ionising radiation. Cytometry 39:151–157, 2000 © 2000 Wiley‐Liss, Inc.
Our study emphasizes the effect of gamma irradiation on intestinal cell membrane fluidity and addresses the potential relationships existing between radiation-induced lipoperoxidation, membrane fluidity, and changes in membrane protein activities. Male Wistar rats were exposed to an 8-Gy total body irradiation (60Co source) and studied 1, 4, and 7 days after irradiation (D1, D4, and D7). Membrane enzyme activities and fluorescence anisotropy were determined on small intestinal crude membrane preparations. The supernatants of membrane preparations as well as plasma were used for malonedialdehyde (MDA) quantification. The effect of carbamylcholine on electrical parameters was estimated on distal ileum placed in Ussing chambers. We observed a decrease in fluorescence anisotropy for at least 7 days, an increase in membrane production of MDA at D4, a decrease in membrane enzyme activities at D4, but an amplification of carbamylcholine-induced increase in short-circuit current at D4 and D7. Furthermore, correlations were observed between the 1,6-diphenyl-1,3,5-hexatriene anisotropy coefficient and sucrase activity and between MDA levels and leucine aminopeptidase activity. Thus, total body irradiation induces changes in intestinal membrane fluidity and an increase in lipoperoxidation. These modifications may have an impact on the activity of membrane proteins involved in intestinal function.
These findings suggest the utility of structural membrane modification measurements as an early bio-indicator of ionizing radiation exposure.
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