Mechanisms of Radiation-induced Hemolysis

Project Summary Increased nuclear energy and industrial radiation use as well as nuclear weapons proliferation have increased the risk for accidental radiation mass casualties or conflict-related radiation exposure. Radiation countermeasures approved by the U.S. Food and Drug Administration (FDA) are limited, primarily targeting the repopulation of white blood cell populations following radiation exposure. Therefore, there is an urgent need to develop countermeasures that address radiation effects in other cell types. Red blood cells (RBC) undergo non-apoptotic hemolysis following radiation exposure. The result of rapid RBC lysis is the sudden release of potentially toxic levels of iron into the serum. Several studies showed that the administration of iron chelators mitigates multi-organ damage following total body irradiation. Although radiation-induced hemolysis is well established, the mechanism of radiation-induced RBC hemolysis is not understood, and the forms of iron released by lysed RBC are not known. Our preliminary data in a murine model of total body irradiation (TBI) show that hemoglobin (Hb) and carbonic anhydrase II (CAII), proteins critical for RBC function, are oxidized and inhibited. Reduced CAII activity affects the acid-base homeostasis in RBC, membrane stiffness, and RBC deformability and fragility. Our data indicate that that ferritin, transferrin, CD71/transferrin receptor and integrin alphaM/Mac-1 (an iron ion receptor) are upregulated in several tissue after TBI, suggesting that free iron and ferritin-bound iron may be increased in the serum. CD163, a high affinity hemoglobin-haptoglobin scavenger, was downregulated. Based on these results, we hypothesize that Hb and CAII oxidation and CA reduced function increase RBC fragility, and that free iron and transferrin-bound iron are increased following radiation-induced RBC hemolysis. This R21 project tests these hypotheses by pursuing two specific aims: 1) Determine the effects of radiation on Hb and CAII function, RBC function, and membrane stiffness; and 2) Identify the forms of iron release from hemolysis following TBI in a murine model. The proposed work is highly innovative because it will address a novel mechanism of radiation injury and means to counteract radiation injury after the exposure has already occurred. Results will be significant because the identification of the mechanism of iron release from RBC will allow the identification of novel targets to mitigate iron-induced multi- organ injury following total body irradiation.