JOYNER-MATOS, J.L.*; BHALLA, R.; DOWNS, C.A.; JULIAN, D.; Univ. of Florida, Gainesville; Univ. of Florida, Gainesville; EnVirtue Biotechnologies, Winchester, VA; Univ. of Florida, Gainesville: Antioxidant Protein Expression during Anoxia-Reoxygenation in Marine Polychaete (Glycera dibranchiata) Coelomocytes

In the tissues of intertidal invertebrates, endogenous production of oxygen-centered free radicals and resulting oxidative damage may occur during tidal cycles due to hypoxia and subsequent reoxygenation. The cellular response to this oxidative damage is poorly understood. In this study, we evaluated the response of coelomocytes from the intertidal polychaete Glycera dibranchiata to hypoxia/reoxygenation in vitro. Coelomocytes were collected from 24 worms, resuspended in buffered seawater, and randomly assigned to one of the following treatments (4 worms per treatment): anoxia (6 h), anoxia (6 h) followed by reoxygenation (6 h), anoxia (6 h) followed by reoxygenation (12 h), and normoxia for 6, 12, and 18 h. We used polyacrylamide gel electrophoresis/Western blotting and immunochemical analysis to quantify the levels of the antioxidant enzyme manganese superoxide dismutase (MnSOD), and heat shock protein 70 (Hsp70). Hsp70 was significantly (p < 0.032) decreased in cells treated with anoxia and with anoxia/reoxygenation, in comparison to normoxia-treated cells. This was likely due in large part to an overall decrease in cellular metabolism during anoxia. In contrast, we found that MnSOD levels decreased significantly (p < 0.04) in anoxia-treated cells, but after 12 hours of reoxygenation, MnSOD levels were indistinguishable from normoxia controls. This upregulation of MnSOD during reoxygenation likely is a response to free radical production. The results of this cellular-level short-term, in vitro study complement those reported by Storey and colleagues, who have conducted longer-term whole-animal incubations with hypoxia-tolerant turtles and intertidal marine gastropods.