Superoxide dismutase activity in Chamelea gallina haemocytes: effects on functional responses of haemocytes during anoxia and reoxygenation

INTRODUCTION: The immune system of the animals consists of cellular and humoral elements involved in defence mechanisms against pathogens, parasites and foreign elements. Concerning to invertebrates, the immune responses are mediated by haemocytes through several internal defence mechanisms. As mammalian monocytes and macrophages, bivalve haemocytes show high phagocytosys activity against non-self molecules and cells. Haemocytes activation determines an increasing oxygen consumption (respiratory burst) resulting in a reactive oxygen species (ROS) generation, that play a very important role in microbiocidal activity. However an excess of ROS production during an infection, can be dangerous for the cells, causing lipid peroxidation and DNA damages. The cell defence against oxidative stress rely on enzymatic and non-enzymatic antioxidant systems. The superoxide dismutase (SOD) is one of the main enzyme involved in antioxidant activity. In this research will be evaluated the production of ROS (indirectly through SOD activity detection) by Chamelea gallina haemocytes during anoxia and following recovery and its role in immune responses. MATERIALS AND METHODS: Specimens of Chamelea gallina were dredged on sandy bottom area of the Emilia-Romagna coast (Italy), acclimated for 1 week to laboratory conditions (salinity of 24‰, temperature of 18°C), before anoxic incubations. Filtered sea water was deoxygenated in a 50 L reservoir through a 2 hours vigorously N2 bubbling and siphoned, under a continuous flow of N2 and a constant water flux of 2L/h, into 10L jars (containing 150 clams each). Clams were maintained in anoxia for 48 hours and then were allowed to recovery in aerated seawater for further 24 hours. Haemolymph of control, anoxic and recovery clams was collected from the anterior adductor muscle with a 1-ml syringe. Pooled haemolymph (10 clams) was centrifuged at 780×g for 10 min; pelleted cells were re-suspended in distillate water, sonicated for a few minutes and centrifuged at 12000 ×g for 15 min. Activity determination of superoxide dismutase (SOD) was performed on the haemocyte lysate. SOD activity was determined spectrophotometrically (UV/Vis Beckman mod. DU 530 Spectrophotometer). CuZnSOD and MnSOD expression have been evaluated in haemocytes by immunoblotting, analysis using two different polyclonal antibody after SDS-PAGE. The results were compared using a t- test. RESULTS AND DISCUSSION: Results of the present research indicated that anoxia exposure caused a significant (p<0.05) decrease in total SOD (and its isoforms) activity in haemocytes with respect to controls. Concerning the reoxygenation we observed a recovery of the MnSOD activity, that showed values higher than in the control ones. The activity values of CuZnSOD, on the contrary, remained very lower during the recovery. By immunoblotting analysis, CuZnSOD and MnSOD immunoreactive bands were identified in all experimental conditions using specific antibodies. The expression levels of both enzymes decreased after 48 h of anoxia and remained low at the end of reoxigenation period (24 h). The decrease of both isoforms activity and expression during anoxic incubation suggested a decreasing superoxide anion generation due to the lack of oxygen. Probably low level of superoxide production affected negatively the microbiocidal effectiveness of the haemocytes and making the animals more sensible to the pathogens attack, according to other immune parameters investigated (THC, phagocytic and lysozyme activity). Concerning reoxygenation period, probably recovery clams suffered a further stress because of a massive spawning. In any case during the recovery, MnSOD showed very high activity (higher than in the control) due to the high inducibility of this isoform (Pinteaux et al, 1998). In fact the reintroduction of the oxygen, causing a higher superoxide generation, enhanced also the SOD activity. This suggestion seemed not be confirmed by our expression data, resulting lower during the recovery. The discrepancy between activity and expression suggested the presence of different regulation mechanisms. On the other hand MnSOD inducibility can be demonstrated with some certainty by MnSOD mRNA quantification. Decreased levels of MnSOD mRNA and activity have been found to be associated with hypoxia; on the contrary, induction of MnSOD mRNA has been correlated with hyperoxia and ROS production (MacMillan-Crow et al, 1996). Increased MnSOD expression represents an appropriate response to oxidative stress as a mechanism to protect against increasing levels of ROS (MacMillan-Crow et al, 1996). The oxidant/antioxidant balance is an important factor in immune cell function, including the control of signal transduction and gene expression, increased level of antioxidant will be needed improving immune response. REFERENCES: Pinteaux E., Perraut M., Tholey G. (1998) Distribution of mitochondrial manganese superoxide dismutase among rat glial cells in culture. Glia 22(4): 408-14. MacMillan-Crow L.A., Crow J.P., Kerby J.D., Beckman J.S., (1996). Nitration and inactivation of manganese superoxide dismutase in chronic rejection of human renal allografts. Proc. Nat. Acad. Sci. USA. 93: 11853-858.