Abstract
Selenium-dependent glutathione peroxidase (SeGPX) forms part of the antioxidant defences, a group of scavenger molecules and enzymes responsible for defending eukaryotic cells against pro-oxidative damage. It catalyses the reduction of hydrogen and lipid peroxides, rendering them nontoxic. Enzyme activity and putative mRNA for SeGPX had been previously detected in the digestive tissues of the mussel Mytilus edulis (Winston et al 1990, Livingstone et al 1992, Goldfarb et al 1989). This study was undertaken to investigate the regulation and function of SeGPX, in M. edulis and also in the scallop Pecten maxim us , the starfish Asterias rubens and the shore crab Carcinus maenas. Sources of pro-oxidant stress include changing oxygen tension and dietary chemicals. The four species were chosen as representing three major invertebrate phyla and different nutritional and respiratory strategies M. edulis (euryoxic, herbivore, Mollusca), P. maximus (stenoxic, herbivore, Mollusca), C. maenas (euryoxic, omnivore, Arthropoda), and A. rubens (stenoxic, carnivore, Echinodermata). Determination of the tissue distribution of SeGPX, total GPX, catalase, superoxide dismutase, glutathione reductase and DT-diaphorase activities was carried out in the four marine invertebrates, with comparison to endogenous potential for reactive oxygen species (ROS) production in the tissues studied. SeGPX enzyme levels were also compared to the putative SeGPX mRNA signal, determined by slot blotting and probing with the human SeGPX gene. Overall, there was a trend for greatest enzyme activity and ROS production potential in the digestive tissues, followed by the respiratory tissues. Comparison to endogenous ROS production potentials indicated that factors such as diet, respiratory strategy and motility were influential in determining the levels of antioxidant enzymes present in each species. Further optimisation of nucleic acid detection resulted in the demonstration of a single copy SeGPX gene in M. edulis genomic DNA and a single SeGPX mRNA species of approximately 1. 2 Kilobases in length in the digestive gland, in agreement with other known SeGPX mRNAs. Western blotting using an antihuman SeGPX polyclonal antibody, detected a 23KDa protein in M. edulis digestive gland. These methods and activity assays for SeGPX and total GPX were then used to investigate the seasonal regulation of SeGPX in M. edulis , the results indicating regulation of SeGPX at the mRNA level, possibly in relation to temperature, reproductive cycle and diet. Total GPX activity mirrored neither SeGPX protein nor SeGPX mRNA levels, indicating the involvement of other enzymes, such as glutathione S-transferases in catalysing GPX activity with organic peroxides. Dietary effects on SeGPX and antioxidant enzymes were investigated by starving M. edulis, A. rubens and C. maenas, for prolonged periods of up to 10 weeks. Differences in induction and repression of antioxidant enzymes were indicated in the three species, SOD activity decreasing possibly due to reduced cellular O2- levels, in all species except A. rubens. Catalase and SeGPX activity in M. edulis and C. maenas showed clear signs of induction after 5 weeks starvation, possibly to counteract increased protein oxidation and lipid peroxidation. Finally, water-borne heavy metal ions, Cu2+ Hg2+ and CH3Hg+, known perturbers of glutathione metabolism in the Mediterranean mussel Mytilus galloprovincialis, were used to study changes in the mRNA, protein and activity levels of SeGPX. Results showed increased mRNA levels for SeGPX in the gill of M. galloprovincialis in response to exposure to 40μg/l Hg2+, possibly indicating an induction response to either ROS mediated damage, or decreased levels of functional SeGPX. SeGPX protein levels often mirrored the changes in mRNA levels. However only in the case of Cu2+ exposure did enzyme activity show corresponding changes with SeGPX mRNA and protein. Levels of SeGPX were seen to return to normal levels for Cu2+ and CH3Hg+ exposure Overall, a single gene and mRNA species have been indicated for M. edulis SeGPX, a similar mRNA also being present in the major tissues of three other marine invertebrates. The SeGPX mRNA levels in M. edulis appear to respond to a number of endogenous and exogenous stimuli, including seasonal regulation, starvation and heavy metal toxicity-induced ROS mediated stress. Control of SeGPX enzyme levels therefore appears to be exerted at the level of SeGPX mRNA transcription or stabilisation.