Applications of 31P-NMR Spectroscopy for the Assessment of Sublethal Toxicity in Marine Organisms

Many important marine species are suffering declines. Release of pollutants into aquatic environments are likely contributors to this decline, and so biochemical methods to study the effects of sublethal toxicant exposure in marine organisms are urgently needed. The goal of this dissertation has been to develop non-invasive 31P-nuclear magnetic resonance (NMR) techniques to evaluate the effects of toxicant exposure on energy metabolism in marine invertebrates. Red abalone (Haliotis rufescens), the basis for an important commercial and sport fishery were chosen to serve as a model for other invertebrates due to the general physiological adaptations, such as their ability to survive extended periods of hypoxia, possessed by these animals, as well as their sedentary nature and large homogeneous foot muscle, which makes them ideal organisms for in vivo NMR spectroscopy.Initial studies examined changes in adenosine triphosphate (ATP), phosphoarginine (PA), inorganic phosphate, intracellular pH, and intracellular free Mg2+ in response to exposure to the mitochondrial stressors hypoxia, pentachlorophenol (PCP), and sodium azide. These studies were also adapted for use with a larval abalone stage, and the effects of PCP exposure was examined in these animals.Further studies refined these techniques to examine the rate of ATP formation from PA via the arginine kinase reaction using a magnetization transfer approach. Stress application increased rates of ATP formation, in contrast to mammalian systems, where declines in ATP formation are generally noted.In the final study the sensitivity of the classical biochemical parameter adenylate energy charge (AEC) was compared with NMR derived measurements of cytosolic adenosine diphosphate (ADP) in response to the three stressors. Cytosolic ADP increased two-three fold in response to stress application while AEC showed little or no change. The effect of increasing ADP on arginine kinase flux is also discussed.The work provided in this dissertation demonstrates some of the utility of in vivo approaches in evaluating the effects of sublethal stress exposure, and suggests future environmental applications in identifying currently unrecognized populations of marine organisms subject to toxicant induced stress