1080 toxicity - in vitro aconitase studies : a dissertation submitted in partial fulfilment of the Degree of Bachelor of Science with Honours at Lincoln University

Abstract

Sodium monofluoroacetate (1080) is a highly effective vertebrate pesticide that is widely used in New Zealand for control of mammalian pests. 1080 toxicity results from its conversion in the mitochondria to fluorocitrate, a competitive inhibitor and an irreversible in activator of aconitase. Blockage of TCA cycle, depletion of cellular energy, and organ failure are the fatal consequences of the inhibition of mitochondrial aconitase. However, being a non-selective toxin it poses high risk to the lives of non-target species as a result of accidental poisoning. Our ultimate goal is to develop an effective antidote that reverses the inhibition of mitochondrial aconitase caused by 1080. This study summarizes the factors I considered while standardizing an assay for measuring aconitase activity including methods devised in overcoming the artefacts, experiments carried out for testing the validity of the established assay procedure, and the stability of the enzyme preparations. I applied techniques that allowed the measurement of aconitase in both mitochondrial and cytoplasmic fractions of rat liver, intactness of isolated mitochondria, and the release of matrix contents by the disruption of mitochondria. Systematic studies using 1080 and fluorocitrate revealed that 1080 has no direct effect on aconitase, and inhibition of aconitase is possible only after the conversion of fluoroacetate to fluorocitrate. I standardized the conditions that effectively bring about the conversion in vitro by using isolated mitochondria. The knowledge and information obtained through this work can now be used in further in vitro and in vivo studies on the development of antidotes. The methods will be particularly useful in screening for compounds with antidotal properties.