A number of serious health problems are associated with exposure to organophosphate (OP) insecticides. The varied biochemical lesions leading to these maladies are not completely understood but most likely involve precursory phosphorylation of certain enzymes leading to chemical and biochemical imbalances. The long term goals of this proposal are: 1. to show that impurities in commercial 0P insecticides (phosphorothiolates) are responsible for certain acute and delayed deleterious health effects and, 2. to develop methods for the assessment and prevention of mammalian poisoning by the [stereoisomers of) phosphorothiolates based upon information gathered from biochemical and chemical investigations with enzymes and enzyme model systems. Within this framework, the following specific aims will be tested: 1. to show that inactivation of acetylcholinesterase by chiral phosphorothiolates occurs via a general mechanism, namely, expulsion of an X-group resulting in the formation of a chiral 0,Sdimethylphosphorothiolated active site serine residue, 2. To determine the degree to which stereochemistry influences the rate and mechanism of cholinesterase inactivation by isomalathion, 3. to show that phosphorothiolate-induced aging reactions are stereo-dependent processes and to characterize the reactions responsible for the aging phenomena using a synthetic model of an inhibited active site portion of human serum acetylcholinesterase, and 4. to show that phosphorothiolates are oxidized preliminarily to S-oxide intermediates. To determine the nature of the leaving group (aim 1), we shall react isomeric, radiolabeled phosphorothiolates with cholinesterase and evaluate the resultant site of radiolabel enrichment. The influence of stereochemistry upon the inhibitory potency of isomalathion will be evaluated by kinetic analysis with specific cholinesterases and by product analysis of chemical reactions. The mechanism of inactivation by isomalathion will be determined with the aid of stable isotope enrichment. Reaction of C-13 enriched isomalathion with cholinesterase will be monitored by NMR and the phosphate ligand connectivity determined by observation of specific signals that are altered or lost. Biochemical aging will be studied with the aid of enzyme models (tripeptides). Experiments designed to specifically determine the rate and extent of thiolester lability will be conducted. The existence of the reactive phosphorothiolate S-oxides will be proved by synthesis of a model phosphorothiolate that is designed to result in an isolable alpha-phosphoryl S-oxide. The collective investigations will provide insight into mechanisms of toxicity due solely to impurities in commercial 0P insecticides.
