Reactive intermediates are proposed to be responsible for both the useful properties and adverse effects of many pesticides and related toxicants. The long-term objectives are to define the mechanism of action and safety criteria for the parent toxicants by understanding the toxicological properties of their reactive intermediates.
The specific aims focus this objective on: sulfoxides, sulfones, sulfonic acids and N-dealkylation products derived from EPTC and other thiocarbamate herbicides and dithiocarbamate fungicides; the sterically-hindered carbodiimide formed from diafenthiuron, a thiourea insecticide; and octachlorofulvalene and glutathione (GSH) adducts from the acaricide dienochlor. The goal is to define the toxicologically-relevant targets and the pesticide-binding site interactions at a molecular level. There are two hypotheses for EPTC and other important mono- and dithiocarbamate pesticides; first, inhibition of aldehyde dehydrogenase involving carbamoylation of a thiol group in its active site, by metabolically-formed reactive intermediates, is both a serious adverse effect and a biological marker for exposure; second, carbamoylation of acyl carrier protein, coenzyme A or other sites in the: fatty acid synthetase complex by bioactivated thiocarbamate herbicides leads to a block in lipid biosynthesis. The hypothesis for diafenthiuron is that oxidation at sulfur leads to formation of its carbodiimide which in turn, as with dicyclohexylcarbodiimide, reacts with the oligomycin- sensitive site of ATPase (or a related enzyme) to form a covalent derivative (possibly at a carboxyl group) and block oxidative phosphorylation. This is the first thiourea insecticide so the mode of action at the organismal, enzyme and molecular level must be adequately defined before broad public exposure. The exceptionally high toxicity and very delayed effects of the diafenthiuron carbodiimide [e.g. mouse intraperitoneal (ip) LD50 0.3 mg/kg] indicate that the action is much broader in scope and of greater concern for health than currently appreciated. The hypothesis for dienochlor (C10-Cl10) (mouse in LD50 5 mg/kg) is that it is converted to octachlorofulvalene (C10-Cl8) and GSH adducts [by sequential dechlorination leading eventually to C10-H2-Cl(SG)5 and C10-H2-(SG)6] which derivatize essential protein thiols (including hemoglobin, albumin and GSH S-transferases) and thereby disrupt cellular functions. An evaluation of these effects is necessary to determine if the current non-food-use restrictions for dienochlor are adequate to ensure human safety. The research design and methods involve: identifying and localizing the toxicological targets by assays of relevant enzymes for in vitro and in vivo inhibition; synthesizing related compounds for essential structure-activity studies to establish relevance; radiolabelling the precursor toxicants and activated intermediates with tritium at high specific activity; radioligand and binding investigations with the activated intermediates at the targets; sequencing the active sites undergoing covalent derivatization.
