Organophosphorus (OP) compounds have found tremendous use as industrial chemicals, pharmaceuticals and chemical warfare agents. The intended toxicity of warfare agents and the unwanted toxicity of industrial and agrochemicals results primarily from inhibition of cholinesterase enzymes, predominantly acetylcholinesterase (AChE). Functionalization of the OP class has led to development of compounds with selective action at various primary protein targets and the identification of numerous secondary and tertiary off target effects. Two OP compounds, glufosinate (GLF) and ethephon, have significant agricultural use as an herbicide and plant growth regulator respectively. They are also characterized by atypical mammalian OP toxicity. This dissertation explores newly characterized aspects of GLF and ethephon mammalian toxicology.
GLF can cause convulsions in mammals without AChE inhibition. The structural similarity of GLF to glutamate and the known excitatory mechanisms of glutamate signaling implicate the glutamatergic system as a target for GLF neurotoxicity. Known glutamate targets which could reasonably be expected to be involved in a convulsive phenotype after modulation were assayed for effect by GLF and related compounds. There appears to be no effect of GLF or the primary metabolite N-acetyl-GLF (N-Ac-GLF) on the urea cycle regulator enzyme CPS-I. However, GLF and N-Ac-GLF can interact directly with the N-methyl-D-aspartate (NMDA) subtype glutamate receptor and the high affinity glutamate reuptake transporter GLT-I. Potency at the NMDAR is in the range of 10-10,000 μM and potency at GLT-I is >1,000 μM. The concentrations required to affect transport through GLT-I are likely not to be attained and therefore not relevant to the neurotoxic mode of action. However, toxicokinetic data from reports of intentional human poisonings suggest that the concentration of GLF in the brain could reach high enough levels after acute exposure to account for the convulsive and memory loss effects. Furthermore, the newly characterized action of N-Ac-GLF at the NMDAR suggests that both the parent compound and metabolite could contribute to neurotoxicity.
Direct radioligand binding experiments are one of the most unbiased approaches to target site identification. However, the preparation of high specific activity radioligands for compounds of interest is specialized and costly. Progress toward the preparation of [3H-Me] GLF is reported with successful synthesis of unlabeled GLF prepared with methyl addition and deprotection as the last synthetic step. Although the synthetic procedure was not scalable for economical radiosynthesis, the method provides a platform for further optimization and eventual radioligand synthesis. Ethephon is well characterized to readily degrade to ethylene, which acts as a regulator of plant growth. Butyrylcholinesterase (BChE) is selectively inhibited after exposure to ethephon.
Ethephon itself does not inhibit BChE, but is degraded to a BChE inhibitor in alkaline solutions. In the present study, 31P NMR monitoring of ethephon degradation over time demonstrates that a transitory intermediate is formed during the degradation process. The chemical shift of the intermediate is as expected for 2-oxo-2-hydroxy-1,2-oxaphosphetane, the proposed BChE inhibitor. The degradation of ethephon proceeds through competitive reactions, forming either phosphate and ethylene directly or the BChE inhibitor. This mechanism is supported by kinetic modeling which is used to generate rate constants and T1/2 values that are in agreement with reported ethephon degradation rates.
OP chemicals are known to interact with a wide variety of biochemical targets. Functionalization has allowed for the preparation of OP chemicals that have surprising species and target site selectivity. However, complete target site specificity is never achieved as most OP chemicals have some secondary or tertiary effects at alternate sites. Furthermore, OP herbicides are not specific for plants. The main goal of toxicological profiling is therefore to identify the type and threshold for activity at all targets. The mechanistic insight obtained through these studies allows for better toxicological evaluation of GLF and ethephon.
|Advisor:||Casida, John E.|
|Commitee:||Bautista, Diana M., Vulpe, Chris D.|
|School:||University of California, Berkeley|
|School Location:||United States -- California|
|Source:||DAI-B 76/08(E), Dissertation Abstracts International|
|Subjects:||Neurosciences, Toxicology, Surgery, Analytical chemistry|
|Keywords:||Butyrylcholinesterase, Ethephon, Glufosinate, Herbicides, Neurotoxicity, Nmda receptor|
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