Detection Quantification and Microlocalisation of Targets of Pesticides Using Microchannel Plate Autoradiographic Imagers

摘要

Organophosphorus (OP) compounds are a diverse chemical group that includes nerve agents and pesticides. They share a common chemical signature that facilitates their binding and adduction of acetylcholinesterase (AChE) within nerve synapses to induce cholinergic toxicity. However, this group diversity results in non-uniform binding and inactivation of other secondary protein targets, some of which may be adducted and protein activity influenced, even when only a relatively minor portion of tissue AChE is inhibited. The determination of individual OP protein binding targets has been hampered by the sensitivity of methods of detection and quantification of protein-pesticide adducts. We have overcome this limitation by the employment of a microchannel plate (MCP) autoradiographic detector to monitor a radiolabelled OP tracer compound. We preincubated rat thymus tissue in vitro with the OP pesticides, azamethiphos-oxon, chlorfenvinphos-oxon, chlorpyrifos-oxon, diazinon-oxon, and malaoxon, and then subsequently radiolabelled the free OP binding sites remaining with ³H-diisopropylfluorophosphate (³H-DFP). Proteins adducted by OP pesticides were detected as a reduction in ³H-DFP radiolabelling after protein separation by one dimensional polyacrylamide gel electrophoresis and quantitative digital autoradiography using the MCP imager. Thymus tissue proteins of molecular weights ~28 kDa, 59 kDa, 66 kDa, and 82 kDa displayed responsiveness to adduction by this panel of pesticides. The 59 kDa protein target (previously putatively identified as carboxylesterase I) was only significantly adducted by chlorfenvinphos-oxon (p < 0.001), chlorpyrifos-oxon (p < 0.0001), and diazinon-oxon (p < 0.01), the 66 kDa protein target (previously identified as serum albumin) similarly only adducted by the same three pesticides (p < 0.0001), (p < 0.001), and (p < 0.01), and the 82 kDa protein target (previously identified as acyl peptide hydrolase) only adducted by chlorpyrifos-oxon (p < 0.0001) and diazinon-oxon (p < 0.001), when the average values of tissue AChE inhibition were 30%, 35%, and 32% respectively. The ~28 kDa protein target was shown to be heterogeneous in nature and was resolved to reveal nineteen ³H-DFP radiolabelled protein spots by two dimensional polyacrylamide gel electrophoresis and MCP autoradiography. Some of these ³H-DFP proteins spots were responsive to adduction by preincubation with chlorfenvinphos-oxon. In addition, we exploited the useful spatial resolution of the MCP imager (~70 μm) to determine pesticide micolocalisation in vivo, after animal dosing and autoradiography of brain tissue sections. Collectively, MCP autoradiographic imaging provided a means to detect targets of OP pesticides, quantify their sensitivity of adduction relative to tissue AChE inhibition, and highlighted that these common pesticides exhibit specific binding character to protein targets, and therefore their toxicity will need to be evaluated on an individual compound basis. In addition, MCP autoradiography afforded a useful method of visualisation of the localisation of a small radiolabelled tracer within brain tissue.