Organophosphorus pesticides are toxic compounds that are used to kill insects and pests that target cultivable crops. Nerve agents are organophosphorus compounds which have been used in the past during the Skripal poisoning in 2018 at Salisbury (UK) as well as recently in 2020 during poisoning of Alexei Navalny (Russia). These organophosphorus compounds (OPs) target an enzyme called acetylcholinesterase (AChE). AChE is a serine hydrolase found in human brain synapses and neuromuscular junctions. AChE hydrolyses the neurotransmitter acetylcholine thereby terminating the neuronal signal. OPs bind covalently to AChE thereby blocking its function which increases acetylcholine levels in the synaptic cleft. This results in seizures and respiratory termination that could lead to death if not treated. To help develop effect countermeasures, it is necessary to determine how OPs are metabolized in the human body. Cytochrome P450 (CYPs) are monooxygenases found in the human body that are responsible for metabolizing the majority of foreign substances that enter the human body and as such are important in studying the metabolism of foreign compounds such as OPs. The purpose of this study is to determine how CYPs metabolize OPs and which OPs are CYP activators or inhibitors. In this study we have used 6 human CYPs CYP1A2, CYP3A4, CYP2C9, CYP2C19, CYP2D6 and CYP2B6. An assay has been developed for each of the CYPs using different fluorogenic substrates based on Michalis-Menten enzyme kinetics. Km and Vmax values have been determined using each CYP-substrate pair namely, CYP1A2 with Ethoxy resorufin; CYP2C9 with Ethoxy resorufin; CYP2D6 with Ethoxy resorufin, CYP2C19 with 3-O-methyl fluorescein, CYP2B6 with Benzyl resorufin ether, and CYP3A4 with Dibenzyl fluorescein. This method was validated by using CYP selective inhibitors reduced the CYP activity in a dose dependent manner. Initially, a 10 µM concentration of OPs was tested to determine their inhibitory potency to reduce CYP activity for different time durations such as 10, 30, 60 minutes. Based on inhibition differences at these durations, we were able to propose which CYPs could be potential bioscavengers for specific OPs. This study is critical to determine the metabolic effects of OPs in humans. This information can then be used to help develop CYP-based or small-molecule CYP inhibitor-based therapies for OP poisoning.

In vitro CYP450 activation/inhibition by organophosphorus pesticides and nerve agent surrogates using fluorometric assays

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