Novel approaches to mitigating parathion toxicity: targeting cytochrome P450-mediated metabolism with menadione

Abstract

Accidental or intentional exposures to parathion, an organophosphorus (OP) pesticide, can cause severe poisoning in humans. Parathion toxicity is dependent on its metabolism by the cytochrome P450 (CYP) system to paraoxon (diethyl 4-nitrophenyl phosphate), a highly poisonous nerve agent and potent inhibitor of acetylcholinesterase. We have been investigating inhibitors of CYP-mediated bioactivation of OPs as a method of preventing or reversing progressive parathion toxicity. It is well recognized that NADPH-cytochrome P450 reductase, an enzyme required for the transfer of electrons to CYPs, mediates chemical redox cycling. In this process, the enzyme diverts electrons from CYPs to support chemical redox cycling, which results in inhibition of CYP-mediated biotransformation. Using menadione as the redox-cycling chemical, we discovered that this enzymatic reaction blocks metabolic activation of parathion in rat and human liver microsomes and in recombinant CYPs important to parathion metabolism, including CYP1A2, CYP2B6, and CYP3A4. Administration of menadione to rats reduces metabolism of parathion, as well as parathion-induced inhibition of brain cholinesterase activity. This resulted in inhibition of parathion neurotoxicity. Menadione has relatively low toxicity and is approved by the Food and Drug Administration for other indications. Its ability to block parathion metabolism makes it an attractive therapeutic candidate to mitigate parathion-induced neurotoxicity.

Keywords: menadione; organophosphorus pesticides; paraoxon; parathion; redox cycling.

Figure 1

Examples of phosphorothioate pesticides and corresponding oxon metabolites generated following CYP-mediated metabolism.

Figure 2

Redox cycling of menadione suppresses parathion metabolism by the P450 system. NADPH–cytochrome P450 reductase catalyzes the one-electron reduction of menadione, generating a semiquinone radical. Under aerobic conditions, these radicals react rapidly with molecular oxygen to form superoxide anion, regenerating menadione in the process. Redox cycling diverts electrons from CYP-mediated parathion metabolism. This inhibits formation of paraoxon and suppresses parathion toxicity.

Figure 3

Menadione redox cycling inhibits CYP enzyme activities. (A) CYP1A2 mediates menadione redox cycling as measured by the formation of hydrogen peroxide (H₂O₂). H₂O₂ formation was assayed using the Amplex Red assay and is presented as mmol/min/mg protein. (B) Menadione causes a concentration-dependent inhibition of the activities of the major CYPs that mediate parathion metabolism. CYP1A2, CYP2B6, and CYP3A4 are recombinant human CYP enzyme preparations prepared from insect cells coexpressing human NADPH–cytochrome P450 reductase. Substrates used to measure CYP1A2, CYP2B6, and CYP3A4 activity were 7-ethoxyresorufin, 7-ethoxymethyloxy-3-cyanocoumarin, and dibenzylfluorescein, respectively. The data points shown the mean ± SE, n = 3. Summary of data from Ref. .

Figure 4

Menadione redox cycling inhibits parathion metabolism. (A) Human liver microsomes were treated with 20 µM parathion (PT) in the absence and presence of 50 µM menadione. At the indicated times, reactions were assayed for paraoxon. Data are mean ± SE (n= 3). Asterisks show that the formation of paraoxon in the presence of menadione was significantly different (P < 0.05) from that in the absence of menadione. (B) The ability of menadione to reverse the inhibitory effects of parathion on AChE activity in reactions with human liver microsomes. Reactions containing human liver microsomes were supplemented with AChE. Incubations with parathion inhibited AChE activity, and this was reversed by menadione. The figures show that menadione redox cycling readily inhibited paraoxon formation. Each point is the mean ± SE (n = 3). Asterisks show that the inhibition of AChE in the presence of menadione was significantly different (p < 0.05) from that in the absence of menadione. Summary of data from Ref. .