In vitro detoxification of cyclosarin in human blood pre-incubated ex vivo with recombinant serum paraoxonases

Abstract

An ex vivo protocol was developed to assay the antidotal capacity of rePON1 variants to protect endogenous acetylcholinesterase and butyrylcholinesterase in human whole blood against OP nerve agents. This protocol permitted us to address the relationship between blood rePON1 concentrations, their kinetic parameters, and the level of protection conferred by rePON1 on the cholinesterases in human blood, following a challenge with cyclosarin (GF). The experimental data thus obtained were in good agreement with the predicted percent residual activities of blood cholinesterases calculated on the basis of the rate constants for inhibition of human acetylcholinesterase and butyrylcholinesterase by GF, the concentration of the particular rePON1 variant, and its k(cat)/K(m) value for GF. This protocol thus provides a rapid and reliable ex vivo screening tool for identification of rePON1 bioscavenger candidates suitable for protection of humans against organophosphorus-based toxicants. The results also permitted the refinement of a mathematical model for estimating the efficacious dose of rePON1s variants required for prophylaxis in humans.

Figure 1

Reactions of GF with human whole blood collected with citrate buffer. Panel A: Titration of 1:10 dilution of whole blood in 50 mM sodium phosphate, pH 8.0, with GF. The concentration of GF was determined by titration of a known amount of TcAChE. Shown are titration curves determined after 10 min (□ - □) and 30 min (○ - ○) of incubation of GF with the enzyme. r²>0.97. Panel B: Effect of spiking whole blood with 2 mM CaCl₂ (□ - □) on the rate of hydrolysis of 100 nM GF by rePON1 variant 4E9. Activity is presented as the percentage of residual blood ChE activity. ○ - ○, whole blood without addition of CaCl₂. Data points are average from duplicates with CV<16% .

Figure 2

The proficiency of 3.6 μM 4E9 (Panel A) and 1 μM VIID2 (Panel B) in human whole blood spiked with increasing concentrations of GF. Protection of AChE + BChE is expressed in terms of residual activity of whole blood ChEs, assayed with ATC at t = ∞. Black bars denote % ChE activity without rePON1 pre-treatment. Data point are average from duplicates with CV<16%