Reaction of Cresyl Saligenin Phosphate, the Organophosphorus Agent Implicated in Aerotoxic Syndrome, with Human Cholinesterases: Mechanistic Studies Employing Kinetics, Mass Spectrometry, and X-ray Structure Analysis

Abstract

The aerotoxic syndrome is assumed to be caused by exposure to tricresyl phosphate (TCP), an anti-wear additive in jet engine lubricants and hydraulic fluids. CBDP (2-(ortho-cresyl)-4H-1,2,3-benzodioxaphosphoran-2-one) is the toxic metabolite of tri-ortho-cresylphosphate, a component of TCP. Human butyrylcholinesterase (BChE; EC 3.1.1.8) and human acetylcholinesterase (AChE; EC 3.1.1.7) are irreversibly inhibited by CBDP. The bimolecular rate constants of inhibition (ki), determined under pseudo first-order conditions, displayed a biphasic time course of inhibition with ki 1.6×108 M−1min−1 and 2.7×107 M−1min−1 for E and E′ forms of BChE. The inhibition constants for AChE were one to two orders of magnitude slower than for BChE. CBDP-phosphorylated cholinesterases are non-reactivatable due to ultra fast “aging”. Mass spectrometry analysis showed an initial BChE adduct with an added mass of 170 Da from cresylphosphate, followed by dealkylation to a structure with an added mass of 80 Da. Mass spectrometry in 18O–water showed that 18O was incorporated only during the final aging step to form phospho-serine as the final “aged” BChE adduct. The crystal structure of CBDP-inhibited BChE confirmed that the phosphate adduct is the ultimate aging product. CBDP is the first organophosphorus agent that leads to a fully dealkylated phospho-serine BChE adduct.

Detection of cresyl phosphate-modified butyrylcholinesterase in human plasma for chemical exposure associated with aerotoxic syndrome

“…Cresyl saligenin phosphate (CBDP) is highly reactive with human butyrylcholinesterase (BChE), an enzyme in blood that captures cresyl saligenin phosphate and makes a permanent bond with it. The reaction rate of CBDP with BChE is among the fastest known, similar to that with nerve agents [15]. Figure 2 shows that cresyl saligenin phosphate reacts with BChE to make a covalent bond on the active site serine 198.…”

Assessment of neurotoxic effects of tri-cresyl phosphates (TCPs) and cresyl saligenin phosphate (CBDP) using a combination of in vitro techniques

“…In comparison to ToCP, the metabolite CBDP exhibits higher affinity towards NTE and AChE as saligenin cyclic phosphorus esters are among the most potent inhibitors of NTE (Glynn, 1999) and human acetylcholinesterase is irreversibly inhibited by CBDP (Carletti, Schopfer, 2011). Based on these mechanistic difference CBDP should be more toxic than ToCP.…”

“…The different positioning of the methyl group might determine the activity at receptors or the reactivity with enzymes. This may explain why ToCP also via its metabolite CBDP, but not the other isomers, causes OPIDN by NTE inhibition (Johnson, 1990, Johnson andGlynn, 1995), and why it is reactive enough to build adducts with a variety of enzymes (Carletti, Schopfer, 2011, Masson et al , 2013, Schopfer et al , 2010. Further studies are needed to address the question whether ToCP directly interacts with the various glutamate receptors in cortical neurons.…”

“…Clinically, OPIDN is characterized by leg weakness and gain abnormalities, and several epidemiological reports of human poisoning due to ToCP contaminated food and beverage showed that this particular TCP isomer can also cause OPIDN (Crandall, 1931, Morgan, 1982. Experimental studies showed that the metabolism of ToCP in the liver by different enzymes is relevant for its toxicity (Carletti et al , 2011). For example, the ToCP metabolite, cresyl saligenin phosphate (CBDP, see Fig.…”

“…For example, the ToCP metabolite, cresyl saligenin phosphate (CBDP, see Fig. 1D) is generated by cytochrome P450-catalyzed oxidation and serum albumin-catalyzed cyclization (Carletti, Schopfer, 2011). With respect to OPIDN, CBDP has been shown to be a very potent inhibitor of NTE (Glynn, 1999), thus potentially increasing the neurotoxic potency of ToCP.…”