The cyclic organophosphorus agent 2-(o-cresyl)-4H-1,3,2-benzodioxaphosphoran-2-one (CBDP), also called cresyl saligenin phosphate, has been implicated in development of organophosphate-induced delayed neuropathy (OPIDN) (1
). OPIDN is a paralytic condition that is distinct from the toxicity associated with organophosphate inhibition of acetylcholinesterase (AChE) (2
). Well known incidents of OPIDN have been attributed to the consumption of Jamaica ginger, a folk medicine, adulterated with tri-o-cresyl phosphate (TOCP) (3
), and to the consumption of cooking oil adulterated with jet engine oil containing TOCP (4
). The toxicity of TOCP has been attributed to its conversion into CBDP, in vivo
). CBDP is formed in vivo
by two consecutive reactions: 1) liver microsomal cytochrome P450-catalyzed oxidation (6
), and 2) serum albumin-catalyzed cyclization of the oxidation product (7
Metabolic activation of TOCP to CBDP
TOCP is a component of tri-cresyl phosphate (TCP) that is a combination of ten tri-cresyl phosphate isomers (tri-ortho, tri-para, tri-meta, and mixtures of the three). TOCP is more toxic than the para and meta isomers (8
). TCP is used as an anti-wear/extreme pressure agent and flame retardant in jet hydraulic fluids and engine oils (8
). Due to its toxicity, the level of TOCP in commercial TCP mixtures has been reduced over time (8
). However, it is unclear whether this precaution has been sufficient to prevent toxic exposure to TOCP because safe levels of exposure are still in dispute (10
Over the past 30 years, an increasing number of reports have appeared documenting the occurrence of neurological signs associated with air travel, both commercial and military (10
). Short-term symptoms include blurred vision, dizziness, confusion, headache, tremors, nausea, vertigo, shortness of breath, increased heart rate, and irritation of the eyes and nose. Long-term symptoms include memory loss, numbness, lack of co-ordination, sleep disorders, severe headaches, nausea, diarrhea, susceptibility to upper respiratory infection, chest pain, skin blisters, signs of immunosuppression, muscle weakness, muscle pain, and fatigue (10
). The term aerotoxic syndrome has recently been coined to describe this condition (10
). Fumes escaping from the engine through leaky oil seals into the bleed air of the aircraft cabin are suspected to be the source of the toxicants that cause aerotoxic syndrome (8
Toxic components of these fumes include hexane, CO, CO2
and TOCP. TOCP after bio-activation to CBDP is suspected of being responsible for the symptoms. A key gap in the evidence trail between fumes and syndrome is a quantitative biomarker for exposure to TOCP. CBDP has long been known as an inhibitor of carboxylesterases (12
) and of neuropathy target esterase (15
). Animal studies have shown that CBDP is also an irreversible inhibitor of both AChE and BChE (12
). However, in vitro
studies have indicated that CBDP reacts slowly with mammalian ChEs (14
). Modifications to the structure of CBDP have led to the development of insecticides, such as salioxon (2-methoxy-4H-1,3,2-benzodioxaphosphorin 2-oxide), that display strong anti-cholinesterase activity (18
). Recently, we established that human BChE reacts with CBDP. The organophosphorylated adduct undergoes two consecutive dealkylation reactions, i.e. aging, forming an ultimate phosphate adduct on the active site serine (Ser198) (1
). This phosphorylated derivative is unique in the study of organophosphate (OP) reactions with BChE, and therefore would be an ideal candidate for use as a biomarker of exposure to TOCP. Interest in BChE as a biomarker for exposure to TOCP has prompted us to investigate the mechanism and kinetics of the reaction of CBDP with BChE and AChE in more detail.
In the present report, we 1) investigated the kinetics of phosphorylation, inhibition and aging of highly purified human AChE and human BChE by CBDP; 2) examined the chemistry for formation of the post-phosphorylation adducts, using mass spectrometry and 18O-water; and 3) determined the X-ray structure of the ultimate aged conjugate of CBDP-phosphoryated human BChE.
The kinetics for the reaction of CBDP with human BChE indicate that CBDP is one of the most potent OP inhibitors for BChE heretofore discovered, and that post-phosphorylation aging is extremely rapid. Because of this high reactivity, BChE very likely plays a role in protection against toxicity of TOCP by scavenging CBDP from the human blood stream. Both X-ray crystallography and mass spectrometry confirm that the ultimate product from the reaction of CBDP with BChE is a novel phosphorylated adduct of the catalytic serine, Ser198.
Kinetics for the reaction of CBDP with human AChE indicate that AChE is significantly sensitive to CBDP, although less than BChE by at least one order of magnitude.