title: Characterization of human cytochrome P450s involved in the bioactivation of tri-ortho-cresyl phosphate (ToCP).
abstract: Tri-ortho-cresyl phosphate (ToCP) is a multipurpose organophosphorus compound that is neurotoxic and suspected to be involved in aerotoxic syndrome in humans. It has been reported that not ToCP itself but a metabolite of ToCP, namely, 2-(ortho-cresyl)-4H-1,2,3-benzodioxaphosphoran-2-one (CBDP), may be responsible for this effect as it can irreversibly bind to human butyrylcholinesterase (BuChE) and human acetylcholinesterase (AChE). The bioactivation of ToCP into CBDP involves Cytochrome P450s (P450s). However, the individual human P450s responsible for this bioactivation have not been identified yet. In the present study, we aimed to investigate the metabolism of ToCP by different P450s and to determine the inhibitory effect of the in vitro generated ToCP-metabolites on human BuChE and AChE. Human liver microsomes, rat liver microsomes, and recombinant human P450s were used for that purpose. The recombinant P450s 2B6, 2C18, 2D6, 3A4 and 3A5 showed highest activity of ToCP-bioactivation to BuChE-inhibitory metabolites. Inhibition experiments using pooled human liver microsomes indicated that P450 3A4 and 3A5 were mainly involved in human hepatic bioactivation of ToCP. In addition, these experiments indicated a minor role for P450 1A2. Formation of CBDP by in-house expressed recombinant human P450s 1A2 and 3A4 was proven by both LC-MS and GC-MS analysis. When ToCP was incubated with P450 1A2 and 3A4 in the presence of human BuChE, CBDP-BuChE-adducts were detected by LC-MS/MS which were not present in the corresponding control incubations. These results confirmed the role of human P450s 1A2 and 3A4 in ToCP metabolism and demonstrated that CBDP is the metabolite responsible for the BuChE inactivation. Interindividual differences at the level of P450 1A2 and 3A4 might play an important role in the susceptibility of humans in developing neurotoxic effects, such as aerotoxic syndrome, after exposure to ToCP.
authors.affiliation: †Division of Molecular Toxicology, Amsterdam Institute for Molecules Medicines and Systems (AIMMS), Faculty of Sciences, VU University Amsterdam, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands.
authors.forename: Jelle
authors.initials: J
authors.lastname: Reinen
authors.affiliation: †Division of Molecular Toxicology, Amsterdam Institute for Molecules Medicines and Systems (AIMMS), Faculty of Sciences, VU University Amsterdam, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands.
authors.forename: Leyla
authors.initials: L
authors.lastname: Nematollahi
authors.affiliation: ‡Department of CBRN Protection, TNO Technical Sciences, P.O. Box 45, 2280 AA Rijswijk, The Netherlands.
authors.forename: Alex
authors.initials: A
authors.lastname: Fidder
authors.affiliation: †Division of Molecular Toxicology, Amsterdam Institute for Molecules Medicines and Systems (AIMMS), Faculty of Sciences, VU University Amsterdam, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands.
authors.forename: Nico P E
authors.initials: NP
authors.lastname: Vermeulen
authors.affiliation: ‡Department of CBRN Protection, TNO Technical Sciences, P.O. Box 45, 2280 AA Rijswijk, The Netherlands.
authors.forename: Daan
authors.initials: D
authors.lastname: Noort
authors.affiliation: †Division of Molecular Toxicology, Amsterdam Institute for Molecules Medicines and Systems (AIMMS), Faculty of Sciences, VU University Amsterdam, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands.
authors.forename: Jan N M
authors.initials: JN
authors.lastname: Commandeur
mesh_terms: D065767:Activation, Metabolic; D000818:Animals; D002091:Butyrylcholinesterase / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D051381:Rats; D014317:Tritolyl Phosphates / Q000378:metabolism / Q000493:pharmacokinetics* / Q000633:toxicity
chemical_list: D014317:Tritolyl Phosphates; D003577:Cytochrome P-450 Enzyme System; D002091:Butyrylcholinesterase; C025541:tri-o-cresyl phosphate