Characterization of Human Cytochrome P450s Involved in the

Feb 23, 2015 - Division of Molecular Toxicology, Amsterdam Institute for Molecules Medicines and Systems (AIMMS), Faculty of Sciences, VU. University ...
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Characterization of Human Cytochrome P450s Involved in the Bioactivation of Tri-ortho-cresyl Phosphate (ToCP) Jelle Reinen,† Leyla Nematollahi,† Alex Fidder,‡ Nico P. E. Vermeulen,† Daan Noort,‡ and Jan N. M. Commandeur*,† †

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 ‡ Department of CBRN Protection, TNO Technical Sciences, P.O. Box 45, 2280 AA Rijswijk, The Netherlands 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 ToCPmetabolites 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 GCMS 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.

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INTRODUCTION Tricresyl phosphate (TCP) is a heterogeneous multipurpose mixture of aryl phosphates that has been extensively used in industry as a lubricant, plasticizer, fuel additive, and flameretardant.1,2 Synthesis of commercial TCP involves the reaction of phosphorus oxychloride with industrial cresol, which is a mixture of cresol isomers (ortho-, meta-, and para-cresol). The composition of the initial reaction mixture in combination with the processing conditions eventually determines the isomeric constituents of the final TCP product.2 Tri-ortho-cresyl phosphate (ToCP) is one of the isomers that can be present in TCP. ToCP has been reported to induce immunotoxicity,3,4 testicular toxicity,5−7 and neurotoxicity in animals.8,9 More importantly, it has been shown that ToCP can cause adverse health effects in humans.1,2,10 Various incidents of ToCP poisoning due to accidental contamination of food,11−13 drinks,14−16 or drugs have been reported.15,17 More recently, the presence of ToCP in jet engine oil has been linked to neurotoxic symptoms in air crew members, an illness referred to as “aerotoxic syndrome”.18,19 The adverse health effects related © XXXX American Chemical Society

to ToCP are caused by inhibition of esterase enzymes, most particularly acetylcholinesterase (AChE) and butryrylcholinesterase (BuChE), and neurotoxic esterases.20 Organophosphateinduced delayed neuropathy (OPIDN) is a neuropathological condition of progressive neuronal damage that can be caused by ToCP-mediated inhibition of neurotoxic esterases and may eventually result in the development of paralysis.21 The mechanism by which ToCP exerts its toxic effects in animals has been studied in detail by various groups. A study by Aldridge showed that in rabbit and chicken metabolic activation ToCP was required to inhibit cholinesterases in vitro.22 In the 1960s, Eto et al. demonstrated that the toxic effects of ToCP in rats were actually caused by metabolic conversion into 2-(orthocresyl)-4H-1,2,3-benzodioxaphosphoran-2-one (CBDP); see also Scheme 1.23,24 More recently, a study by Baker et al. reported that in rat liver microsomes conversion of ToCP into CBDP is NADPH-dependent.25 These results clearly show that Received: November 27, 2014

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Measurement of BuChE activity. The activity of BuChE was measured by the method of Ellman.32 Butyrylthiocholine (BTC) iodide and 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) were added to a final concentration of 330 μM and 1 mM, respectively. Kinetic data were acquired for 10 min using a BioTek Powerwave X340 (Bio-Tek instruments, USA) at a wavelength of 405 nm. Only linear reaction rates were used for analyses. Microsomal Bioactivation of ToCP. All incubations had a final volume of 200 μL and were performed in 100 mM potassium phosphate (KPi) buffer at pH 7.4 to which 2.5 mM MgCl2 was added. Rat liver and human liver microsomes were added to a final concentration of 20 and 32 μg/mL, respectively. Stock solutions of ToCP were made in ACN at different concentrations (10 nM−10 mM), then diluted in KPi buffer prior to addition to the reaction mixture. Final ACN concentrations in the incubations were always below 1% (v/v). eBuChE was added to a final concentration of 0.05 U/mL. Reactions were initiated by the addition of an NADPH regenerating system (NRS; final concentrations, 100 μM NADPH, 5 mM glucose-6-phosphate, and 1 U/mL glucose-6phosphate dehydrogenase). Bioactivation proceeded for 1 h at 37 °C after which eBuChE activity was determined. Enzymatic Incubations with Commercial Human P450s. All incubations to evaluate the involvement of recombinant human P450s in the bioactivation of ToCP had a final volume of 200 μL and consisted of KPi containing 2.5 mM MgCl2, 5 μM ToCP, and 0.1 U/mL eBuChE, and final ACN concentrations were always below 1% (v/v). Commercial human P450s (P450 1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4, and 3A5) were added to final concentrations of 5 nM and 500 pM. Reactions were initiated by the addition of NRS and were allowed to proceed for 1 h at 37 °C after which eBuChE activity was determined. The incubations to evaluate the activity of P450 1A2 and 3A4 at varying ToCP concentrations (5 nM to 5 μM) had a final volume of 200 μL and consisted of KPi containing 2.5 mM MgCl2, 0.1 U/mL eBuChE, and final ACN concentrations were always below 1% (v/v). Commercial human P450s 1A2 and 3A4 were added to a final concentration of 10 nM. Reactions were initiated by the addition of NRS and were allowed to proceed for 30 min at 37 °C after which eBuChE activity was determined. Inhibition of ToCP Bioactivation in Incubations with Pooled Human Liver Microsomes and Specific P450 Inhibitors. The contribution of individual P450s in ToCP bioactivation was studied by incubating ToCP with pooled human liver microsomes in the presence or absence of specific inhibitors of individual P450 enzymes. The final concentration of human liver microsomes was 10 μg/mL, and incubations were performed in a total volume of 200 μL of KPi containing 2.5 mM MgCl2. eBuChE was added to a final concentration of 0.1 U/mL. P450-selective inhibitors ketoconazole (KTZ, 2 μM), sulfaphenazole (SUL, 10 μM), quinidine (QUI, 2 μM), ticlopidine (TIC, 5 μM), α-naphtoflavone (αNF, 10 μM), tranylcypromine (TRA, 25 μM), and diethyldithiocarbonate (DDC, 20 μM) were used to investigate the involvement of P450 3A, 2C9, 2D6, 2B6, 1A2, 2A6, and 2E1, respectively. These inhibitors and inhibitor concentrations have been previously shown to offer selective inhibition.33 The concentration of ToCP was 5 μM, and the final concentration of ACN (used for stock solutions of ToCP) was less than 1% (v/v). Incubations containing the mechanism-based inhibitors TRA and DDC were preincubated for 15 min in the presence of NRS before addition of ToCP. For all other inhibitors, reactions were initiated by the addition of NRS. Bioactivation proceeded for 1 h at 37 °C after which eBuChE activity was determined. GC- and LC-MS Analysis of Enzymatic Incubations with Recombinant Human P450s. All incubations were performed in glass reaction tubes, had a final volume of 1 mL, and consisted of KPi containing 2.5 mM MgCl2 and 50 μM ToCP. Recombinant human P450s were added to a final concentration of 100 nM. Reactions were initiated by the addition of NRS and were allowed to proceed for 30 min at 37 °C. Reactions were stopped by placing the tubes on ice. Samples were extracted by vortexing with 2 mL of ethyl acetate for 20 s. Subsequently, samples were centrifuged at 4000g for 5 min to separate phases. About 1.5 mL of the organic layer was freeze-dried using a vacuum centrifuge, and samples were reconstituted in 200 μL of ACN and analyzed by GC-MS and LC-MS. For GC-MS, the extracts were

Scheme 1. Cytochrome P450-Mediated Bioactivation of ToCP to Form CBDP

in animals P450s are involved in the bioactivation of ToCP into its toxic metabolite CBDP. However, until now no studies have been performed that investigated the bioactivation of ToCP in human liver microsomes. Furthermore, until now no detailed study has been performed to identify the specific human P450s that are involved in the metabolic activation of ToCP. Such a study would be very valuable since it is known that amounts and activities of the different P450s vary among individuals and that this variation may have great consequences for the susceptibility to toxic effects caused by ToCP.26 The formed CBDP can react with BuChE to form an organophosphorylated adduct, which undergoes two consecutive hydrolysis reactions which eventually leads to the formation of an ultimate phosphate adduct on the active site serine (Ser198).27,28 Therefore, the aim of the present study was to identify the human P450s which are involved in the hepatic bioactivation of ToCP. The formation of the toxic metabolite CBDP was determined indirectly by assessing the inhibitory potential of metabolic incubations of ToCP using BuChE as a biomarker esterase.25,29 Metabolic incubations were performed with rat liver microsomes, human liver microsomes, and recombinant human P450s. These incubations included experiments with individual recombinant P450s and with pooled human liver microsomes with P450-selective inhibitors. Finally, incubations were performed with recombinant human P450s in the presence of human BChE, and these incubations were analyzed by LC-MS/ MS to demonstrate the formation of P450-mediated CBDPBuChE-adducts.

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MATERIALS AND METHODS

Caution: CBDP and ToCP are highly toxic organophosphorus compounds. Handling requires suitable personal protection, training, and facilities. These requirements are the same as those for other organophosphorus compounds. Chemicals and Enzymes. ToCP (96% purity) was purchased from Acros Organics (Geel, Belgium). CBDP was custom synthesized at TNO and was 98% pure. All working solutions of ToCP and CBDP were made in acetonitrile (ACN) and stored at −80 °C. BuChE from equine serum (eBuChE) was obtained from Sigma-Aldrich (Zwijndrecht, The Netherlands). BuChE from human serum was kindly donated by Dr. Saxena (WRAIR, Silver Spring, MD, USA). Commercially available recombinant human P450 enzymes (Supersomes) were purchased from BD Gentest (San Jose, USA). P450 1A1, 1A2, 1B1, 2C18, and 2D6*1 were coexpressed with human P450 oxidoreductase. P450 2A6, 2B6, 2C8, 2C9*1, 2C19, 2E1, 2J2, 3A4, and 3A5 were coexpressed with human P450 oxidoreductase and cytochrome b5. Pooled human liver microsomes (20 mg/mL) were from Xenotech (lot no. 0710619). Rat liver microsomes were prepared according to the protocol already used in our laboratory.30 The plasmids BMX100/h1A2 and pCWh3A4 with human cytochrome P450 NADPH reductase were kindly donated by Dr. M. Kranendonk (Lisbon, Portugal). Recombinant human P450s 1A2 and 3A4 were expressed and isolated following the protocol previously used in our laboratory.31 All other reagents and chemicals were of analytical grade and purchased from standard commercial suppliers. B

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Chemical Research in Toxicology analyzed using a Shimadzu GC-MS-QP2010 Plus equipped with an AOC-20i autoinjector and an AOC-20S autosampler. Samples were separated on a ZB-1 GC-column (30 m × 0.25 mm; Phenomenex, Amstelveen, The Netherlands). Ionization was done by electron impact (EI) at 70 eV. The injector temperature was maintained at 280 °C. The temperature program for the GC was as follows: 2 min stationary at 80 °C, ramped up at 20 °C min−1 to 300 °C, and maintained at 300 °C for 5 min. For LC-MS, extracts were separated by reversed phase chromatography using a C18 column (Luna C18(2), 5 μm, 4.6 × 150 mm i.d.; Phenomenex, Amstelveen, The Netherlands) at a flow rate of 0.5 mL/min. The gradient was composed of solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in methanol (MeOH)). The gradient was applied as follows: constant at 40% B for 1 min, linear increase from 40% B to 95% B in 19 min, constant at this percentage of B for 5 min, and linear decrease to 40% B in 0.5 min, and the column was allowed to re-equilibrate for 14.5 min at 40% B. Samples were analyzed on an Agilent 1200 Series Rapid resolution LC equipped with a time-offlight (TOF) Agilent 6230 mass spectrometer (Agilent technologies, Waldbronn, Germany). The mass spectrometer was operated at a capillary voltage of 3500 V with nitrogen as drying gas (12 L/min) and nebulizer gas (pressure 60 psig). The gas temperature was 350 °C during operation. The TOF was used in the positive mode, and data were acquired using the Mass Hunter workstation software (version B.06.00). Mass Spectrometry of CBDP-BuChE-Adducts. All incubations had a final volume of 250 μL and consisted of KPi containing 2.5 mM MgCl2, 5 μM ToCP, and 20 U/mL hBuChE, and final ACN concentrations were always below 1% (v/v). Recombinant human P450 1A2, 3A4, human, and rat liver microsomes were added to a final concentration of 100 nM, 100 nM, 0.2 mg/mL, and 0.2 mg/mL, respectively. Reactions were initiated by the addition of NRS and were allowed to proceed for 2 h at 37 °C after which hBuChE activity was determined. Immunomagnetic separation (IMS) of hBuChE from the incubation mixtures was performed according to the method developed by Sporty et al.34 Dynabeads Protein G (4 × 100 μL, 30 mg/mL, Invitrogen, Carlsbad, CA, USA) were washed by vortexing in three aliquots of 200 μL phosphate buffered saline (PBS). After removing the final wash solution, 380 μL of PBS and 20 μL of an antibody solution containing anti-BuChE antibody were added. The beads were incubated in the antibody solution at room temperature overnight (18 h) in an Eppendorf thermomixer (Hamburg, Germany). After removing and discarding the supernatant, the beads were washed with two volumes of 200 μL of a 200 mM triethanolamine buffer. Antibodies were then crosslinked to the beads by incubating at room temperature for 30 min with rotation in 200 μL of dimethyl pimelidate dihydrochloride (DMP) in triethanolamine buffer (0.54 mg/mL). The supernatant was removed, and the beads were washed with three aliquots of 200 μL of tris buffered saline (TBS). Finally, the beads were restored in water (4 × 100 μL). For IMS of BuChE from the incubation mixtures, a Kingfisher mL magnetic processor system from Thermo Scientific (Breda, The Netherlands) was used. Forty microliters of the beads solution was transferred to 250 μL of incubation mixture after which the whole mixture was incubated for 2 h at room temperature with rotation. Beads were then washed in two aliquots of 1 mL of PBS and transferred to a well containing 100 μL of water. For digestion of hBuChE, the beads were resuspended in 75 μL of a pepsine solution (0.25 mg/mL pepsin in 0.63% formic acid) and incubated in a water bath at 37 °C for 1.5 h. After incubation, the supernatant was removed and filtered at 3000 rpm for 60 min using a Millipore MultiScreen Ultracel-10, 10 kDa molecular weight cutoff filter (Fisher Scientific, Fair Lawn, NJ) to remove large peptides and proteins and active pepsin. The analysis was performed on a TSQ Quantum Ultra mass spectrometer (Finnigan, Thermo Electron Corporations, San Jose, USA), an Acquity Sample Manager, and Binary Solvent Manager (Water, Milford, USA). For LC-MS/MS experiments, the liquid chromatograph was connected to the mass spectrometer source via the Sample Manager equipped with a 10 L loop and an Acquity HSS T3 column (1.8 μm particles, 2.1 × 100 mm; Waters, Milford, USA). The liquid chromatography system was run with a 20 min linear gradient from 100% A to 90% B (A: 0.2% formic acid in water; B: 0.2% formic acid in ACN) at a flow rate of 0.1 mL/min. The TSQ Quantum Ultra mass spectrometer was operated with a spray voltage of 3 kV, a skimmer

offset of 5 V, a sheath gas pressure of 30 A.U., aux gas pressure of 0 A.U., and a capillary temperature of 350 °C. Positive electrospray product ion SRM spectra were recorded at an indicated collision energy of 31 to 26 V and using argon as the collision gas at a pressure of 1.5 mTorr.

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RESULTS Optimization of the in Vitro ToCP Bioactivation Assay. Initial experiments to validate the BuChE activity assay were performed using various concentrations of eBuChE in combination with a range of CBDP concentrations in order to determine the inhibitory potential of CBDP. IC50 values of 17.9, 20.0, and 19.6 nM were obtained using 0.025, 0.05, and 0.1 U/ mL eBuChE, respectively. As shown in Figure 1, an increase of

Figure 1. Concentration-dependent inhibition of eBuChE by CBDP measured at three different eBuChE concentrations. Incubations were performed in duplicate for 30 min at 37 °C after which eBuChE activity was determined.

the absorbance was observed at the lower CBDP concentrations when more eBuChE was used. The next step was to test if human and rat liver microsomes could be used to bioactivate ToCP and thereby cause eBuChE inhibition. Rat and human liver microsomes were incubated at different ToCP concentrations with and without the addition of NRS (control) after which the remaining eBuChE activity was determined. As can be seen from Figure 2A, a concentration dependent inactivation of eBuChE was observed. For rat liver microsomes, an IC50 value of 76 ± 17 nM was determined, whereas for human liver microsomes an IC50 value of 710 ± 230 nM was determined. These results demonstrate that the bioactivation assay was suitable to be used for further experiments. During optimization, it was also investigated if CBDP could be further metabolized by rat liver microsomes. For this purpose, a 1 h incubation was performed at 37 °C at various CBDP concentrations. As can be seen from Figure 2B, incubating CBDP with rat liver microsomes has no significant effect on the IC50 value. All determined IC50 values were in the range between 23 and 29 nM. Also addition of the NADPH regenerating system (NRS) did not influence inhibition by CBDP. These results indicate that CBDP is not further metabolized by rat liver microsomes. Bioactivation of ToCP by Commercial Human P450s. In order to get more information about the human P450s involved in the bioactivation of ToCP, commercial recombinant human P450s were incubated in the presence and absence (control) of NRS, and the remaining eBuChE activity was subsequently analyzed. The activities of 14 major P450s, i.e., 1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, 2J2, 3A4, and 3A5 were tested. The experiment was performed at two different P450 concentrations (5 nM and 500 pM), and insect microsomes and microsomes expressing only reductase and C

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Figure 2. (A) Concentration-dependent inhibition of BuChE activity by rat (RLM) and human liver microsomal (HLM) incubations with ToCP. Data are expressed as % of BuChE activity compared to the control where no NADPH regenerating system (NRS) was added. Data represent the mean of duplicate determinations. (B) Concentration-dependent inhibition of eBuChE (0.05 U/mL) by CBDP after preincubation with rat liver microsomes. Incubations were performed in duplicate for 60 min at 37 °C with (RLM + NRS) and without NRS (RLM) after which eBuChE activity was determined. As additional controls, incubations where only NRS was added and incubations with only buffer and CBDP (0) were added were performed.

cytochrome b5 were used as controls. As can be seen from Figure 3, at a high P450 concentration most of the enzymes are capable

Figure 4. Effect of P450-inhibitors on the bioactivation of ToCP by human liver microsomes. P450-selective inhibitors ketoconazole (KTZ, 2 μM), sulfaphenazole (SUL, 10 μM), quinidine (QUI, 2 μM), ticlopidine (TIC, 5 μM), α-naphtoflavone (αNF, 10 μM), tranylcypromine (TRA, 25 μM), and diethyldithiocarbonate (DDC, 20 μM) were used to investigate the involvement of P450 3A, 2C9, 2D6, 2B6, 1A2, 2A6, and 2E1, respectively. Data are expressed as % of inhibition of BuChE activity compared to the control where no NRS was added. Data represent the mean of duplicate determinations.

Figure 3. Evaluation of recombinant human P450s involved in the bioactivation of ToCP. Microsomes expressing reductase and cytochrome b5 (reductase) and control microsomes (insect) are indicated. Data are expressed as % of inhibition of BuChE activity compared to the control where no NRS was added. Data represent the mean of duplicate determinations.

clearly shows that eBuChE inactivation is inhibited when αNF and KET are used as inhibitors, which demonstrates that P450 1A2 and 3A4 play a role in the hepatic bioactivation of ToCP. Additional experiments were performed with recombinant human P450 1A2 and 3A4 to investigate the inactivation of eBuChE at various ToCP concentrations. As shown in Figure 5, for P450 1A2 and 3A4 IC50-values of 1.02 ± 0.25 μM and 0.31 ± 0.02 μM, respectively, were found, suggesting that the more abundant P450 3A4 will probably have the highest contribution in ToCP-bioactivation. GC- and LC-MS Analysis of Enzymatic Incubations with Recombinant Human P450s. In the previous experiments, it was demonstrated that P450 enzymes play a key role in the bioactivation of ToCP and thereby form product(s) that inhibit eBuChE activity. P450 1A2 and 3A4 were shown to be mainly involved in the microsomal biotransformation of ToCP, and as expression systems were available in-house to produce these two enzymes in large quantities (>150 nmol), it was decided to use

of inhibiting eBuChE activity. Only P450 2A6, 2C9, 2E1, and 2J2 display an inhibition below 40%. At a lower P450 concentration, P450 2B6, 2C18, 2D6, 3A4, and 3A5 display the highest inhibition. At a concentration of 100 pM, none of the P450s was capable of inhibiting eBuChE activity (data not shown). These results therefore clearly indicate that P450 2B6, 2C18, 2D6, 3A4, and 3A5 are involved in the bioactivation of ToCP into a metabolite that inhibits eBuChE activity. Effect of Isoenzyme-Selective Inhibitors on the Bioactivation of ToCP by Pooled Human Liver Microsomes. Figure 4 shows the effect of the enzyme-selective inhibitors on the bioactivation of ToCP by pooled human liver microsomes. The results obtained are expressed as % of control incubation where no NRS was added. Figure 4 also shows the control incubation in which no inhibitor was added. When compared to the control incubation without inhibitor, the incubations in which 2 μM KET and 10 μM αNF were added display a significant decrease in eBuChE inhibition. Figure 4 D

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ion traces of CBDP (m/z 276.1) from ToCP incubations performed with recombinant 100 nM P450 3A4. Two peaks (at 11.10 and 12.51 min) are present in the incubation where NRS was added, while in the control incubation only the peak with a retention time of 12.51 min is present. The corresponding mass spectra of both peaks are also depicted. Reference standards of ToCP and CBDP were analyzed by GC-MS (data not shown). The ToCP reference standard eluted at 12.51 min and displayed a mass spectrum similar to the one depicted in Figure 7C, which corresponds very well with the mass spectrum of ToCP described by De Nola et al.18 The CBDP reference standard eluted at 11.10 min and displayed a mass spectrum similar to the one depicted in Figure 7B. It was therefore confirmed that the product formed in the incubation was CBDP. Incubations for longer time frames (60 and 90 min) were also performed, and in these incubations, the amount of CBDP was below the detection level in all cases. This observation might be due to the instability of CBDP in buffer. The LC- and GC-MS analyses of the incubations with human recombinant P450 1A2 showed results similar to those of the P450 3A4 incubations. It was observed that CBDP was formed, and the amount of product was similar to that in the P450 3A4 incubations. On the basis of the CBDP reference standards, in an incubation containing 5 μM ToCP and 100 nM P450, ∼360 and ∼630 nM CBDP is formed by P450 1A2 and 3A4, respectively. In the current study, the generated LC- and GC-MS traces were also analyzed for the presence of the intermediate metabolite hydroxymethyl-ToCP (or di-o-cresyl mono-o-hydroxymethylphenyl phosphate)23 by extracting the expected molecular ion. However, the formation of hydroxymethyl-ToCP could not be detected by either LC-MS or GCMS. In Vitro Generation of CBDP-hBuChE Adducts by Recombinant Human P450s. It has been confirmed in the previous experiments that P450 1A2 and P450 3A4 can bioactivate ToCP into CBDP which causes inhibition of eBuChE. In this experiment, it was investigated if P450-mediated bioactivation of ToCP resulted in the formation of CBDPhBuChE adducts by LC-MS/MS. ToCP incubations were performed with rat and human liver microsomes, human recombinant P450 1A2 and 3A4 in the presence of hBuChE. As a control, incubations in which no NRS was added were

Figure 5. Concentration-dependent inhibition of BuChE activity by commercial human P450 1A2 and 3A4 with ToCP. Data are expressed as % of BuChE inhibition compared to the control where no NADPH regenerating system (NRS) was added. Incubations were performed in duplicate for 30 min at 37 °C after which eBuChE activity was determined.

human recombinant P450 1A2 and 3A4 to identify the products formed during ToCP bioactivation. Experiments were performed in the presence and absence (control) of NRS, and the incubations were analyzed by both LC-MS and GC-MS. The results of the analysis of the incubations with 100 nM P450 3A4 are depicted in Figure 6, which shows the ion trace of the product found with the corresponding mass trace. It can be seen in this figure that in the incubation with NRS a peak elutes at 16.194 min, which is not present in the control incubation. This peak shows two clear molecular ions in the corresponding mass spectrum: one molecular ion with an m/z value of 277.0630 and a molecular ion with an m/z value of 299.0450. The difference between these m/z values is 21.982, which indicates that the second molecular ion is an in-source sodium adduct of the first ion. On the basis of literature, at least one of the expected products should be CBDP (calculated m/z value of 277.0624).23 Analysis of CBDP reference standards using identical analytical conditions resulted in a chromatogram with a peak eluting at the same retention time, and the corresponding mass spectrum showed two molecular ions with m/z values of 277.0639 and 299.0461. It was therefore concluded that the product formed in the incubation was CBDP. Figure 7A depicts extracted GC-MS

Figure 6. Extracted LC-MS ion traces of CBDP (m/z 277.06) from ToCP incubations performed with recombinant P450 3A4. The black trace shows a complete incubation (30 min at 37 °C; 100 nM P450 3A4), whereas the gray trace shows a control incubation where no NRS was added. The inset shows the mass spectrum of the peak at 16.194 min corresponding to CBDP (277,0630 [M + H]+ and 299,0450 [M + Na]+), which was formed during the incubation. E

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Figure 7. (A) Extracted GC-MS ion traces of CBDP (m/z 276.1) from ToCP incubations performed with recombinant P450 3A4. The black trace shows a complete incubation (30 min at 37 °C; 100 nM P450 3A4), whereas the gray trace shows a control incubation where no NRS was added. Insert (B) depicts the electron-impact mass spectrum of the peak at 11.100 min which indicates that CBDP was formed during the incubation. Insert (C) depicts the electron-impact mass spectrum of the peak at 12.515 min corresponding to residual ToCP at the end of the incubation.

53000) is much higher than the intensity of the saligeninphospho-hBuChE adduct (Figure 8D; approximately 4450). This suggests that during the aging reactions, the release of saligenin mainly precedes the release of the o-cresyl moiety. It can, however, also imply that the stability of the saligeninphospho-hBuChE adduct is lower, which makes it more difficult to detect. Overall, the results in Table 1 show that rat and human liver microsomes can bioactivate ToCP into CBDP, which can covalently bind to the active site serine (Ser198) of hBuChE. More importantly, these results confirm that P450 1A2 and 3A4 are involved in the bioactivation of ToCP to ultimately form CBDP-hBuChE adducts.

performed. Subsequently, the remaining hBuChE in the different incubations was measured after which hBuChE was isolated and digested using IMS.34 The obtained protein adducts were then analyzed by LC-MS/MS. The results of the LC-MS/MS analyses and the hBuChE activity measurements are listed in Table 1. The Table 1. P450-Mediated Formation of CBDP-BuChEAdducts

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DISCUSSION It has been shown in various studies that the neurotoxic effects related to ToCP are not caused by the compound itself but are a result of metabolic bioactivation.22−25 CBDP has been proposed as the metabolite responsible for the neurotoxic effects as it has been shown that this compound can covalently bind to inhibit AChE and BuChE.35 It has been stated by among others Eto et al. that ToCP is metabolized by liver microsomal P450s and serum albumin into CBDP.23,24,36 Baker et al. reported that in rat liver microsomes ToCP bioactivation is inhibited by naringenin.25 They hypothesized that P450s 19, 2C9, 2C19, and 3A might be involved. However, since this study was performed with rat rather than human liver microsomes and because naringenin is not considered to be a very selective P450 inhibitor, these results are not conclusive with respect to the human P450s involved in ToCP bioactivation. The aim of the present study therefore was to study the human P450-mediated bioactivation of ToCP in more detail. The first step of this study was to develop an assay to measure the P450-mediated biotransformation of ToCP into CBDP. Experiments were performed to investigate the stability of CBDP in water, ACN, and KPi buffer (data not shown), and it was found that in buffer the amount of CBDP was decreased by more than 90% after 30 min. This observation is in agreement with the findings by Carletti et al., who have reported that CBDP is unstable in water.27 During these experiments, it was also observed that it was very difficult to detect CBPD both by LC-

a

For each condition, the BuChE activity of the corresponding control (incubation without NRS) was set at 100%. bFor CBDP, the sample containing only ToCP and hBuChE was used as the control. cRLM: incubation with rat liver microsomes. dHLM: incubation with human liver microsomes.

LC-MS/MS results for rhP450 3A4 are shown in Figure 8. Figure 8B shows two products with molecular ion M − H+ 876.3 Da, which corresponds to the phosphorylated peptide FGESAGAAS, which probably result from the sequential hydrolysis reactions shown in Scheme 2. However, the peak at 8.54 min is also formed in the absence of NRS and apparently results from an as yet unidentified non-P450 dependent pathway. As shown in Figure 8D, two peaks with molecular ion 982.3 Da were detected in the incubation with rhP450 3A4, which were not present in the incubation in the absence of NRS. The peak at 8.08 min was not present in the incubation of hBuChE with CBDP and did not show the presence of all three fragment ions (data not shown). It has to be noted that the intensity of the ocresyl-phospho-hBuChE adduct (Figure 8C; approximately F

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Figure 8. (A) Total ion chromatographic traces from ToCP incubations performed with recombinant P450 3A4 in the presence of hBuChE as analyzed by LC-MS/MS in SRM mode (fragments at 602.3−673.3−778.3). The black trace shows a complete incubation (2 h at 37 °C; 100 nM P450 3A4), whereas the gray trace shows a control incubation where no NADPH regenerating system (NRS) was added. Panels B, C, and D display the corresponding extracted ion traces of the phospho-hBuChE adduct (M − H+: 876.3), the o-cresyl-phospho-hBuChE adduct (M − H+: 966.3), and the saligenin-phospho-hBuChE adduct (M − H+: 982.3), respectively.

CBDP in buffer. The IC50 value found for rat liver microsomal incubations during these experiments is lower than the one reported by Baker et al. (∼330 nM).25 Possible explanations for this discrepancy are that Baker et al. used phenobarbital-induced rat liver microsomes (whereas in this study noninduced rat liver microsomes were used), they incubated at a lower temperature (25 instead of 37 °C), and they used human BuChE which they added only after 25 min to the bioactivation mixture. After optimization of the in vitro ToCP bioactivation assay, a set of recombinant human P450s was screened, and it was found that P450s 2B6, 2C18, 2D6, 3A4, and 3A5 were most active at a P450 concentration of 500 pM. Second, human liver microsomes were incubated with P450-specific inhibitors to determine which inhibitors were able to cause a decrease in BuChE inhibition. It was found that both KET and αNF caused significant BuChE inhibition. KET is a very selective inhibitor of P450 3A when

and GC-MS. It was therefore decided to use a bioactivation assay based on the measurement of BuChE activity to indirectly detect P450-mediated formation of CBDP. First, a BuChE activity assay was set up and validated by measuring the inhibitory potential of CBDP over a range of concentrations. The IC50 values obtained during these experiments (17.9−20.0 nM) corresponded very well with the value of 25 nM as was reported by Baker et al.25 Second, a bioactivation assay was developed which was mainly based on the protocol described by Baker et al.25 However, it was decided to perform bioactivation at 37 °C (instead of 25 °C) and to add eBuChE from the start of the incubation (instead of adding it after 25 min of bioactivation). The temperature was increased to 37 °C to better mimic the human and rat in vivo situation, whereas eBuChE was added from the start of the incubation in order to trap the formed CBDP as efficiently as possible to overcome the problems related to the poor stability of G

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Chemical Research in Toxicology Scheme 2. Products formed by Reaction of CBDP with Serine 198 of hBuChEa

a Masses (M − H+) refer to the expected molecular ions of modified peptide FGESAGAAS, formed after the digestion of modified BuChE with pepsin.

and 3A4 toward ToCP by performing incubations with recombinant human P450s at varying substrate concentrations. As shown in Figure 5, P450 3A4 appears to have a higher affinity because half maximal inhibition was found at 0.31 μM ToCP, whereas half maximal inhibition by P450 1A2 was found at 1.02 μM ToCP. The identification of the roles of P450 1A2 and 3A4/ 3A5 in ToCP bioactivation is highly relevant as it is known that differences in levels of these P450s can cause significant variations in susceptibility between individuals for toxic effects caused by P450-mediated bioactivation of xenobiotics.40,41 Genetic polymorphisms have been reported for P450s 1A2, 3A4, and 3A5,41 and these can lead to a variation in the expression and activity of these enzymes. In addition, it has been reported that the expression of the P450 1A2 and 3A4/3A5 genes is highly inducible by numerous xenobiotics.41 These factors have to be certainly taken into account when evaluating the susceptibility of individuals to ToCP for deleterious health effects such as the aerotoxic syndrome. Exposure to ToCP can occur by various routes, i.e., by oral ingestion, vapor inhalation, or absorption through the skin. Two human P450s (i.e., 2B6 and 3A5) that were found to be involved in ToCP bioactivation are known to be present in the skin.42 It would be interesting to investigate the P450-mediated ToCP metabolism in the skin in more detail to establish if this could possibly also be a route of exposure that needs to be considered for risk assessment. Furthermore, it has been demonstrated in this study that rat and human P450s play an important role in the bioactivation of ToCP. As already mentioned earlier, ToCP is one of the isomers that can be formed during commercial synthesis of TCP.2 During this synthesis, also other isomers containing o-cresol are formed, and it has been reported that the three mono-o-cresyl isomers of TCP and the two di-o-cresyl phosphate species are regarded as being 10 times and 5 times, respectively, more toxic than ToCP.43 It has not been clarified if bioactivation is required for this toxicity, and it would be very valuable to investigate this in more detail using a similar approach as presented in this study. The mechanism by which CBDP inhibits hBuChE has been extensively studied and proceeds through a three-step reaction mechanism.27,28,35 During the first step, the active site serine of hBuChE (Ser198) is organophosphorylated to form the ringopened CBDP-hBuChE adduct. The organophosphorylated adduct then undergoes so-called aging by two consecutive hydrolysis reactions resulting in the formation of the ultimate phospho-hBuChE adduct. The reaction of CBDP with hBuChE is summarized in Scheme 2. Theoretically, four different hBuChE

used at low micromolar concentrations, and the results thus showed that P450 3A enzymes play a role in hepatic bioactivation of ToCP. Four members of the P450 3A subfamily have been described in humans (P450 3A4, 3A5, 3A7, and 3A43),37 and P450 3A enzymes account for almost 30% of the total amount of P450s present in human liver.38 P450 3A4 is the most abundant P450 3A isoform, followed by P450 3A5,39 which is a polymorphic enzyme and in many cases metabolizes the same substrates as P450 3A4 does, although at slower rates. In the human liver, P450 3A4 and 3A5 cannot be readily distinguished from each other by specific inhibition, which makes it very difficult to determine the individual contribution of the two isoforms to the hepatic bioactivation of ToCP. αNF is a potent inhibitor of P450 1A2, which accounts for almost 13% of the total amount of P450s present in the human liver,38 and has good selectivity against other major P450s. αNF can also inhibit P450 1A1 and 1B1 at submicromolar concentrations, but in this experiment with human liver microsomes, this is not a problem as both these enzymes are extrahepatic.23 From the latter screening, it was thus concluded that P450 1A2 and 3A4/3A5 are mainly involved in hepatic biotransformation of ToCP. Addition of specific inhibitors for P450 2B6 (TIC) and 2D6 (QUI) to human liver microsomal incubations did not lead to a significant decrease of BuChE inhibition. The experiment with human liver microsomes was performed at a concentration of 10 μg/mL protein. On the basis of the study of Shimada et al., the average P450 concentrations in the human liver microsomal incubations are approximately 420, 10, 50, and 960 pM for P450 1A2, 2B6, 2D6, and 3A4, respectively.38 With the commercial human P450s, we observed that none of the P450s was active at a concentration below 100 pM. The fact that P450 2B6 and 2D6 were not found active in hepatic biotransformation of ToCP is therefore explainable. Using a higher human liver microsomal concentration during the experiment was not an option as the increased activity of P450 3A4 and 1A2 probably would have overcompensated for the loss of metabolic activity due to the inhibition of P450 2B6 and 2D6. It has previously been suggested that plasma albumin acts as a catalyst in the cyclization of diaryl o-(o-hydroxy)tolyl phosphates.36 Therefore, the effect of human serum albumin (HSA) on CBDP formation has been investigated as well (data not shown). It was found that P450 1A2 and 3A4 were capable of forming CBDP in the absence of HSA and that the presence of HSA did not affect CBDP formation. It was therefore concluded that HSA is not required for ToCP bioactivation. An additional experiment was performed to evaluate the affinity of P450 1A2 H

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used in animal studies to investigate the effects of P450 induction on the formation of hBuChE-adducts. In conclusion, the results in the present study show that the human P450s 2B6, 2C18, 2D6, 1A2, and 3A4/3A5 are involved in the bioactivation of ToCP. P450 1A2 and 3A4/3A5 are mainly involved in the microsomal metabolism of ToCP, and CBDP is the hP450-mediated metabolite responsible for hBuChE inactivation. Furthermore, a novel method to monitor P450mediated formation of CBDP-hBuChE-adducts by in situ trapping of the hBuChE-adducts formed has been presented, and this method clearly showed that P450 1A2 and 3A4 are involved in the generation of the reactive CBDP intermediate. Interindividual differences at the level of P450 1A2 and P450 3A4, caused by genetic or environmental factors, might be important factors which determine the susceptibility of humans in developing aerotoxic syndrome after exposure to ToCP.

adducts can be formed, i.e., the ring-opened CBDP-hBuChE adduct (M-H+: 1056.3), the o-cresyl-phospho-hBuChE adduct (M − H+: 966.3), the saligenin-phospho-hBuChE adduct (M − H+: 982.3), and the phospho-hBuChE adduct (M − H+: 876.3). During LC-MS/MS analysis of the protein digests (FGES*AGAAS, where the asterisk indicates the serine to which the CBDP-adducts are bound), it was decided to check for the presence of all four of these adducts. The presence of the ringopened CBDP-hBuChE adduct was not detected in any of the samples. This is in agreement with the findings of Carletti et al. and Schopfer et al., who also have been unable to detect this adduct and reported that this adduct probably decays faster than it forms.28,35 The presence of the other three adducts was confirmed in the incubations with rat and human liver microsomes, rhP450 1A2, and rhP450 3A4 to which NRS was added and in the control incubation with CBDP. In the mechanistic study by Carletti et al., it was suggested that during the aging reactions release of saligenin always precedes the release of the o-cresyl moiety.27 The identification of the saligenin-phospho-hBuChE adduct (Figure 8D) indicates that this can also occur the other way around (release of the o-cresyl moiety precedes the release of saligenin) and agrees with the results of the study by Schopfer et al.28 In the present study, three approaches have been used to detect exposure to the organosphosphate ToCP. The first method employed measurement of the decrease in BuChE activity after in vitro bioactivation of ToCP by rat liver microsomes, human liver microsomes, and recombinant human P450s. Although similar approaches are widely used, the method has some drawbacks. The exact identity of the compound responsible for the inhibition cannot be determined, while the assay format is also less suitable for retrospective exposure detection.44 The second approach used is based on the analysis of formed hydrolysis products after bioactivation, and it was shown that ToCP was metabolized into CBDP by recombinant human P450s. A major drawback of this method is the poor stability of the formed CBDP, which was subsequently difficult to detect accurately by both GC- and LC-MS. This drawback also limits the use of this approach for retrospective detection of exposure. It has been shown in various studies that phosphorylated BuChE is one of the most important biomarkers to verify a retrospective exposure to xenobiotic organophosphates such as pesticides45 and chemical warfare agents.46 The third approach used in this study is therefore based on LC-MS/MS analysis of the peptide containing the active site serine that results after pepsin digestion of BuChE isolated from plasma.34 This method enables analysis of both nonaged and aged phosphyl moieties bound to serine and provides a more detailed insight in the origin of the formed adducts.47 Recently, several groups have reported the development of assays for the detection of CBDP-hBuChE adducts in human plasma for chemical exposure associated with aerotoxic syndrome based on the fourth approach.28,48,49 In the present study, the method of Sporty et al. to detect the formation of hBuChE-adducts was adapted to demonstrate for the first time that P450-mediated metabolism of ToCP in the presence of hBuChE leads to CBDPadduct formation.34 It would be very valuable to use a similar approach to study plasma samples obtained from flight crew members who might have been exposed to ToCP in order to investigate if there exists a link between the amounts of the phosphorylated hBuChE-adducts found and the levels of hP450s present in the study objects. Additionally, the method can be

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AUTHOR INFORMATION

Corresponding Author

*Phone: +31 205987595. Fax: +31 205987610. E-mail: j.n.m. [email protected]. Funding

This study was financed by the Department of Chemistry and Pharmaceutical Sciences of the Faculty of Sciences, VU University Amsterdam. Notes

The authors declare no competing financial interest.

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ABBREVIATIONS AChE, acetylcholinesterase; ACN, acetonitrile; αNF, α-naphtoflavone; BTC, butyrylthiocholine; BuChE, butyrylcholinesterase; CBDP, 2-(ortho-cresyl)-4H-1,2,3-benzodioxaphosphoran-2one; P450, cytochrome P450; DDC, diethyldithiocarbonate; DMP, dimethyl pimelidate dihydrochloride; DTNB, 5,5′dithiobis(2-nitrobenzoic acid); HLM, human liver microsomes; HSA, human serum albumin; IMS, immunomagnetic separation; KPi, potassium phosphate; KTZ, ketoconazole; MeOH, methanol; NRS, NADPH regenerating system; OPIDN, organophosphate-induced delayed neuropathy; QUI, quinidine; RLM, rat liver microsomes; SUL, sulfaphenzaole; TBS, tris buffered saline; TCP, tricresyl phosphate; TIC, ticlopidine; ToCP, triortho-cresyl phosphate; TRA, tranylcypromine

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