Biomarkers of organophosphorus (OP) exposures in humans
Highlights
► Tricresyl phosphate (TCP) can enter aircraft cabins and cause aerotoxic syndrome. ► Biomarkers of exposure: butyrylcholinesterase (BChE) and acylpeptide hydrolase (APH). ► One-step purification protocols for BChE and APH using immunomagnetic beads. ► Characterization of the active site serine adducts of BChE by mass spectrometry. ► Detection of cresyl phospho-serine and phospho-serine from in vitro inhibited BChE.
Introduction
Tricresyl phosphates (TCP) are organophosphate (OP) compounds used as anti-wear additives in jet engine lubricants. Their fire resistant properties are also beneficial (Solbu et al., 2007). Commercial TCPs are mixtures of triaryl phosphate (TAP) isomers (De Nola et al., 2008). The tri-ortho isomer (ToCP) was thought to be responsible for the 10–50,000 cases of paralysis resulting from the consumption of TCP-adulterated ethanolic extracts of ginger during Prohibition in the US (Smith et al., 1931). Henschler demonstrated that the mono-ortho isomer of TCP was approximately 10-times more toxic than the ToCP isomer, with the di-ortho isomer exhibiting intermediate toxicity (Henschler, 1958).
Over the past 25 years, there have been numerous reports worldwide of aircrew and passengers reporting short or long-term symptoms such as dizziness, cognitive problems, disorientation and uncontrolled tremors following exposure to oil-contaminated cabin air. These events have been described in a number of publications (Hale and Al-Seffar, 2009, Montgomery et al., 1977, Murawski and Supplee, 2008, Ross, 2008, van Netten and Leung, 2001, Winder, 2006, Winder and Michaelis, 2005, Winder and Balouet, 2002, Winder et al., 2002). The ill health effects have been referred to as aerotoxic syndrome (Balouet and Winder, 1999, Winder and Balouet, 2000).
A major aim of our research is to develop protocols for determining whether an individual has been exposed to TCPs. Aldridge reported that bioactivation by liver was required to convert TCP to highly toxic metabolites (Aldridge, 1954). In 1961, Casida et al. (1961) reported the structure of the bioactivated metabolite of tri-orthocresyl phosphate as 2-(ortho-cresyl)-4H-1,3,2-benzodioxaphosphoran-2-one, also known as cyclic saligenin cresyl phosphate (CBDP). Bioactivated TAPs inhibit many different serine active site enzymes including carboxylesterases (CESs), lipases [e.g., neuropathy target esterase (NTE)], and cholinesterases (Casida and Quistad, 2004, Glynn et al., 1994). The serine at the active site of these enzymes is covalently modified by OPs, and these modifications can be identified by mass spectrometry (MS). The use of OP-labeled enzymes as biomarkers of exposure is useful because unlike the parent OPs, they can remain in circulation for weeks (Kim et al., 2010).
Human butyrylcholinesterase (BChE, accession # P06276) is an 85 kDa tetrameric glycoprotein that is synthesized by the liver and has a 11-day half-life in plasma (Ostergaard et al., 1988). BChE can play an important role in the detoxification of ingested or inhaled OPs by stoichiometrically binding OP molecules (Masson and Lockridge, 2010). Inhibition or modification of BChE is commonly used as a biomarker of OP exposure (Schopfer et al., 2010, Sun and Lynn, 2009, Wieseler et al., 2006).
Acylpeptide hydrolase (APH, accession # P13798) is a serine peptidase that cleaves N-acetylated peptides. The enzyme possesses both acylpeptide and esterase activities and is present in many tissues. APH is localized in red blood cells (RBCs), and has a lifespan of around 120 days. APH was also proposed as a sensitive biomarker of OP exposure (Quistad et al., 2005). OP-modified APH can possibly be detected several weeks following exposure since the half-life of RBCs is ≈33 days (Umlas et al., 1991).
This study is focused on the rapid purification from blood samples of TCP metabolite-modified BChE and APH from in vitro exposed samples.
Section snippets
Sample collection
The samples used for this study came from an institutional review board-approved project investigating organophosphate exposure. Blood samples were collected in lithium heparin tubes. RBCs were separated from plasma by centrifugation at 1285 × g for 10 min at 4 °C. Plasma samples were stored at −80 °C until use. The RBCs were washed 3 times with PBS and then frozen at −80 °C. The experiments reported here made use of blood fractions obtained from non-exposed control subjects. The samples were exposed
Target proteins isolation
One of our aims was to develop protocols for fast and accurate purification of the biomarker proteins BChE and APH. We were able to obtain high levels of purity of BChE and APH in single steps (Fig. 2). The samples obtained from the IMB protocol, when digested and analyzed by MS, provided modified target peptides that were easily identified.
MS analysis of adducted target proteins
The chymotryptic sequence of the BChE active site peptide, GESAGAASVSLH, is shown in Fig. 3a. A peptide database search of the acquired data identified
Discussion
OP-modified enzymes are more stable in the organism than intact OPs, due to the rapid elimination of free OPs. For this reason, new methods to identify and quantify the degree of modification of biomarker proteins need to be developed.
An ideal protein purification/enrichment strategy would be amenable to high-throughput scale-up. Affinity chromatography is currently the most commonly used technique for the isolation of peptides and proteins. The main disadvantages are the large amount of sample
Conclusions
We report here a rapid and efficient method for purifying two protein biomarkers of OP exposure, BChE and APH, and MS protocols for identifying the sequence modifications. These protocols are readily adaptable for automated high-throughput assessment of OP exposures.
Conflict of interest
The authors declare that there are no conflicts of interest.
Acknowledgments
Supported by U.S. Army Medical Research and Materiel Command (W81XWH-07-2-0034), the NIH (U01 NS058056, P30CA36727, R01ES09883, P41 RR011823, and P42ES04696), and funding from Pilot and Flight Attendant Unions, the Royal Australian Air Force, the Norwegian Union of Energy Workers (SAFE), and NYCO S.A. JM was supported by a Beatriu de Pinós postdoctoral fellowship (2008 BP A 00166) from Comissionat per a Universitats i Recerca del Departament d’Innovació, Universitats i Empresa, Catalunya,
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