Biomarkers of organophosphorus (OP) exposures in
J Marsillach 1,2, RJ Richter 1,2, JH Kim3, RC Stevens 1,2, MJ MacCoss2, D Tomazela 2, SM Suzuki 1,2, LM Schopfer 4, O Lockridge 4, CE
Univ. of Washington, Departments of 1Medicine (Div. Medical Genetics), 2Genome Sciences, 3Anesthesiology, Seattle, WA, University of Nebraska Medical Center, Omaha, NE
The Problem The Problem
Background / Aims Nature of the Problem
Oxidized by cytochromes P450 by cytochromes P450
There have been a number of events where Triaryl phosphates (TAPs) are added to jet engine lubricants as anti-
Oxidized by cytochromes P450
aircraft engine bleed air has become wear additives. Over the past 20 years, there have been numerous O
O P O CH3 O
O To CNS
contaminated with engine lubricating oil that reports worldwide of air crew and passengers affected by exposure
P O P O CH3 CH3 O
O PP O
O O CH3
CH3 CH3 O O
O P O
contains tricresyl phosphate (TCP), the to TAPs (Fig. 1), when they enter the aircraft cabin via the bleed air
due to engine seal malfunction or design problems (Fig. 2). There
molecule that caused thousands of cases of
Monoorthocresyl phosphate Monoorthocresyl O
paralysis during prohibition when it was has been an ongoing controversy as to whether individuals in the
Saligenin cyclic cresyl
phosphate phosphate C
aircraft cabin experience a significant exposure to TAPs. A major
used to adulterate ginger extracts. The Triaryl phosphate metabolism
bleed air enters the aircraft cabins aim of our research is to develop protocols for determining whether Saligenin cyclic cr
P O CH3 O
To PNS cresyl O
phosphate P O
unfiltered. It has been known since 1954 an individual has been exposed to a TAP. Bioactivated OPs (Fig. 3) Triaryl phosphate metabolism
that the ortho-isomers of TCP are inhibit many different serine active site enzymes including Triaryl phosphate metabolism
metabolized into neurotoxic metabolites. It carboxylesterases (CESs), lipases [e.g. neuropathic target esterase Figure 1. Channel 36 Charlotte News story about a cabin air Figure 2. Origin of the engine oil fumes when the engine seals To PNS
is now known that the bioactivation is (NTE)], and cholinesterases. Plasma butyrylcholinesterase (BChE, incident where the pilot was taken off the plane on a stretcher, fail. When the engine seals fail, it is possible for the TAP-
Figure 3. Bioactivation of tricresylphosphate to the
carried out primarily by liver cytochromes E.C. 220.127.116.11) is an 85 kDa tetrameric glycoprotein that reacts with creating concerns about both the health and safety of passengers containing oil mist to enter the cabin air via a “bleed stream”
highly toxic metabolite saligenin cyclic cresyl phosphate
and air crew (http://www.wcnc.com/home/Plane-with-sick- from the partially compressed engine air. About one-half of the
P450. Since these enzymes vary greatly OPs, forming a covalent bond with the active site serine. Inhibition passengers-had-toxic-fume-incident-before-82127832.html). air supplied to the cabin comes from the bleed air.
among individuals in response to diet and of BChE has been used as a biomarker of OP exposure. Acylpeptide
medications as well as from genetic hydrolase (APH), also known as oxidized protein hydrolase or OPH,
differences, some individuals are much more (E.C. 18.104.22.168) is a serine protease that cleaves N-acetyl peptides. Results
susceptible to neurological damage from APH was proposed as a sensitive biomarker of LP exposure by
TCP exposure. Mass spectrometric protocols Quistad et al. (Toxicol Sci 2005, 86:291).
Target protein isolation MS analysis of adducted target proteins F.GES*AGAASVSLH.L *: CBDP / +80
are being developed to determine the
percentage of “biomarker proteins” that are We have developed single step immunomagnetic bead-based BChE
modified by such exposures. Both plasma protocols for purifying both plasma BChE and red blood cell (RBC)
butyrylcholinesterase (BChE) and red cell APH to a high level of purity. OP inhibited samples have been used -
to identify and characterize the adducted active site peptides. The
O P OH
acylpeptide hydrolase (APH) are readily CBDP O
inhibited by cresyl saligenin cyclic phosphate aim of the MS protocols is to detect and quantify active site serine -49 (x 2 = 98 = H3PO4)
Molecular Formula = HO3P
(CBDP) or phenyl saligenin cyclic phosphate modifications in people affected by this toxic airline syndrome. Monoisotopic Mass = 79.966878 Da
(PSP) and have the potential to provide
information about both the level of exposure
and the P450 status of an exposed Experimental Design and Methods
individual. We have characterized the active Our initial aims were to label candidate biomarker proteins with
site serine adducts and have developed bioactivated (active metabolite form) of specific OP compounds. In Chymotrypsin digestion
immunomagnetic bead-based single step addition to bioactivated TAPs, we also labeled the active site serine F.GES*AGAASVSLH.L *: CBDP / +170
purification protocols for both BChE and of the biomarker proteins with dimethyl and diethyl insecticide
APH. Sensitive individuals have experienced metabolites.
tremors for a number of years following an H3C
exposure. This work was supported by the The second aim was to develop immunomagnetic bead protocols for Figure 5. A: BChE purification. B: APH purified from APH
lysed red cells with the single-step IMB protocol and the 156 Da
U.S. Army Medical Research and Materiel the rapid isolation of the specific biomarker proteins BChE and APH O
phenyl cyclic saligenin phosphate adduct. Abbreviations:
O P O
Command (W81XWH-07-2-0034), the (Fig. 4). MW, molecular weight marker; P, plasma.
(Mike MacCoss) -85 (x 2 = 170 = C7H7O3P) SERINE
National Institutes of Health (U01 The third aim was to identify and characterize the modified serine
Molecular Formula = C7H7O3P
NS058056, P30CA36727, R01ES09883, and
Monoisotopic Mass = 170.013829 Da
active site peptides of the biomarker proteins that had been
P42ES04696), and funding from pilot unions,
flight attendant unions, the Royal Australian
adducted with the different bioactivated (or synthesized) OP Conclusions
metabolites by mass spectrometry (MS). The data were acquired in
Air Force, the Norwegian Union of Energy an LC-MS/MS system nanoAcquity/LTQ-FT. SEQUEST was used for The IMB protocol provides highly pure biomarker proteins for MS analysis.
Workers (SAFE), and NYCO S.A. the databank search using the following dynamic modifications: S
The tricresyl phosphate and phenyl phosphate adducts were as expected.
(Serine): 79.9668 and 170.0138.
References Target BChE has been purified from as little as 100 microliters of plasma, and target APH from 400 microliters or RBCs.
1. Schopfer LM, Furlong CE, Lockridge O. Figure 4. Rapid
Development of diagnostics in the search for an isolation of labeled The aging all the way to phosphoserine was unexpected and to our knowledge has not been observed previously.
explanation of toxic airline syndrome. Anal Biochem. and unlabeled BChE
2010, In press. by the IMB protocol.
From the frequency of cabin air events, it is evident that something should be done to improve the quality of cabin air and reduce
2. Kim JH, Stevens RC, Maccoss MJ, Goodlett DR,
Scherl A, Richter RJ, Suzuki SM, Furlong CE.
the danger of exposure to triaryl phosphates capable of being converted by cytochromes P450 into highly toxic metabolites. These
Identification and characterization of biomarkers of conversions are readily observed in vitro in the laboratory.
organophosphorus exposures in humans. Adv Exp
Med Biol. 2010;660:61-71. These exposures raise concerns about their impact on both health and the flight safety of passengers and crew (Fig. 1 and link).