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					                                           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
                                                                                                                                                 Furlong 1,2.

   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
                                                                                                                                                                                                                                                                                                                                The Problem
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
                                                                                                                                                                                                                                                                                                                                                                                                            To CNS
                                                                                                                                                                                                                                                                                                     O   O
                                                                                                                                                                                                                                                                                                         P O P O     CH3       CH3                          O
                                                                                                                                                                                                                                                                         O                               O
                                                                                                                                                                                                                                                                                                                                                      O PP O
                                                                                                                                                                                                                                                                                                                                                      O   O                 CH3
                                                                                                                                                                                                                                                                                                                                                                                                  To CNS
                                                                                                                                                                                                                                                                   CH3                             CH3       O                                              O
                                                                                                                                                                                                                                                                                                                                                                                                                          O P O
                                                                                                                                                                                                                                                                                                    CH3                                             CH2OH

contains tricresyl phosphate (TCP), the                   to TAPs (Fig. 1), when they enter the aircraft cabin via the bleed air
                                                                                                                                                                                                                                                                                                                                                  CH2OH                                                                         O

                                                          due to engine seal malfunction or design problems (Fig. 2). There
                                                                                                                                                                                                                                                                                           Monoorthocresyl phosphate
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
                                                                                                                                                                                                                                                                                                                                                                P O


                                                          aircraft cabin experience a significant exposure to TAPs. A major

used to adulterate ginger extracts. The                                                                                                                                                                                                                                                                              Triaryl phosphate metabolism
                                                                                                                                                                                                                                                                                                                                                                    O                                     Saligenin
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
                                                                                                                                                                                                                                                                                                                                                            C                                             phosphate
                                                                                                                                                                                                                                                                                                                                                                                                          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
                                                                                                                                                                                                                                                                                                                                               To PNS

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. 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 (                 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. 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
                                                                                                                                                                                                                                                                                                             Chymotrypsin digestion
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).