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Biomarkers of organophosphorus (OP) exposures in humans 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 4 The Problem The Problem Background / Aims Nature of the Problem Abstract Oxidized 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 O To CNS contaminated with engine lubricating oil that reports worldwide of air crew and passengers affected by exposure O To CNS O O P O P O CH3 CH3 O O O O PP O O O CH3 CH3 To CNS CH3 CH3 O 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 CH3 CH2OH CH3 due to engine seal malfunction or design problems (Fig. 2). There Monoorthocresyl phosphate molecule that caused thousands of cases of CH3 CH3 CH3 CH Monoorthocresyl phosphate Monoorthocresyl O paralysis during prohibition when it was has been an ongoing controversy as to whether individuals in the O Saligenin cyclic cresyl Monoorthocresyl phosphate phosphate C P O O CH3 phosphate aircraft cabin experience a significant exposure to TAPs. A major H2 used to adulterate ginger extracts. The Triaryl phosphate metabolism O O Saligenin cyclic 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 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 H2 C H2 O 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. 184.108.40.206) 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. (CBDP) 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. 220.127.116.11) 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 - O 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 SERINE (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: - GGS*HGGFISCHLIGQYPETY O P O Command (W81XWH-07-2-0034), the (Fig. 4). MW, molecular weight marker; P, plasma. O (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).
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