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2007 Flight International Crew Management Conference

2007 Flight International Crew Management Conference. Biomarkers of Exposure to TCP Clement E. Furlong Departments of Medicine (Div. Medical Genetics) & Genome Sciences clem@u.washington.edu.

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2007 Flight International Crew Management Conference

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  1. 2007 Flight InternationalCrew ManagementConference Biomarkers of Exposure to TCP Clement E. Furlong Departments of Medicine (Div. Medical Genetics) & Genome Sciences clem@u.washington.edu

  2. Status ReportToday, I would like to present a progress report on research related to two of the issues that are important in exploring questions related to exposures in aircraft cabins and flight decks that result from engine seal failure. • Identifying biomarkers of tricresyl phosphate (TCP) exposure • Understanding the effects of TCP exposure on gene expression in cells- cultured monocytes (sensitive carboxylesterase)- glial cells- neurons Preliminary Data

  3. Why were these cell lines chosen? • Monocytes were chosen for three reasons - known effects of OP exposure on the immune system - they possess an OP sensitive carboxyl esterase - they are readily accessible with a blood draw • Neuronal and glial cells were chosen as cells of the nervous system affected by OP exposures.

  4. Discussions at two conferences on cabin air quality (London – 2005; Boeing, Everett, WA - 2004) pointed to the urgent need for developing a method to determine whether or not an individual had been exposed to toxic organo-phosphorus (OP) compounds (e.g. TCP) during a fume event

  5. Molecules of Interest Tricresyl phosphate isomers are present in jet engine lubricants The methyl groups can be: ortho or para meta

  6. Why are these isomers of interest?A Very Brief History of TCP Exposures • 1930s-TOCP was identified as the cause of paralysis in Ginger Jake Syndrome

  7. THE HISTOPATHOLOGY OF TRIORTHOCRESYL PHOSPHATE POISONING. Smith, ML and Lillie RD. Arch Neurol Pshchiat, Chicago 26:976 (1931) “The histology of the nervous system in paralysis due to adulterated fluidextract of ginger in man has been studied and compared with the effects produced by triorthocresyl phosphate in experimental animals. The results indicate that the multiple neuritis of this paralysis is essentially a degeneration of the myelin sheaths of the peripheral nerves, with a variable amount of relatively moderate central degenerative changes affecting the anterior horn cells throughout the spinal cord, but more often in the lumbar and cervical regions.Essentially similar lesions were observed in experimental animals in which partial paralysis was produced by means of triorthocresyl phosphate.”

  8. Why are these isomers of interest?A Very Brief History of TCP Exposures • 1930-TOCP identified as the cause of paralysis in Ginger Jake Syndrome • 1955-TOCP has to be converted to a toxic metabolite (probably in the liver)

  9. Metabolism of triaryl phosphates in Rodents DK Meyers, JBJ Rebel, C Derger, A Kemp, EGL Simmons Nature, 176:259-260 (1955) “Considerable interest is attached to the metabolism of the compound tri-o-cresyl phosphate, which has been shown to inhibit various esterases in vivo and which is capable of producing demyelination and paralysis in certain species of animals1. Pure tri-o-cresyl phosphate exhibits little inhibitory activity against esterases in vitro and the compound appears to be converted into an active inhibitor in the animal: this conversion can also be effected by incubation with liver slices in vitro. This conversion requires the genetically and environmentally variable cytochrome P450 enzymes This may explain some of the individual variability in sensitivity

  10. Why are these isomers of interest?A Very Brief History of TCP Exposures • 1930-TOCP identified as the cause of paralysis in Ginger Jake Syndrome • 1955-TOCP has to be converted to toxic metabolite (probably in the liver) • 1961-Structure of toxic metabolite determined (cyclic saligenin phosphate) by John Casida

  11. Tricresyl Phosphate, a Toxicant of Interest Para Ortho Meta Saligenin cresyl phosphate Casida J et al. Nature191:1396 (1961)

  12. Other Important Observations Neuropathic target esterase (NTE)-Related References • Johnson, M.K., 1969. The delayed neurotoxic action of some organophosphorus compounds. Identification of the phosphorylation site as an esterase. Biochem. J. 114, 711–717. • Johnson, M.K., 1977. Improved assay of neurotoxic esterase for screening organo-phosphates for delayed neurotoxicity potential. Arch. Toxicol. 37, 113–115. Other important esterases are targets of PSP and CSP • Read DJ et al. 2007. Phospholipase B activity and organophosphorus compound toxicity in cultured neural cells. Toxicology and Applied Pharmacology 219 (2007) 190–195. The study Showed that PSP and CSP (10µ), but not mipafox and phenyl dipentyl- phosphinate—NTE inhibitors—killed differentiated PC12 neuroblastoma cells. “the identity of the target of this action of PSP –– presumably a serine hydrolase –– is clearly of neurobiological interest.i.e. – Other important esterases are targets of PSP and CSP • Richards PG, Johnson MK, and Ray DE. 2000. Identification of Acylpeptide Hydrolase as a Sensitive Site for Reaction with Organophosphorus Compounds and a Potential Target for Cognitive Enhancing Drugs. Mol Pharmacol 58:577–583 Acylpeptide hydrolase was found to be potently inhibited by the organophosphorus compoundschlorpyrifosmethyl oxon, dichlorvos, and diisopropylfluorophosphate (20-min IC50 values of 18.3, 118.7, and 22.5 nM, respectively). The in vitro sensitivity of acylpeptide hydrolase toward these compounds is between six and ten times greater than that of acetylcholinesterase (AChE),… Bottom Line: Many proteins and cellular functions are affected by OP exposures.

  13. How do you know if you have been exposed to something harmful?

  14. One way is to have data from continuous monitoring of the individual’s environment.While this would be very useful, it is seldom, if ever, done.

  15. Other ways to Determine Exposure • Measure residues on clothing or skin • Measure residues in:- Blood- Urine All Short Lived- Saliva • Analyze proteins modified by exposure

  16. One Example of Analyzing a Protein Biomarker as Proof of Concept

  17. Proof of concept: Use multidimentional protein identification technology (MudPIT) to identify TCP modifications to carboxylesterase (CaE) CaE • Inhibition of Porcine Liver CaE by TCP CaE active site

  18. HN HO Modified Protein Biomarkers of Exposure . O Modified Protein Protein Aged residue Digest with specific proteases Un-Aged residue Separate Fragments O To mass spectrometer

  19. Modified Carboxylesterase Peptides

  20. Searching for Useful Biomarkers inHuman Blood Samples Uninhibited +TCP +PSP Uninhibited +TCP +PSP Uninhibited +TCP +PSP Acetyl Cholinesterase ~120 d ½ life Monocyte Carboxylesterase ½ life unknown Butyryl Cholinesterase ~11 d ½ life PSP is a very potent inhibitor of esterases

  21. General Approach • Look at all red cell membrane protein peptides for modification

  22. PSP Modified Peptides • glycophorin A • K.KSPSDVKPLPSPDTDVPLS*.S • K.KSPSDVKPLPSPDTDVPLSS*VEIENPETSD.Q • K.SPSDVKPLPSPDTDVPLSS*VEIENPETSD.Q

  23. KSPSDVKPLPSPDTDVPLSS*VEIENPETSD

  24. What Are the Physiological Consequences of OP Exposures ? • One way to examine the effects of OP exposures is to quantify the changes in gene expression in the presence and absence of specific OP compounds.- in animals - in cultured cells

  25. Grow Cells Design of Gene Expression Experiment • Cultured human cell lines • Immune system Cells • (monocytes) • Neuronal Cells • Glial cells Divide Cells Expose Cells 48-h No OP 10 ng/ml TCP 10 ng/ml TIPP No OP 100 ng/ml TIPP 100 ng/ml TCP Extract , label and process RNAs Bind to microarray slides (Affymetrix Whole Genome Arrays )

  26. Exposure Analysis(764,885 gene probes/slide=28,869 genes) 2 color array DATA ANALYSIS Increased expression = red dot Decreased expression = green dot 1 color array

  27. Example of data output from an OP exposure p < 0.01; >2-fold (mouse brain cortex – CPO exposure) Red signals = increased expression Green signals = decreased expression Highly Expressed Genes

  28. Baseline is Average of Controls Gene changes at both doses (10 ng/ml & 100 ng/ml) = 379 Gene changes at 10 ng/ml for TCP and TIPP = 177 (p value< .01)

  29. FatiGO analysis: P<0.001; 23 genes in TIPP10, TIPP100, TCP10, or TCP100 11 annotated Genes

  30. Research Needs • Proper epidemiological studies of exposed individuals - Pilots - Crew - Passengers • Methods for documenting and quantifying exposures • Animal model studies of consequences of exposure • Animal and cell culture determinations of effects of exposure on gene expression Immediate Needs • Eliminate or drastically reduce exposures

  31. Resources/Collaborators Research supported by: RAAF GCAQE Unions & othersSpecial thanks to: David Learmount Operations & Safety Editor Flight International Primary Laboratory-Principal Investigator Clement E. Furlong Mass Spec Analyses Mike MacCoss Cell cultures / Exposures / RNA preparation Toby B. Cole Sarah Park Chip Analysis Laboratories-UW (RNA labeling / hybridization / scanning) Deborah Nickerson laboratory Joshua Smith (Illumina Chips) Fred Farin (core facility) Theo Bammler (Affymetrix chips) Richard Beyer(Statistics & Analysis) Additional Data analysis (no charge to project) Mette Peters (Rosetta Inpharmatics) Tim Glennon

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