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2007 Flight International Crew Management Conference Biomarkers of Exposure to TCP Clement E. Furlong Departments of Medicine (Div. Medical Genetics) &

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Presentation on theme: "2007 Flight International Crew Management Conference Biomarkers of Exposure to TCP Clement E. Furlong Departments of Medicine (Div. Medical Genetics) &"— Presentation transcript:

1 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

2 Status Report Today, 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. Status Report Today, 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) exposureIdentifying biomarkers of tricresyl phosphate (TCP) exposure Understanding the effects of TCP exposure on gene expression in cells - cultured monocytes (sensitive carboxylesterase) - glial cells - neuronsUnderstanding 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 reasonsMonocytes were chosen for three reasons - known effects of OP exposure on the immune system - known effects of OP exposure on the immune system - they possess an OP sensitive carboxyl esterase - they possess an OP sensitive carboxyl esterase - they are readily accessible with a blood draw - they are readily accessible with a blood draw Neuronal and glial cells were chosen as cells of the nervous system affected by OP exposures.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 meta or para

6 Why are these isomers of interest? A Very Brief History of TCP Exposures 1930s1930s -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) fluidextract of ginger effects produced by triorthocresyl phosphate “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 19301930 -TOCP identified as the cause of paralysis in Ginger Jake Syndrome 19551955 -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 animals 1. 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 19301930 -TOCP identified as the cause of paralysis in Ginger Jake Syndrome 19551955 -TOCP has to be converted to toxic metabolite (probably in the liver) 19611961 -Structure of toxic metabolite determined (cyclic saligenin phosphate) by John Casida

11 Tricresyl Phosphate, a Toxicant of Interest Ortho Para 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 Acylpeptide Hydrolase as a Sensitive Site for Reaction with Organophosphorus Compounds Acylpeptide hydrolasepotently inhibited by the organophosphorus compounds sensitivitysix and ten times greater than that of acetylcholinesterase (AChE ),…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 compounds chlorpyrifosmethyl 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 skinMeasure residues on clothing or skin Measure residues in: - Blood - Urine All Short Lived - SalivaMeasure residues in: - Blood - Urine All Short Lived - Saliva Analyze proteins modified by exposureAnalyze 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) Inhibition of Porcine Liver CaE by TCP CaE active site

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

19 Modified Carboxylesterase Peptides

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

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

22 PSP Modified Peptides glycophorin A –K.KSPSDVKPLPSPDTDVPLS*.S –K.KSPSDVKPLPSPDTDVPLSS*VEIENPETS D.Q –K.SPSDVKPLPSPDTDVPLSS*VEIENPETSD. Q

23 KSPSDVKPLPSPDTDVPLSS*VEI ENPETSD

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

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

27 Example of data output from an OP exposure p 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 individualsProper epidemiological studies of exposed individuals - Pilots - Pilots - Crew - Crew - Passengers - Passengers Methods for documenting and quantifying exposuresMethods for documenting and quantifying exposures Animal model studies of consequences of exposureAnimal model studies of consequences of exposure Animal and cell culture determinations of effects of exposure on gene expressionAnimal and cell culture determinations of effects of exposure on gene expression Immediate Needs Eliminate or drastically reduce exposuresEliminate or drastically reduce exposures

31 Resources/Collaborators 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 Research supported by: RAAF GCAQE Unions & others Special thanks to: David Learmount Operations & Safety Editor Flight International


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