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Exposures to Tri-Aryl Phosphates

Exposures to Tri-Aryl Phosphates. 2009 GCAQE ANNUAL INFORMATION EXCHANGE AND UPDATE April 28-29, 2009 Clement E. Furlong, Research Professor Departments of Medicine - Division of Medical Genetics & Genome Sciences.

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Exposures to Tri-Aryl Phosphates

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  1. Exposures to Tri-Aryl Phosphates 2009 GCAQE ANNUAL INFORMATION EXCHANGE AND UPDATE April 28-29, 2009 Clement E. Furlong, Research Professor Departments of Medicine - Division of Medical Genetics & Genome Sciences

  2. Discussions at two conferences on cabin air quality (London – 2005; Boeing, Everett, WA - 2004) pointed to the urgent need for developing molecular methods to determine whether or not an individual has been exposed to toxic organophosphorus (OP) compounds (e.g.,TCP) during a fume event We’ve been working on this issue since then. Continued exposures emphasize the urgent need for documentation of exposures.

  3. The following slide/TV footage is an excerpt from a London Channel 4 News report by Julian Rush, Science Correspondent.It is not to be reproduced or copied without permission from Channel 4 News London. It is presented here with permission.

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

  5. 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 (Smith et al.) • 1954-TOCP has to be converted to toxic metabolite (probably in the liver - Aldridge) • 1961- Structure of toxic metabolite (cyclic saligenin phosphate) determined by John Casida

  6. Oxidized by cytochromes P450 The Problem Monoorthocresyl phosphate Saligenin cyclic cresyl phosphate Triaryl phosphate metabolism

  7. Update on Exposed Individual April 15, 2009, Exposure February 12, 2008 “Hi Clem, Thank you for your concern, it means a lot to me to have people care. Harborview discharged me last Thurs and we came home, I am doing fine just have to take it easy, which isnt an easy task. I still have the tremors in my hands, face and teeth but not the severe ones. I got an e-mail from Susan before I went in for surgery about doing a documentary for Australia, I could do that but my recovery time is at least 3 months according to Dr. Starnes. Thanks for caring, Angie”

  8. Need a “Canary Test” Exposure X

  9. HN HO Biomarkers of Exposure(Molecular Canaries) O X

  10. HN HO Modified Protein Biomarkers of Exposure . O Modified Protein Protein This also holds for insecticide exposures | O=P-OEt (unaged) | OEt | O=P-OEt (aged) | O- Aged residue Digest with specific proteases Un-Aged residue Separate Fragments O To mass spectrometer (Mike MacCoss, Dave Goodlett)

  11. Modified Carboxylesterase Peptides(Mike MacCoss)

  12. Tricresyl Phosphate, a Toxicant of Interest -Setting up a Test System Para Ortho Rat Liver Microsomes Meta Inhibit esterases (BChE, APH) Saligenin cresyl phosphate Casida J et al. Nature 191:1396 (1961)

  13. Searching for Useful Biomarkers inHuman Blood Samples Uninhibited +TCP +PSP Uninhibited +TCP +PSP Uninhibited +TCP +PSP Acetyl Cholinesterase ~35 d ½ life Monocyte Carboxylesterase ½ life unknown Butyryl Cholinesterase ~11 d ½ life PSP is a very potent inhibitor of esterases

  14. Molecular Canary Test of P450 Conversion of Pre-toxicant to Toxicant with Pooled Human Liver Microsomes

  15. 156 Da GGS*HGGFISCHLIGQYPETY(Mike MacCoss) Purification of an OP sensitive esterase from RBCs A B C 15N-APH has been expressed and purified from E. coli. (Acylpeptide hydrolase – D Goodlett et al.) (Rick Stevens, Stephanie Suzuki, Becky Richter & Jerry Kim)

  16. Immuno Magnetic Bead Separation (IMS)

  17. Immuno Magnetic Bead Separation (IMS) BChE

  18. Rapid Isolation of Human RBC-APH N15-E. coli APH Insecticide inhibited Uninhibited APH purified from red cells of exposed individual PSP inhibited Purified RBC APH Mag Bead Purification of APH from RBC Extracts, Not Exposed and Exposed Mag Bead Purification of APH from Exposed Individual

  19. Ion funnel equipped LTQ (MacCoss lab.)Increases Sensitivity 6-10X

  20. MS Analysis of IMB-Purified APH Active site serine

  21. Simplifying the MS Sample with a 2-Step Purification Approach IMB-purify target protein Protease digest target protein 15N IMB-purify active site peptide with specific antibody-IMBs TO MS

  22. Advantages of the two-step protocol • This rapid isolation of the active site peptide will greatly simplify the MS analyses. • It will also shorten the chromatography time from ~90 min to ~10 min. • This approach will also allow for the rapid identification of the properties of the OP modified peptide for both the normal and 15N-standard peptides.

  23. Rapid Purification of Mouse RBC-APH with anti-Human APH Mag Beads Mouse red cell extract Molecular WeightStandards Mag Bead Purified Mouse RBC-APH This experiment means that we now have the capability to examine the effects of tri-aryl phosphate inhalation exposure on APH in brain in a mouse model.

  24. Target Goal A target goal of this research is to develop protocols that will allow the assessment of exposure from a drop of blood. For the APH analysis, the current protocol uses only 0.4 mL of blood. The Neonatal blood drop card will be a good starting point. We have preserved antibodies on the surfaces of biosensors for more than a year by drying under a carbohydrated glass (dextran/trehalose).

  25. Oxidized by cytochromes P450 Why are some people more sensitive than others? Monoorthocresyl phosphate Saligenin cyclic cresyl phosphate Triaryl phosphate metabolism

  26. Where are we? • Identify/Characterize OP sensitive biomarkers-BChE (purified, antibodies generated (rabbit, chicken), IMBs tested-APH (purified, antibodies generated (rabbit), IMBs tested -Cbx (Inhibition verified, use as alternative if necessary)-AChE (Inhibition verified, possible alternative) • MS analysis-APH (ID verified, active site adduct with PSP verified) • BChE 15N-standard-Expressed in E. coli-Partially purified • APH 15N-standard-Expressed in E. coli-Purified

  27. Immediate next steps • Generate anti active site peptide antibodies-BChE -APH • Validate IMB step with peptides • Characterize peptide OP adducts with MS • Test intentionally exposed samples-PSP-Human microsomal generated products from oil additives • Test samples from exposed individuals • Implement 96-well IMB protocols-Manual magnets-Robot system • Initiate blood spot card tests or alternative spot collection impregnated filter paper

  28. Presentations to Increase Awareness of the Cabin Air Issue Furlong CE, Soelberg SD, Chinowsky T, Geiss G, Spinelli CB, Stevens R, Near S, Kauffman P, Yee S. 2005. A surface plasmon resonance (SPR)-based sensor system for real-time monitoring of analytes that range in size from small organics to whole cells. Sensors for the aircraft cabin environment. FAA Center of Excellence for Airliner Cabin Environmental Research. 2005. Workshop on: Sensors for the Aircraft Cabin Environment. Boeing, Everett, WA January 19th & 20th Furlong CE. Biomarkers for exposure to organophosphorus (OP) compounds. Contaminated Air Protection Conference. Imperial College, London, England, April 21 & 22, 2005. Furlong, CE. Biomarkers of Exposure and Sensitivity to Organophosphorus [OP] Compounds. The Annual International Aircraft Cabin Safety Symposium, February 12-15, 2007. Torrance, CA. Furlong, CE. Biomarkers of Exposure and Sensitivity to Organophosphorus [OP] Compounds and Consequences of Exposure. Global Cabin Air Quality Executive Meeting 5&6 March, 2007, University College, London, UK (Invited Speaker) Furlong, CE. NIEHS. Adventures in Research Translation. EHS Core Center Annual Meeting & Translational Research Workshop. Oregon State University, Corvallis, OR. October 16th, 2007 (Invited Speaker) Furlong, CE. Adventures in Research Translation. SBRP 20th Anniversary Celebration, Washington Duke Inn, Durham, NC. Dec. 3-5, 2007. Presentation available at: http://tools.niehs.nih.gov/sbrp/events/index.cfm?id=301 Furlong, CE. Biomarkers of Exposure to TCP. 2007 Flight International Crew Management Conference. Brussels, December 6, 2007. Furlong, CE. Meetings with members of Parliament, London, UK, December 11, 12. Discussions of OP (TCP) exposures in pilots, crew and passengers. See House of Lords Report: http://www.publications.parliament.uk/pa/ld/ldsctech.htm; Written comments provided: http://www.publications.parliament.uk/pa/ld200708/ldselect/ldsctech/7/7we10.htm Furlong CE. Exposures and disease risks associated with genetic variability in human plasma paraoxonase (PON1) and biomarkers of exposure. Environmental and Occupational Health Sciences, Superfund Basic Research Project (SBRP). January 17th, 2008. University of Washington. Furlong CE. Exposures to tricresylphosphate. SBRP-WETP Technical Workshop on Reducing Risk and Protecting Public Health through Research and Training. April 3-4, 2008. NIH, Bethesda, MD. Furlong CE. Exposures to Tricresyl Phosphate. GCAQE Annual Information Exchange and Update. May 20-21, 2008. London, UK. Furlong, CE. Biomarkers of Exposure & Paraoxonase 1 as a Biomarker of Susceptibility to Environmentally-Induced Diseases. ATSDR, Atlanta, GA, October 15, 2008. (Invited Speaker) CE Furlong, M MacCoss, D Goodlett, AC Sherl, TB Cole, RJ Richter, JH Kim. Blood tests for documenting exposures to pyrolyzed jet engine oils. Aerospace Medical Association 80th Annual Scientific Meeting, Westin Bonaventure Hotel, Los Angeles, CA, May 3-7, 2009 Furlong CE. Exposures to Tri Aryl Phosphates. GCAQE Annual Information Exchange and Update. April 28-29, 2008. London, UK.

  29. Other Activities Continued - Grants Requesting Additional Support for Biomarker Research • NIH (NIEHS) RO1 (Pending) • NIH (NIEHS) Super Fund Basic Research Grant (Finding Started April 1 2009). • NIH (NIEHS) Childrens Health Center UW (to be submitted 4/09) Television Interviews/Segments etc. • Channel 4 News, London • http://www.channel4.com/news/articles/world/fears+over+cabin+crew+poisoning/166630 • Channel 5, Seattle • First clip http://www.king5.com/video/?z=y&nvid=221600 • Second clip http://www.king5.com/video/healthlink-index.html?nvid=228629 • Channel 7 News Australia (to air 5/09) Papers published/ in press • Furlong CE, Cole TB, MacCoss M, Richter R, Costa LG. 2005. Biomarkers for Exposure and of Sensitivity to Organophosphorus [OP] Compounds. Proceedings of the BALPA Air Safety and Cabin Air Quality International Aero Industry Conference. Reports in Safety and Environmental Science, School of Safety Science, The University of New South Wales. • Kim JH, Stevens RC, MacCoss MJ, Goodlett DR, Scherl A, Richter RG, Suzuki SM, Furlong CE. Identification and Characterization of Biomarkers of Organophosphorus (OP) Exposures in Humans. In press for inclusion in the volume “Paraoxonases in Inflammation, Infection and Toxicology”. Humana Press.

  30. Genetics, purification & bacterial expressionRebecca J Richter Stephanie Suzuki Rick Stevens Jerry Kim Proteomics/Mass SpectometryRick Stevens, Mike MacCossDave Goodlett, Alex Sherl P450sAlan Rettie, Yvonne Lin Toby Cole, Ken Thummel Acknowledgements Supported by: Pilot and Crew Unions, RAAF, NYCO Special thanks to Tristan Loraine Susan Michaelis, Judith Murawski and Chris Witkowski and the participating crews and pilots

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