Exposures to Tri-Aryl Phosphates 2009 GCAQE ANNUAL INFORMATION EXCHANGE AND UPDATE April 28-29, 2009 Clement E. Furlong, Research Professor Departments.

Exposures to Tri-Aryl PhosphatesExposures 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

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 eventWe’ve been working on this issue since then.

Continued exposures emphasize the urgent need for documentation of exposures.

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.

Molecules of Interest

CH3

P

O

O

OCH3

O

CH3

Tricresyl phosphate isomers are

present in jet engine lubricants

The methyl groups can be:The methyl groups can be:

orthoortho metameta or paraor para

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 (Smith et al.)

• 19541954-TOCP has to be converted to toxic metabolite (probably in the liver - Aldridge)

• 19611961- Structure of toxic metabolite (cyclic saligenin phosphate) determined by John Casida

Triaryl phosphate metabolism

O P O

CH3

O

CH3

CH3

O

O P O

O

CH3

CH3

O

CH2OH

O

OO

P

OCH2

CH3

The Problem

Monoorthocresyl phosphate

Oxidized by cytochromes P450

Saligenin cyclic cresyl phosphate

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 timeis at least 3 months according to Dr. Starnes.Thanks for caring,Angie”

X

Need a “Canary Test”

Exposure

Biomarkers of Exposure (Molecular Canaries)

HO

HN

CH3

P

O

O

OCH3

CH3

P

O

O

OCH3

O

CH3

O

X

Modified Protein Biomarkers of Exposure

HO

HN

O

.

CH3

P

O

O

OCH3

CH3

P

O

O

OCH3

O

CH3

Protein Modified Protein

Digest with specific proteases

O

CH3

P

O

O

OCH3

Separate Fragments

CH3

P

O

O

OCH3

To mass spectrometer (Mike MacCoss, Dave Goodlett)

CH3

P

O

O

OCH3

Aged residueAged residue

Un-Aged residueUn-Aged residue

This also holds for insecticide This also holds for insecticide exposuresexposures | | O=P-OEt (unaged) | OEt

|

O=P-OEt (aged) | O-

HO

HN

HO

HN

Modified Carboxylesterase Peptides(Mike MacCoss)

Tricresyl Phosphate, a Toxicant of Interest -

Setting up a Test System

O P O

CH3

O

CH3

CH3

O

O P O

O

CH3

CH3

O

O

OO

P

OCH2

CH3

P450

CH2OHOrtho

Para

Meta

Saligenin cresyl phosphate

Casida J et al. Nature 191:1396 (1961)

Rat Liver Microsomes

Inhibit esterases

(BChE, APH)

Searching for Useful Biomarkers inHuman Blood Samples

Butyryl Cholinesterase

~11 d ½ life

Acetyl Cholinesterase

~35 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

Monocyte Carboxylesterase

0%

20%

40%

60%

80%

100%

0 5 10 15 20 25

Triaryl phosphate (mg/ml)

BC

hE a

ctiv

ity (

% o

f con

trol

)

TOCP (- NADPH)TPP (- NADPH)TOCP (+ NADPH)TPP (+ NADPH)

Molecular Canary Test of P450 Conversion of Pre-toxicant to Toxicant

with Pooled Human Liver Microsomes

Protocols for I solating Biomarker Proteins

Purify Protein

Inject purified protein into: Rabbit, Chicken

Purify antibodies from rabbit serum Purify antibodies from chicken egg yolk

Attach purified antibodies to magnetic beads Use antibody-bead complexes to rapidly purify target biomarker proteins from RBCs or plasma.

Purification of an OP sensitive esterase from RBCs

Inhibition by Organophosphates

0

10

20

30

40

50

aNB

act

ivit

yA

B C

(Rick Stevens, Stephanie Suzuki, Becky Richter & Jerry Kim)

(Acylpeptide hydrolase – D Goodlett et al.)

156 Da

GGS*HGGFISCHLIGQYPETY(Mike MacCoss)

15N-APH has been expressed and purified from E. coli.

Immuno Magnetic Bead Separation (IMS)

Immuno Magnetic Bead Separation (IMS)

BChE

Rapid Isolation of Human RBC-APH

1 2 3 4 5 6

A

B 1 2

Mag Bead Purification of APH from Exposed Individual

Mag Bead Purification of APH from RBC Extracts, Not Exposed and Exposed

Insecticide inhibited

PSP inhibited

N15-E. coli APH

Purified RBC APH

Uninhibited

APH purified from red cells of exposed individual

Ion funnel equipped LTQ (MacCoss lab.)

Increases Sensitivity 6-10X

MS Analysis of IMB-Purified APH

Active site serine

Simplifying the MS Sample with a 2-Step Purification Approach

O

CH3

P

O

O

OCH3

HO

HN

HO

HN

IMB-purify target protein Protease digest target protein

CH3

P

O

O

OCH3

CH3

P

O

O

OCH3

CH3

P

O

O

OCH3

CH3

P

O

O

OCH3

CH3

P

O

O

OCH3

IMB-purify active site peptidewith specific antibody-IMBs

15N

TO MS

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.

Rapid Purification of Mouse RBC-APH with anti-Human APH Mag Beads

1 2 3 Mouse red cell

extractMolecular Weight Standards

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.

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).

Triaryl phosphate metabolism

O P O

CH3

O

CH3

CH3

O

O P O

O

CH3

CH3

O

CH2OH

O

OO

P

OCH2

CH3

Why are some people more sensitive than others?

Monoorthocresyl phosphate

Oxidized by cytochromes P450

Saligenin cyclic cresyl phosphate

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

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

Presentations to Increase Awareness of the Cabin Air IssueFurlong 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.

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.

• Genetics, purification & bacterial expression Rebecca J Richter Stephanie Suzuki

• Rick Stevens Jerry Kim

• Proteomics/Mass Spectometry Rick Stevens, Mike MacCoss Dave Goodlett, Alex Sherl

• P450s Alan 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