2007 Flight International2007 Flight InternationalCrew ManagementCrew Management

ConferenceConference

Biomarkers of Exposure to Biomarkers of Exposure to TCPTCP

Clement E. Furlong

Departments of Medicine (Div. Medical Genetics) & Genome Sciences

clem@u.washington.edu

Status ReportStatus Report

Today, I would like to present a progress report on research related to 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 two of the issues that are important in exploring questions related to exposures in aircraft cabins and flight decks that result from engine exposures in aircraft cabins and flight decks that result from engine seal failure.seal failure.

• Identifying biomarkers of tricresyl phosphate (TCP) Identifying biomarkers of tricresyl phosphate (TCP) exposureexposure

• Understanding the effects of TCP exposure on Understanding the effects of TCP exposure on gene expression in cellsgene expression in cells

- cultured monocytes (sensitive carboxylesterase)- cultured monocytes (sensitive carboxylesterase)- glial cells- glial cells- neurons- neurons Preliminary Data

Why were these cell lines Why were these cell lines chosen?chosen?

• Monocytes were chosen for three reasonsMonocytes were chosen for three reasons

- known effects of OP exposure on the- known effects of OP exposure on the immune system 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 Neuronal and glial cells were chosen as cells of the

nervous system affected by OP exposures.nervous system affected by OP exposures.

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

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

• 1930s1930s -TOCP was identified as the cause of paralysis in Ginger Jake Syndrome

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 gingerfluidextract of ginger in man has been studied and compared with the effects produced by triorthocresyl phosphateeffects 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.”

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)

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

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

Tricresyl Phosphate, a Toxicant of Interest

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. Nature191:1396 (1961)

Other Important ObservationsOther Important ObservationsNeuropathic 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 Acylpeptide Hydrolase as a Sensitive Site for Reaction with Organophosphorus CompoundsOrganophosphorus Compounds and a Potential Target for Cognitive Enhancing Drugs. Mol Pharmacol 58:577–583

Acylpeptide hydrolaseAcylpeptide hydrolase was found to be potently inhibited by the organophosphorus compoundspotently 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 sensitivitysensitivity of acylpeptide hydrolase toward these compounds is between six and tensix and ten times greater than that of times greater than that of acetylcholinesterase (AChEacetylcholinesterase (AChE),…),…

Bottom Line: Many proteins and cellular functions are affected by OP exposures.

How do you know if you have How do you know if you have been exposed to something been exposed to something

harmful?harmful?

One way is to have data from One way is to have data from continuous monitoring of the continuous monitoring of the individual’s environmentindividual’s environment..

While this would be very useful, it isWhile this would be very useful, it is seldom, if ever, done seldom, if ever, done..

Other ways to Determine Other ways to Determine ExposureExposure

• Measure residues on clothing or skinMeasure residues on clothing or skin

• Measure residues in:Measure residues in:- Blood- Blood- Urine - Urine All Short LivedAll Short Lived - Saliva- Saliva

• Analyze proteins modified by exposureAnalyze proteins modified by exposure

One Example of Analyzing a Protein Biomarker as Proof of

Concept

Proof of concept: Use multidimentional protein identification technology (MudPIT) to identify TCP modifications to carboxylesterase (CaE)

Time (secs)

0 50 100 150 2000

0.5

1

1.5

2

Well A4 B4 C4 D4 E4

Vmax 193.07 368.06 10.392 17.986 3.325

R^2 1.000 1.000 0.997 0.998 0.999

Vmax Points = 17

CE (50 or 100 ng)

+ TCP (25 M)

CE 100 ng

CE 50 ng

Time (secs)

0 50 100 150 2000

0.5

1

1.5

2

Well A4 B4 C4 D4 E4

Vmax 193.07 368.06 10.392 17.986 3.325

R^2 1.000 1.000 0.997 0.998 0.999

Vmax Points = 17

CE (50 or 100 ng)

+ TCP (25 M)

CE 100 ng

CE 50 ngCH3

P

O

O

OCH3

O

CH3

Inhibition of Porcine Liver CaE by TCP

CaE

CaEactivesite

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

CH3

P

O

O

OCH3

Aged residueAged residue

Un-Aged residueUn-Aged residue

Modified Carboxylesterase Peptides

Searching for Useful Biomarkers inHuman 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

General ApproachGeneral Approach

• Look at all red cell membrane protein Look at all red cell membrane protein peptides for modificationpeptides for modification

PSP Modified PeptidesPSP Modified Peptides

• glycophorin A – K.KSPSDVKPLPSPDTDVPLS*.S– K.KSPSDVKPLPSPDTDVPLSS*VEIENPETS

D.Q– K.SPSDVKPLPSPDTDVPLSS*VEIENPETSD.

Q

KSPSDVKPLPSPDTDVPLSS*VEIENPETSD

What Are the Physiological What Are the Physiological Consequences of OP Exposures ?Consequences of OP Exposures ?

• One way to examine the effects of OP One way to examine the effects of OP exposures is to quantify the changes in exposures is to quantify the changes in gene expression in the presence and gene expression in the presence and absence of specific OP compounds.absence of specific OP compounds.

- in animals- in animals- in cultured cells- in cultured cells

Design of Gene Expression ExperimentGrow Cells

Expose Cells 48-h

10 ng/ml TCP

100 ng/ml TCP

10 ng/ml TIPP

100 ng/ml TIPP

No OP

No OP

Divide Cells

Extract , label and process RNAsExtract , label and process RNAs

Bind to microarray slides (Affymetrix Whole Genome Arrays )Bind to microarray slides (Affymetrix Whole Genome Arrays )

Cultured human cell linesCultured human cell lines•Immune system CellsImmune system Cells (monocytes)(monocytes)• Neuronal Cells Neuronal Cells • Glial cells Glial cells

Exposure Analysis(764,885 gene probes/slide=28,869 genes)

DATA ANALYSISIncreased expression = red dot

Decreased expression = green dot

2 color array

1 color array

Example of data output from an OP exposurep < 0.01; >2-fold (mouse brain cortex – CPO exposure)

Red signals = increased expression

Green signals = decreased expression

Highly Expressed Genes

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

FatiGO analysis: P<0.001; 23 genes in TIPP10, TIPP100, TCP10, or TCP100

11 annotated Genes

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 Animal and cell culture determinations of effects of

exposure on gene expressionexposure on gene expression

Immediate Needs

• Eliminate or drastically reduce exposuresEliminate or drastically reduce exposures

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