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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

2007 Flight InternationalCrew ManagementConference

Biomarkers of Exposure to TCP

Clement E. Furlong

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

clem@u.washington.edu


2007 flight international crew management conference

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


Why were these cell lines chosen

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.


2007 flight international crew management conference

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

Molecules of Interest

Tricresyl phosphate isomers are

present in jet engine lubricants

The methyl groups can be:

ortho

or para

meta


Why are these isomers of interest a very brief history of tcp exposures

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


2007 flight international crew management conference

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.”


Why are these isomers of interest a very brief history of tcp exposures1

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)


2007 flight international crew management conference

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 exposures2

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


Tricresyl phosphate a toxicant of interest

Tricresyl Phosphate, a Toxicant of Interest

Para

Ortho

Meta

Saligenin cresyl phosphate

Casida J et al. Nature191:1396 (1961)


Other important observations

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.


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

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


2007 flight international crew management conference

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.


Other ways to determine exposure

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


One example of analyzing a protein biomarker as proof of concept

One Example of Analyzing a Protein Biomarker as Proof of Concept


2007 flight international crew management conference

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


Modified protein biomarkers of exposure

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


Modified carboxylesterase peptides

Modified Carboxylesterase Peptides


Searching for useful biomarkers in human blood samples

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


General approach

General Approach

  • Look at all red cell membrane protein peptides for modification


Psp modified peptides

PSP Modified Peptides

  • glycophorin A

    • K.KSPSDVKPLPSPDTDVPLS*.S

    • K.KSPSDVKPLPSPDTDVPLSS*VEIENPETSD.Q

    • K.SPSDVKPLPSPDTDVPLSS*VEIENPETSD.Q


Kspsdvkplpspdtdvplss veienpetsd

KSPSDVKPLPSPDTDVPLSS*VEIENPETSD


What are the physiological consequences of op exposures

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


Design of gene expression experiment

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 )


Exposure analysis 764 885 gene probes slide 28 869 genes

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


2007 flight international crew management conference

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


2007 flight international crew management conference

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)


Fatigo analysis p 0 001 23 genes in tipp10 tipp100 tcp10 or tcp100

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

11 annotated Genes


Research needs

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


Resources collaborators

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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