Molecular Responses of the Skin to JP8 and Some Components

1
Molecular Responses of the Skin to JP-8 and Some
Components

• James N. McDougal
• Wright State University
• Tucson AZ, January 2007

2
Overview

• JP-8 concentration in epidermis
• Time course of gene expression with JP-8
• Component concentrations in skin
• Gene Expression comparison of pure components to
  JP-8
• Undecane, Tetradecane, Dimethyl naphthalenes,
  Trimethyl benzene

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Patch and Harness
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Exposure Protocol
Skin Samples
Skin exposure
Time (hours)
0
1
4
6
8
(5 treated rats and 5 sham rats at each time
point)

• Carefully clip fur on rats back
• Apply chemicals (500 µl) to back in a HillTop
  chamber (2.5 cm diameter)
• Secure with Lomir rodent harness
• Euthanize rat, excise skin, take biopsy punches,
  snap freeze
• Isolate epidermis using cryotome

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Epidermis
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JP-8 Concentrations in Epidermis
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Heatmap
1 hour
4 hours
8 hours
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Principal Component Analysis
4-hr
8-hr
1-hr
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Genes Changed
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Ingenuity Pathways Analysis

• Commercial software application to help identify
  biological mechanisms, pathways and functions of
  genes
• Curated by scientists unlike other pathway
  programs that mine PubMed for relationships
  between key terms
• Web-based
• Updated a couple of times a year
• Drug information (includes phase I II
  candidates)

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Signaling Pathways 1hr
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Signaling Pathways 4hr
13
Metabolic Pathways 8hr

• No signaling pathways significantly changed
• 16 metabolic pathways significantly changed
• All pathways had more genes decreased than
  increased except
• Sterol Biosynthesis
• Synthesis and degradation of ketone bodies

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Genes in Common
1 hour
4 hour
1 hour
4 hour
7
1
58
68
9
26
27
1
3
72
14
82
204
397
8 hour
8 hour
INCREASED
DECREASED
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27 Upregulated Genes All Times
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Conclusions

• Magnitude of change in gene expression increases
  with time after the 1-hour exposure
• Specific signaling pathways can be identified
  (i.e. inflammation, apoptosis, growth and
  proliferation)
• Trigger may be physical disruption of membranes
  and oxidative or osmotic balance

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Trigger?
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Can specific components of JP-8 be identified
that are responsible for the early changes in
gene expression?
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Literature

• Lower boiling hydrocarbons more irritating than
  higher boiling
• Aromatic components more irritating than
  aliphatic
• Aromatic reactivity increases with the number of
  rings
• Tetradecane seems to be the most irritating
  alkane over a period of days

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Component Methods

• 1-hr Exposure to NEAT chemicals and JP-8 for
  comparison with sham treated
• Skin samples (epidermis) taken only at the end of
  the 1-hr exposure
• Analytical measurements using Gas
  Chromatography/Mass spectrometry

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Component Characteristics
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Epidermal Concentrations
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Gene Expression Methods

• GeneChip RG 230-2 (31,099 genes) vs RGU-34A
  (8,798 genes)
• Normalization Robust Multichip Average (RMA) vs
  Affymetrix default normalization
• 4 chips were run for each treatment
• 3 chips determined most alike by GeneSpring
  Find Similar Samples were carried on for
  analysis

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Result of Normalization
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Changes in Gene Expression
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Post Hoc Analysis
TMB UND JP-8 DMN TET
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Gene Ontology (GO)

• Consortium of 14 data bases or knowledge
  bases contributes gene annotations to a public
  data base
• Biological phrases applied to genes (i.e. Protein
  kinase, apoptosis, membrane) using a hierarchical
  structure
• Allows inferences across large numbers of genes
  without researching each one individually
• 3 Categories of GO terms (cellular component,
  molecular function biological process)
• Incorporated into GeneSpring GX
• GO slim is a selected subset of the 17,700 terms

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GO Biological Process
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GO Molecular Function
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Significant signaling pathways with JP-8
20 pathways changed compared to sham









9 of 10 Pathways from the older method were
changed in the new one
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Genes and Pathways

• IPA recognized 95 genes to be significantly
  changed in 20 signaling pathways
• Pathways were categorized as either
• inflammatory
• or growth, proliferation and apoptosis
• Genes for each category changed with JP-8 were
  evaluated back in GeneSpring for changes with
  each component

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Ingenuity Pathway AnalysisSignaling Gene
Pathways for JP-8

• Inflammation related (8)
• IL-6
• Chemokine
• Toll-like
• IL-10
• Complement
• IL-2
• T cell
• B cell
• Miscellaneous (1)
• Xenobiotic

• Growth related (11)
• EGF
• PDGF
• VEGF
• IGF-1
• PPAR
• Neurotrophin/Trk
• P38 MAPK,
• ERK/MAPK
• Neuroregulin
• Insulin
• Integrin

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Inflammatory genes
Extracellular Space
Plasma Membrane
Cytoplasm
Nucleus
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Inflammatory genes
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Growth Genes
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Growth Genes
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Conclusions

• Simple aliphatic and aromatic categories dont
  explain the responses of JP-8 mixture
• Component responses are not totally explained by
  concentrations in the epidermis
• Short chain aliphatic more response than long
  chain aliphatic
• Substituted napthalene more response than
  substituted benzene

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Acknowledgements

• Funding AFOSR
• Technical Assistance
• Carol Garrett
• Thomas Kannanayakal
• Carol Amato
• Collaboration
• Steve Berberich (WSU)
• James Rogers (Battelle)
• Rory Conolly (US EPA)

Teamwork Peter Wichman
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Questions?