Molecular Responses of the Skin to JP-8 and Some Components: Molecular Responses of the Skin to JP-8 and Some Components James N. McDougal Wright State University Tucson AZ, January 2007


Overview: 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


Patch and Harness: Patch and Harness


Exposure Protocol: Exposure Protocol Carefully clip fur on rat’s 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 Time (hours) 0 1 4 6 8 Skin Samples Skin exposure (5 treated rats and 5 sham rats at each time point)


Epidermis: Epidermis


JP-8 Concentrations in Epidermis: JP-8 Concentrations in Epidermis


Heatmap: Heatmap 1 hour 4 hours 8 hours


Principal Component Analysis: Principal Component Analysis 4-hr 8-hr 1-hr


Genes Changed : Genes Changed


Ingenuity Pathways Analysis: 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)


Signaling Pathways – 1hr: Signaling Pathways – 1hr


Signaling Pathways – 4hr: Signaling Pathways – 4hr


Metabolic Pathways – 8hr: 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


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


Slide15: 27 Upregulated Genes All Times


Conclusions: 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


Trigger?: Trigger?


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


Literature: 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


Component Methods: 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


Slide21: Component Characteristics


Epidermal Concentrations: Epidermal Concentrations


Gene Expression Methods: 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


Result of Normalization: Result of Normalization


Slide25: Changes in Gene Expression


Post Hoc Analysis: Post Hoc Analysis TMB UND JP-8 DMN TET


Gene Ontology (GO): 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


GO Biological Process: GO Biological Process


GO Molecular Function: GO Molecular Function


Significant signaling pathways with JP-8: 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 *


Genes and Pathways: 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


Ingenuity Pathway AnalysisSignaling Gene Pathways for JP-8: Ingenuity Pathway Analysis Signaling 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


Slide33: Extracellular Space Plasma Membrane Cytoplasm Nucleus Inflammatory genes


Inflammatory genes: Inflammatory genes


Slide35: Growth Genes


Growth Genes: Growth Genes


Conclusions: Conclusions Simple aliphatic and aromatic categories don’t 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


Acknowledgements: 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


Slide39: Questions?