1. Vascular Endothelial Injury by Chlorpyrifos: Relationship to Brain Metastasis A. Hirani, S. Kang, M. Ehrich, Y.W. Lee Virginia Polytechnic Institute and State University Blacksburg, VA

2. Release from the primary tumor & Invasion of the surrounding tissues Entry into vascular or lymphatic circulation “Intravasation” Transit in the circulation Arrest in the capillary bed of a distant organ Transendothelial migration from the circulation “Extravasation” Growth at selective sites that are distant from the original tumor site

4. Extravasation and Vascular Endothelium Oxidative stress-induced endothelial damage promotes the metastasis of circulating cancer cells Inflammatory environments within vascular endothelium facilitate the metastasis of tumor cells Endothelial dysfunction through up-regulation of pro- inflammatory mediators such as cytokines, chemokines and adhesion molecules plays a crucial role in cancer metastasis Cells derived from solid tumor impair endothelium integrity by inducing endothelial cell apoptosis The vascular injury via inflammatory and apoptotic pathways can play an active role in the extravasation process of cancer metastasis

5. Chlorpyrifos (CPF) Broad spectrum Organophosphate Pesticide (OP) Broad spectrum Organophosphate Pesticide (OP) 1 of most widely used insecticides in US 1 of most widely used insecticides in US Developmental neurotoxicant Developmental neurotoxicant

6. Goal of Study To investigate the molecular mechanisms of CPF-mediated injury to the brain microvascular endothelium

7. IL-6 upregulated in dose-dependent manner at 4 h CPF exposure

8. IL-6 upregulated in dose-dependent manner at 8 h CPF exposure

9. IL-6 upregulated in dose-dependent manner at 12 h CPF exposure

10. IL-6 upregulated in dose-dependent manner at 24 h CPF exposure

11. Dose-dependence Upregulation of TNF- 

12. Dose-dependence Upregulation of MMP-2

13. CPF-induced Endothelial Cell Death

14. Summary CPF exposure resulted in a significant increase in mRNA expression of pro-inflammatory cytokine interleukin-6 (IL-6) in a time- and dose-dependent manner CPF exposure resulted in a significant increase in mRNA expression of pro-inflammatory cytokine interleukin-6 (IL-6) in a time- and dose-dependent manner CPF exposure markedly and dose-dependently up-regulated expression of tumor necrosis factor-  (TNF-  ) and matrix metalloproteinase-2 (MMP-2) CPF exposure markedly and dose-dependently up-regulated expression of tumor necrosis factor-  (TNF-  ) and matrix metalloproteinase-2 (MMP-2) Viability of BMEC is unaffected by CPF treatment at ≤ 100 nM Viability of BMEC is unaffected by CPF treatment at ≤ 100 nM Higher concentrations of CPF (≥ 1  M) caused endothelial cell death in a time- and dose-dependent manner Higher concentrations of CPF (≥ 1  M) caused endothelial cell death in a time- and dose-dependent manner CPF-induced brain vascular injury through pro- inflammatory pathways and endothelial cell death pathway may contribute to the BBB disruption and cancer metastasis to the brain.

15. Acknowledgements Dr. YongWoo Lee Dr. YongWoo Lee Dr. Marion Ehrich Dr. Marion Ehrich Sungkwon Kang Sungkwon Kang VMRCVM New Initiative Grants