POTENTIAL ROLE OF CYTOCHROME P450 3A4 (CYP3A4) IN THE PCB104-MEDIATED BARRIER DYSFUNCTION OF HUMAN MICROVASCULAR ENDOTHELIAL CELLS Yong Woo Lee 1, Sung Yong Eum 1, Bernhard Hennig 2, Michal Toborek 1 Departments of Surgery 1 and Animal Sciences 2, University of Kentucky, Lexington, KY Recent evidence has demonstrated that polychlorinated biphenyls (PCBs) can cause endothelial cell injury via cytochrome P450-dependent mechanisms and can thus be implicated in the diseases that involve dysfunction of the vascular endothelium, such as cancer metastasis. The present study was designed to determine the regulatory mechanisms of PCB-mediated barrier dysfunction in human microvascular endothelial cells (HMEC-1) and the potential involvement of specific cytochrome P450 isoforms in this process. A significant and dose-dependent increase in endothelial permeability was observed in HMEC-1 treated with 2,2’,4,6,6’-pentachlorobiphenyl (PCB104). PCB104 also dramatically decreased electrical resistance of HMEC-1 monolayer in a dose- dependent way. To elucidate the role of cytochrome P450 in the PCB104-mediated barrier dysfunction, a variety of cytochrome P450 inhibitors, such as proadifen, metyrapone,  - naphthoflavone, ketoconazole and sulfaphenazole, were employed in the present study. Endothelial cell apoptosis was selectively inhibited when HMEC-1 were treated with PCB104 in the presence of ketoconazole, a specific inhibitor of CYP3A4. Furthermore, pretreatment with ketoconazole significantly reversed the increased permeability and decreased electrical resistance of PCB-treated HMEC-1 monolayer. These results suggest that CYP3A4 may be involved, at least in part, in PCB104-induced barrier dysfunction of HMEC-1 monolayer. ABSTRACT Metastasis and Vascular Endothelium Oxidative stress-induced endothelial damage promotes the metastasis of circulating cancer cells to the lung. The free radical-mediated endothelial damage facilitate the metastasis of pancreatic tumor cells. Linoleic acid induces endothelial dysfunction through overexpression of chemokines and adhesion molecules that play a crucial role in cancer metastasis. Cells derived from solid tumor impair endothelium integrity by inducing endothelial cell apoptosis. The vascular endothelium can play an active role in the extravasation process of cancer metastasis. Polychlorinated biphenyls (PCBs) A class of polychlorinated aromatic hydrocarbons composed of 209 discrete congeners. Serious global environmental pollutants [high lipophilicity & high stability of these compounds from degradation] Extensive toxic effects : Neurotoxicity, Hepatotoxicity, Carcinogenicity, Immunotoxicity, Cardiotoxicity, etc. This work was supported by NIH/NIEHS, P42 ES ACKNOWLEDGEMENTS To investigate the potential role of cytochrome P450 3A4 (CYP3A4) in the 2,2’,4,6,6’-pentachlorobiphenyl (PCB104)- induced barrier dysfunction of human microvascular endothelial cells The aim of the present study PCB104 induces endothelial barrier dysfunction Effects of 2,2’,4,6,6’-pentachlorobiphenyl (PCB104) on the permeability and electrical resistance of human microvascular endothelial cell (HMEC-1) monolayer. HMEC-1 cells were grown to confluence on fibronectin-coated filter inserts for 7 days and then exposed to increasing concentrations of PCB104 (1.0, 10 and 20  M) for 24 h. The endothelial permeability in response to PCB104 exposure was evaluated with fluorescein isothicyanate-labeled dextran (FITC-Dextran 40) as a permeable tracer that passes across endothelial monolayers (left panel). The electrical resistance across endothelial monolyer was measured using EVOM volt-ohm meter with “chopstick” electrodes (right panel). Data are means ± SEM of 4 determinations. *Statistically significant compared with the control group (P<0.05). PCB104 induces endothelial cell apoptosis M PCB104 (  M) Effects of 2,2’,4,6,6’-pentachlorobiphenyl (PCB104) on the viability and DNA fragmentation of human microvascular endothelial cells (HMEC-1). HMEC-1 cells were exposed to increasing concentrations of PCB104 (1.0, 10 and 20  M) for 24 h. The cell viability was measured by the MTT conversion assay and expressed as the percentage of untreated control cell cultures. Data shown are the means ± SD of 6 determinations. *Statistically significant compared with the control group (P<0.05) (left panel). The DNA was extracted, fractionated by 2% agarose gel electrophoresis, and visualized using phosphoimaging technology. M, molecular weight marker (100-bp DNA ladder) (right panel). Inhibitors of cytochrome P450 isoforms  -Naphthoflavone [CYP1A2] Metyrapone [CYP2B1/B2] Proadifen [General] Ketoconazole [CYP3A4] Sulfaphenazole [CYP2C9] CYP3A4 inhibitor specifically blocks PCB104-induced apoptosis M CTL PCB SKF MET ANF KET SUL M CTL PCB + KET (  M) PCB + CYP inhibitor (50  M) Effects of cytochrome P450 inhibitors on the PCB104-induced apoptosis of human microvascular endothelial cells (HMEC-1). HMEC-1 cells were pretreated with 50  M cytochrome P450 inhibitors, such as proadifen (SKF),  -naphthoflavone (ANF), metyrapone (MET), sulfaphenazole (SUL) and ketoconazole (KET), for 1 h and then exposed to 20  M PCB104 for 24 h (left panel). The cells were pretreated with increasing concentrations of ketoconazole (KET; 1, 5 and 50  M), a specific inhibitor of CYP3A4, for 1 h and then incubated with 20  M PCB 104 for 24 h (right panel). The DNA was extracted, fractionated by 2% agarose gel electrophoresis, and visualized using phosphoimaging technology. M, molecular weight marker (100-bp DNA ladder); CTL, control; PCB, PCB104. CYP3A4 inhibitor attenuates PCB104-mediated barrier dysfunction Effects of cytochrome P450 inhibitors on the PCB104-induced apoptosis of human microvascular endothelial cells (HMEC-1). HMEC-1 cells were grown to confluence on fibronectin-coated filter inserts for 7 days, pretreated with 50  M ketoconazole for 1 h, and then exposed to 20  M PCB104 for 24 h. The endothelial permeability in response to PCB104 exposure was evaluated with fluorescein isothicyanate-labeled dextran (FITC-Dextran 40) as a permeable tracer that passes across endothelial monolayers (left panel). The electrical resistance across endothelial monolyer was measured using EVOM volt-ohm meter with “chopstick” electrodes (right panel). Data shown are the means ± SEM of 4 determinations. *Statistically significant compared with the control group (P<0.05). #Values in the groups treated with PCB plus KET are significantly different from the PCB-treated group (P<0.05). PCB, PCB104; KET, ketoconazole. The present study suggests that cytochrome P450 3A4 may be involved, at least in part, in the PCB104-induced barrier dysfunction of human microvascular endothelial cell monolayer. These data may contribute to understanding the cellular and molecular mechanisms of PCB-mediated vascular toxicity, which is critical for the extravasation of tumor cells, and development of therapeutic strategies for PCB-induced cancer metastasis. CONCLUSION 1.PCB104 induces barrier dysfunction of human microvascular endothelial cell monolayer through activation of endothelial cell apoptotic pathways. 2. CYP3A4 inhibitor, ketoconazole, specifically blocks the PCB104- induced apoptosis of human microvascular endothelial cells. 3. CYP3A4 inhibitor, ketoconazole, attenuates the PCB104- mediated barrier dysfunction of human microvascular endothelial monolayer SUMMARY