POTENTIAL ROLE OF CYTOCHROME P450 3A4 (CYP3A4) IN THE PCB104-MEDIATED BARRIER DYSFUNCTION OF HUMAN MICROVASCULAR ENDOTHELIAL CELLS

Yong Woo Lee1, Sung Yong Eum1, Bernhard Hennig2, Michal Toborek1

Departments of Surgery1 and Animal Sciences2, University of Kentucky, Lexington, KY 40536

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 07380.

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

En

doth

elia

l Per

mea

bil

ity

(% C

ontr

ol)

0

50

100

150

200

250

300

350

400

450

*

*

0 1.0 10 20

2,2',4'6,6'-Pentachlorobiphenyl (M)

Ele

ctri

cal R

esis

tanc

e (%

Con

trol

)

0

20

40

60

80

100

120

*

*

0 1.0 10 20

2,2',4'6,6'-Pentachlorobiphenyl (M)

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 0 1.0 10 20

PCB104 (M)

Cel

l Via

bil

iy (

% C

ontr

ol)

0

20

40

60

80

100

120

*

*

0 1.0 10 20

2,2',4'6,6'-Pentachlorobiphenyl (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 0 1.0 5.0 50

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

End

othe

lial P

erm

eabi

lity

(% C

ontr

ol)

0

50

100

150

200

250

300

350

400

450

500

550

*

*

Control PCB KET+PCB

#

Ele

ctri

cal R

esis

tanc

e (%

Con

trol

)

0

20

40

60

80

100

120

*

* #

Control PCB KET+PCB

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