2,3,7,8-tetrachlorodibenzo-p-dioxin increases reactive oxygen species production in human endothelial cells via induction of cytochrome P4501A1.
Studies in our laboratory have demonstrated that subchronic 2,3,7,8,-tetrachlorodibenzo-p-dioxin (TCDD) exposure of adult mice results in hypertension, cardiac hypertrophy, and reduced nitric oxide (NO)-mediated vasodilation. Moreover, increased superoxide anion production was observed in cardiovascular organs of TCDD-exposed mice and this increase contributed to the reduced NO-mediated vasodilation. Since cytochrome P4501A1 (CYP1A1) can contribute to some TCDD-induced toxicity, we tested the hypothesis that TCDD increases reactive oxygen species (ROS) in endothelial cells by the induction of CYP1A1. A concentration-response to 24h TCDD exposure (10pM-10nM) was performed in confluent primary human aortic endothelial cells (HAECs). Oxidant-sensitive fluorescent probes dihydroethidium (DHE) and 2',7'-dichlorofluorescin diacetate (DCFH-DA), were used to measure superoxide anion, and hydrogen peroxide and hydroxyl radical, respectively. NO was also measured using the fluorescent probe diaminofluorescein-2 diacetate (DAF-2DA). These assessments were conducted in HAECs transfected with siRNA targeting the aryl hydrocarbon receptor (AhR), CYP1A1, or CYP1B1. TCDD concentration-dependently increased CYP1A1 and CYP1B1 mRNA, protein, and enzyme activity. Moreover, 1nM TCDD maximally increased DHE (Cont=1.0+/-0.3; TCDD=5.1+/-1.0; p=0.002) and DCFH-DA (Cont=1.0+/-0.2; TCDD=4.1+/-0.5; p=0.002) fluorescence and maximally decreased DAF-2DA fluorescence (Cont=1.0+/-0.4; TCDD=0.68+/-0.1). siRNA targeting AhR and CYP1A1 significantly decreased TCDD-induced DHE (siAhR: Cont=1.0+/-0.1; TCDD=1.3+/-0.2; p=0.093) (siCYP1A1: Cont=1.0+/-0.1; TCDD=1.1+/-0.1; p=0.454) and DCFH-DA (siAhR: Cont=1.0+/-0.2; TCDD=1.3+/-0.3; p=0.370) (siCYP1A1: Cont=1.0+/-0.1; TCDD=1.3+/-0.2; p=0.114) fluorescence and increased DAF-2DA fluorescence (siAhR: Cont=1.00+/-0.03; TCDD=0.97+/-0.03; p=0.481) (siCYP1A1: Cont=1.00+/-0.03; TCDD=0.92+/-0.03; p=0.034), while siRNA targeting CYP1B1 did not. These data suggest that TCDD-induced increase in ROS is AhR-dependent and may be mediated, in part, by CYP1A1 induction.
Kopf
P G
PG
Department of Pharmaceutical Science, University of New Mexico Health Sciences Center, Albuquerque, NM 87131, USA. pkopf@mcw.edu
Walker
M K
MK
D001189:Aryl Hydrocarbon Hydroxylases / Q000378:metabolism; D019363:Cytochrome P-450 CYP1A1 / Q000378:metabolism*; D065633:Cytochrome P-450 CYP1B1; D042783:Endothelial Cells / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D004790:Enzyme Induction / Q000187:drug effects; D006801:Humans; D009589:Nitrogen Oxides / Q000378:metabolism; D000072317:Polychlorinated Dibenzodioxins / Q000633:toxicity*; D017382:Reactive Oxygen Species / Q000378:metabolism*; D018336:Receptors, Aryl Hydrocarbon / Q000378:metabolism
D009589:Nitrogen Oxides; D000072317:Polychlorinated Dibenzodioxins; D017382:Reactive Oxygen Species; D018336:Receptors, Aryl Hydrocarbon; D001189:Aryl Hydrocarbon Hydroxylases; C585496:CYP1B1 protein, human; C585498:Cyp1b1 protein, mouse; D019363:Cytochrome P-450 CYP1A1; D065633:Cytochrome P-450 CYP1B1