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JJ Stegeman, ME Hahn, R Weisbrod, BR Woodin, JS Joy, S Najibi and RA Cohen
Biology Department, Woods Hole Oceanographic Institution, Massachusetts 23543.
Endothelium is a single-cell layer lining blood vessels and constituting capillaries and could be a primary site of chemical effects in the cardiovasculature and systemically. Cytochrome P4501A1 (CYP1A1) is strongly inducible in vertebrate endothelium in vivo by aryl hydrocarbon receptor (AhR) agonists [Mol. Pharmacol. 36:723-729 (1989); Mol. Pharmacol. 41:1039-1046 (1992)]. We investigated CYP1A expression and activity in porcine aorta endothelial cells (PAEC) exposed in culture to the AhR agonists 2,3,7,8-tetrachlorodibenzo-p- dioxin (TCDD), 3,3',4,4'-tetrachlorobiphenyl (TCB), benzo[a]pyrene (BP), or beta-naphthoflavone (BNF). Immunoblotting with monoclonal anti- CYP1A1 and polyclonal anti-CYP1A1 and anti-CYP1A2 antibodies showed that CYP1A1 was induced in cultures exposed to TCDD, TCB, BP, or BNF but was not detectable in untreated or dimethylsulfoxide-exposed cultures. CYP1A1 was strongly induced at intermediate concentrations (0.1 microM or 1.0 microM) of TCB, BP, or BNF, but induction was suppressed by higher concentrations, a response not due to general toxicity; cell viability (trypan blue exclusion) was > 97% with BNF or TCB at up to 10 microM. CYP1A1 induction by TCDD was maximal at 0.3-1.0 nM. ED50 values for induction of CYP1A1 by TCDD, TCB, and BP were 0.016 nM, 3-10 nM, and 180 nM, respectively. Immunohistochemical analysis confirmed CYP1A1 induction in PAEC but also showed that only some cells in the cultures were induced. Subcellular fractionation, marker enzyme analysis, and immunoblot analysis showed that PAEC had a typical complement of microsomal electron-transport components. NADPH- cytochrome P450 reductase showed comparable rates (approximately 40 nmol/min/mg) in induced and control cultures. Cultures maximally induced by 0.1 microM TCB had microsomal CYP1A1 [ethoxyresorufin-O- deethylase (EROD)] activity averaging 25 pmol/min/mg. Addition of purified rat reductase to PAEC microsomes increased the EROD rates 3- fold. EROD rates measured in intact cells maximally induced by BP, TCB, or TCDD ranged from 15 to 30 pmol/min/mg of whole-cell protein. Methoxyresorufin O-demethylase activity induced by TCDD was 2 pmol/min/mg, i.e., < 10% of the EROD activity. In cultures in which CYP1A1 was strongly induced, CYP1A2 was not detectably expressed. The CYP1A2 inducer acenaphthylene did not induce EROD or methoxyresorufin O- demethylase in intact cells. The results show that CYP1A1 but not CYP1A2 is strongly induced in mammalian endothelial cells in culture and that CYP1A1 is active in intact cells, although the catalytic rates are low.(ABSTRACT TRUNCATED AT 400 WORDS)
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