INDUCTION OF CYTOCHROME P4501A1 BY ARYL-HYDROCARBON RECEPTOR AGONISTSIN PORCINE AORTA ENDOTHELIAL-CELLS IN CULTURE AND CYTOCHROME P4501A1 ACTIVITY IN INTACT-CELLS

Endothelium is a single-cell layer lining blood vessels and constituti ng capillaries and could be a primary site of chemical effects in the cardiovasculature and systemically. Cytochrome P4501A1 (CYP1A1) is str ongly inducible in vertebrate endothelium in vivo by aryl hydrocarbon receptor (AhR) agonists [Mel. Pharmacol. 36:723-729 (1989); Mel. Pharm acol. 41:1039-1046 (1992)]. We investigated CYP1A expression and activ ity in porcine aorta endothelial cells (PAEC) exposed in culture to th e AhR agonists 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 3,3',4,4'-t etrachlorobiphenyl (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 cu ltures exposed to TCDD, TCB, BP, or BNF but was not detectable in untr eated or dimethylsulfoxide-exposed cultures. CYP1A1 was strongly induc ed at intermediate concentrations (0.1 mu M Or 1.0 mu M) Of TCB, BP, o r BNF, but induction was suppressed by higher concentrations, a respon se not due to general toxicity; cell viability (trypan blue exclusion) was >97% with BNF or TCB at up to 10 mu M. CYP1A1 induction by TCDD w as maximal at 0.3-1.0 nM. ED(50) values for induction of CYP1A1 by TCD D, TCB, and BP were 0.016 nM, 3-10 nM, and 180 nM, respectively. Immun ohistochemical analysis confirmed CYP1A1 induction in PAEC but also sh owed that only some cells in the cultures were induced. Subcellular fr actionation, marker enzyme analysis, and immunoblot analysis showed th at PAEC had a typical complement of microsomal electron-transport comp onents. NADPH-cytochrome P450 reductase showed comparable rates (simil ar to 40 nmol/min/mg) in induced and control cultures. Cultures maxima lly induced by 0.1 mu M TCB had microsomal CYP1A1 [ethoxyresorufin-O-d eethylase (EROD)I activity averaging 25 pmol/min/mg. Addition of purif ied rat reductase to PAEC microsomes increased the EROD rates 3-fold. EROD rates measured in intact cells maximally induced by BP, TCB, or T CDD ranged from 15 to 30 pmol/min/mg of whole-cell protein. Methoxyres orufin 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 i nduced, CYP1A2 was not detectably expressed. The CYP1A2 inducer acenap hthylene did not induce EROD or methoxyresorufin O-demethylase in inta ct cells. The results show that CYP1A1 but not CYP1A2 is strongly indu ced in mammalian endothelial cells in culture and that CYP1A1 is activ e in intact cells, although the catalytic rates are low. Endothelial C YP1A1 could alter the outcome of drug therapy, activate promutagens co ntributing to atherogenesis, alter the metabolism of endogenous compou nds in the endothelium, and perhaps act as a binding protein, sequeste ring and slowly metabolizing AhR agonists/CYP1A substrates.