Metabolism-based polycyclic aromatic acetylene inhibition of CYP1B1 in 10T1/2 cells potentiates aryl hydrocarbon...

We have used polycyclic aromatic hydrocarbon (PAH) alkyne metabolism-based inhibitors to test whether CYP1B1 metabolism is linked to aryl hydrocarbon receptor (AhR) activation in mouse embryo fibroblasts (MEF). 1-ethynylpyrene (1EP) selectively inactivated CYP1B1 dimethylbenzanthracene (DMBA) metabolism in C3H10T1/2 MEFs; whereas 1-(1-propynyl)pyrene (1PP) preferentially inhibited CYP1A1 activity in Hepa-1c1c7 mouse hepatoma cells (Hepa). In each cell type >90% inhibition of DMBA metabolism after 1 h treatment with each inhibitor (0.1 microM) was progressively reversed and then increased to levels seen with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induction (fourfold stimulation). It was found that 0.1 microM 1EP and 1PP maximally induce CYP1B1 and CYP1A1 mRNA levels in10T1/2 and Hepa cells, respectively, after 6 h. 1-Ethylpyrene (EtP), which lacks the activatable acetylene moiety, was far less effective as an inhibitor and as an inducer. AhR activation is essential for 1EP induction as evidenced by the use of AhR antagonists and AhR-deficient MEFs and absence of induction following inhibition of DMBA metabolism with carbon monoxide (CO). Inhibition of CYP1B1 was linked to enhanced AhR activation even at early stages prior to significant ligand depletion. 1EP and EtP were similarly effective in stimulating AhR nuclear translocation, though 5-10 times slower compared with TCDD, and produced no significant down-regulation of the AhR. TCDD activated AhR/Arnt complex formation with an oligonucleotide xenobiotic response element far more extensively than 1EP or EtP, even at concentrations of 1EP that increased CYP1B1 mRNA to similar levels. CO did not influence these responses to EtP, event hough CO treatment potentiated EtP induction of CYP1B1 mRNA. These differences suggest a fundamental difference between PAH/AhR and TCDD/AhR complexes where CYP1B1 metabolic activity regulates the potency, rather than the formation of the AhR/Arnt complex.