To determine the basis for unexpected differences in CYP1A1 inducing potencies and efficacies for the dietderived indole derivative, indolo[3,2-b]carbazole (ICZ) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), we conducted a systematic analysis of events involved in the induced expression of CYP1A1 in murine hepatoma-derived cell lines (Hepa-1). In contrast to the effects of TCDD, induction kinetics and CYP1A1 mRNA half-life were dependent on ICZ concentration, and the response from low doses of inducer was transient due to rapid clearance of ICZ. TCDD and ICZ produced the same maximum response (i.e. equal efficacies) from a TCDDresponsive CAT reporter construct in Hepa-1 cells. When measured by the immediate responses associated with CYP1A1 expression, including cellular uptake of inducer, receptor transformation and binding to DRE (gel mobility shift assay), initiation of transcription (nuclear run-on assay), and short-term accumulation of mRNA (Northern blot assay), ICZ also exhibited an efficacy equal to that of TCDD and a potency that corresponds to its receptor affinity. ICZ is a potent and selective noncompetitive inhibitor of ethoxyresorufin O-deethylase activity (K i ‫؍‬ 1.5 nM). Taken together these results indicate that ICZ is a bifunctional modulator of CYP1A1 expression with intrinsic efficacy equal to that of TCDD. Indolo[3,2-b]carbazole (ICZ)1 (Structure 1) is a compound of dietary origin present in the gastrointestinal tract of rodents and humans. ICZ is produced in vivo and in vitro as one of the acid-catalyzed reaction products of non-nutritive indoles such as indole-3-carbinol and glucobrassicin that are present in cabbage and Brussels sprouts and other plants of the Brassica genus (1-4). ICZ is also produced, presumably from the nutritive indole, tryptophan, as a metabolic product of intestinal bacteria (5).ICZ is similar in several respects to the potent environmental pollutant, TCDD. Both compounds have immunosuppressive activity in murine fetal thymus organ culture and both substances exhibit potent antiestrogenic activities including inhibition of estrogen-dependent growth of cultured breast tumor cells (6, 7). Additionally, both ICZ and TCDD induce CYP1A1 activity in animals and in cultured cells (1). CYP1A1 is a phase I enzyme involved in the metabolism of many drugs and carcinogens. CYP1A1 is also the enzyme thought to be primarily responsible for the inactivation of estradiol in breast tumor cells (8).Perhaps key to these similarities in activities is the fact that ICZ and TCDD are nearly isosteric and both compounds are potent Ah receptor agonists (9, 10). The Ah receptor is a widely occurring, ligand-activated transcription factor that mediates the activation of CYP1A1, CYP1A2, glutathione S-transferase Ya, and quinone reductase genes. Binding to this receptor is thought also to be responsible for most of the toxic effects of TCDD, including tumor promotion, teratogenesis, and lethal anorexia with wasting, that appear to result mechanistically from effects beyond simple induction of...

Several studies have reported a low inducibility of hepatic cytochrome P4501A (CYP1A) activity in European flounder (Platichthys flesus) following exposure to mixtures of polychlorinated biphenyls (PCBs). Here we report on mechanistic studies toward understanding this low CYP1A inducibility of flounder, involving molecular characterization of the Ah receptor (AhR) pathway as well as inhibition of the CYP1A catalytic activity by PCB congeners. Hepatic cytosolic AhR levels in flounder were determined using hydroxylapatite, protamine sulfate adsorption analysis, or velocity sedimentation on sucrose gradients. AhR levels in flounder (approximately 2-7 fmol/mg protein) were much lower than observed generally in rodents (approximately 50-300 fmol/mg protein). Molecular characterization of the flounder AhR was provided by first-strand cDNA synthesis and amplification of flounder hepatic poly(A)+ RNA using RT-PCR. A 690-bp product was found, similar in size to a Fundulus AhR cDNA. The specificity of the 690-bp band was established by Southern blotting and hybridization with a degenerate AhR oligonucleotide. The deduced amino acid sequence of the flounder AhR fragment was 59-60% identical to mammalian AhR sequences. Although the AhR is present in flounder cytosol, we were unable to demonstrate detectable amounts of inducible TCDD-AhR-DRE complex in gel-retardation assays. High induction levels of CYP1A protein and associated EROD activity have been previously found in flounder following exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In contrast, the induction of CYP1A catalytic activity by PCB mixtures remains unexpectedly low. Therefore, we further characterized the inhibitory potential of PCB congeners on CYP1A activity in flounder and compared this with inhibitory effects of PCB congeners on rat CYP1A activity. Analysis in vitro demonstrated that 3,3',4,4'-tetraCB, 3,3',4,4',5-pentaCB, 2,2',4,4',5,5'-hexaCB, 3,3',4,4',5,5'-hexaCB, and the commercial PCB mixture Clophen A50 are potent competitive inhibitors of hepatic microsomal CYP1A catalytic activity in flounder and rat. The K(m) for ethoxyresorufin (0.095 microM) in flounder is strikingly close to Ki's found for the tested PCBs. This emphasizes the possible involvement of PCB congeners in inhibition of EROD activity in PHAH exposed fish. Finally, our data indicate that flounder CYP1A is more efficient in metabolizing ethoxyresorufin than that of rat CYP1A.