Regulation of CYP1A1 by Indolo[3,2-b]carbazole in Murine Hepatoma Cells

Abstract

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...

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