Rapid and transient induction of CYP1A1 gene expression in human cells by the tryptophan photoproduct 6-formylindolo[3,2-b]carbazole

Abstract

Studies to assess the induction of CYP1A1 gene expression by tryptophan derived oxidation products which are suggested as endogenous ligands for the Ah receptor are described. For the two high affinity Ah receptor ligands produced from tryptophan, the chemical structure was recently identified as 6-formylindolo[3,2-b]carbazole (FICZ) and 6,12-diformylindolo[3,2-b]carbazole (dFICZ), respectively. Therefore these two compounds show a close similarity to the indolecarbinol-derived condensation product indolo[3,2-b]carbazole (ICZ). Incubation of cells from a human keratinocyte (HaCaT) cell line together with ICZ, FICZ, dFICZ and some structurally related indole compounds was performed. The compound with the highest affinity to the Ah receptor, FICZ, was found to be the most efficient inducer of CYP1A1 gene expression in short time incubation (0.5 h) experiments. With longer incubation times (24 h) ICZ was the most efficient inducer. The two most active compounds, FICZ and ICZ, caused increased mRNA levels already at a concentration of 100 pM. FICZ was also shown to increase CYP1A1 mRNA levels in fresh human peripheral blood cells at the same low concentration. FICZ and ICZ were furthermore compared with regard to their capacity to inhibit cDNA-expressed human CYP1A1 enzyme and FICZ was found to be the most potent inhibitor. The inhibition was, however, transient in character indicating that FICZ is also an exceptionally good substrate for the CYP1A1 enzyme. The results showing the potent and transient effect of these formylindolocarbazoles, thus emphasize their important properties as signal substances in the Ah receptor pathway. This makes the most potent compound, FICZ, a good candidate for the endogenous ligand of the Ah receptor necessary for normal development and for the basal expression of Ah receptor-dependent genes.

Introduction

Intensive research in many laboratories has been devoted to the elucidation of the mechanism of action of toxic environmental chemicals such as the dioxin 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) via the aryl hydrocarbon (Ah) receptor system. The Ah receptor mediated interactions and the transcriptional activation processes have been characterized in great detail [1]. Upon binding of dioxin to the transcriptionally inactive Ah receptor, associated with the heat shock protein 90 (hsp90) chaperone, the hsp90 is released and the activated receptor heterodimerizes with its partner, the Ah receptor nuclear translocator (Arnt). The liganded Ah–Arnt complex recognizes and binds to specific DNA target elements (DREs), upstream of the Ah receptor regulated genes. The components of this molecular pathway have been conserved in evolution and functional elements have been identified in all mammalian species investigated [2]. Based on the typical induction of detoxification enzymes, including those encoding cytochrome P450 as well as UDP-glucuronosyl- and glutathione transferases, the Ah receptor system has been considered a defense system against toxic chemicals. A new role for the Ah receptor system is however emerging as it is being demonstrated that the receptor-activated genes, the Ah battery, comprises the immediate early proto-oncogenes [3], growth factors and other proteins 4, 5, 6, in addition to the phase I and phase II type biotransformation enzymes. TCDD has been found to stimulate various signal transduction processes, including activation of phospholipase A2 [7], protein kinase C [8]and Ca²⁺ signaling 9, 10, 11, and furthermore, Ah receptor binding results in an increased rate of oxidative DNA damage [12].

It was earlier postulated that the Ah receptor functions as a receptor for oxidized tryptophan and as such has a significant role in receptor mediated cellular responses to oxidation stress, such as proliferative responses [13]. Tryptophan is the most strongly near-UV absorbing amino acid and it was while utilizing tryptophan as a photosensitizer in experiments directed towards dimerization of purines that we observed that the photosensitizer alone upon irradiation formed potent Ah receptor agonists. UV-oxidation of tryptophan was found to give rise to substances with remarkably high Ah receptor affinity, when tested for their ability to compete with ³H-labeled dioxin [13]. Based on their high binding affinity, with K_d values of 0.44 and 0.07 nM, for the two most active substances respectively, compared to a K_d of 0.48 nM for TCDD itself under the same conditions, we suggested that oxidized amino acids are the endogenous ligands for the Ah receptor. The compounds act as potent inducers and inhibitors of cytochrome P-4501A1 enzyme activity 14, 15. The most potent compound also induces sister chromatid- and gene conversions in yeast, Saccharomyces cerevisiae strain RS112, and slightly increases cell survival or cell division in yeast strains RS112 and D7 but neither of the compounds induces gene mutations in the yeast strains or in Salmonella typhimurium strains TA98 and TA100 15, 16. The formation of photoproducts of tryptophan with high affinity for the Ah receptor and with potent aryl hydrocarbon hydroxylase activity (AHH) has been reproduced by other groups 17, 18. Recently, the two most active Ah receptor binding compounds produced from oxidized tryptophan were identified as the symmetrical 6,12-diformylindolo[3,2-b]carbazole (dFICZ) and the monosubstituted 6-formylindolo[3,2-b]carbazole (FICZ) [19](Fig. 1).

As indicated from the studies on the purified tryptophan photoproducts mentioned above, the active compounds, FICZ and dFICZ, bind to the Ah receptor with higher affinity than TCDD. This high affinity is however not reflected in an extremely high induction of AHH activity compared to TCDD or polycyclic aromatic hydrocarbon type inducers. FICZ induces AHH activity in rat hepatoma cells in a concentration range of 10–50 nM and the peak induction is not very high [14]. In the first set of experiments studying the effects of FICZ on the expression of CYP1A1 using 24 h exposure of human keratinocytes we observed a modest induction of CYP1A1 mRNA at a 10 nM concentration which is approximately three orders of magnitude higher than the concentration required for CYP1A1 expression by TCDD in murine hepatoma cells [20]. The induction of CYP1A1 enzyme activity, the induction of CYP1A1 mRNA and the Ah-receptor binding in a pure system by the tryptophan photoproduct FICZ was observed at approximately 10, 10 and 0.1 nM respectively. Thus, FICZ is obviously very rapidly inactivated in metabolically competent systems.

In this study, the kinetics of the FICZ dependent induction of CYP1A1 mRNA expression in human cells were characterized and its activity compared with that of the unsubstituted analog indolo[3,2-b]carbazole (ICZ) as well as dFICZ and three other high affinity Ah receptor ligands; 6,12-dimethylindolo[3,2-b]carbazole (dMICZ), rutaecarpine (RC) and 7,8-dehydrorutaecarpine (DRC). We have also studied the metabolism of FICZ by the use of microsomes prepared from a human lymphoblast cell line with cDNA expressed human CYP1A1. The results obtained demonstrate that the 6-formyl substituted indolo[3,2-b]carbazole is an extremely potent inducer of CYP1A1 gene expression in cultured human keratinocytes and in fresh human lymphocytes. The results also emphasize different rates of metabolism and gene induction between FICZ and ICZ. The tryptophan derived compound, FICZ, was shown to be a better substrate for the CYP1A1 enzyme than ICZ, thereby explaining the more rapid and transient induction of CYP1A1 observed with FICZ compared to ICZ.