Vol. 31, Issue 2, 233-241, February 2003

Characterization of in Vitro Metabolites of the Aryl Hydrocarbon Receptor Ligand 6-Formylindolo[3,2-b]carbazole by Liquid Chromatography-Mass Spectrometry and NMR.

Linda Bergander, Niklas Wahlström, Tomas Alsberg, Jan Bergman, Agneta Rannug, and Ulf Rannug

Department of Genetic and Cellular Toxicology (L.B., U.R.) and Laboratory for Analytical Environmental Chemistry, Institute of Applied Environmental Research (T.A.), Stockholm University, Stockholm; Unit for Organic Chemistry, Department of Biosciences at Novum, Karolinska Institutet, Novum Research Park, Huddinge (N.W., J.B.); Södertörn University College, Huddinge (N.W., J.B.); and Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden (A.R.).

The tryptophan photoproduct 6-formylindolo[3,2-b]carbazole (FICZ) exhibits the highest aryl hydrocarbon receptor (AhR) binding affinity reported so far. In different cells, in vitro, both extracts of UV-irradiated tryptophan and the synthesized pure compound FICZ induce a rapid and transient expression of AhR-regulated genes. The transient induction suggests that the biotransformation gene battery induced by AhR activation takes part in a metabolic degradation of the ligand, whereby a low steady-state level is regained. The down-regulation of AhR-regulated gene expression was previously shown to be dependent on cytochrome P450 1A1 (CYP1A1). Metabolism of FICZ generates five major metabolites, which appeared as three peaks (M1-M3) in the high performance liquid chromatography. The aim of the present study was to use rat liver S9 from Aroclor-pretreated rats to produce large enough quantities of FICZ metabolites for structure characterization and to determine their product precursor relationship. NMR analysis of large combined fractions of the metabolites indicated that M3 and M2 contained 2 isomers, respectively. By means of liquid chromatography-mass spectrometry (negative ion electrospray mode) and NMR spectroscopy (by ¹H-NMR, correlation spectroscopy, and nuclear Overhauser effect spectroscopy techniques) five metabolites of FICZ were identified, and their structures were elucidated. The molecular weights of the two M3 isomers were 300 and both M2 and M1 compounds demonstrated molecular weights of 316, corresponding to addition of one (M3) and of two oxygen (M2 and M1), respectively. The structures were assigned as 2- and 8-hydroxy (M3), 2,10- and 4,8-dihydroxy (M2) and 2,8-dihydroxy derivatives of indolo[3,2-b]carbazole-6-carboxaldehyde (6-formylindolo[3,2-b]carbazole).