Abstract 5419: The plant alkaloid ellipticine is detoxified by cytochrome P450 1A1/2 (CYP 1A1/2) but activated and forms DNA adducts if CYP1A1 or 1A2 is combined with cytochrome b5: an explanation for an in vivo/in vitro discrepancy

Ellipticine (5,11-dimethyl-6H-pyrido[4,3-b] carbazole) is an alkaloid with anti-cancer activity elicited by interfering with many cellular functions. One of these is the formation of DNA adducts upon activation by CYP or peroxidases. The metabolites responsible for detoxication or DNA adduct formation have been characterised and CYP3A1 and the othologous human enzyme CYP3A4 are the most efficient activating enzymes in microsomes from either species (Stiborova M et al. Cancer Res 64 (2004) 8374-8380). In vivo also CYP1A effectively activate ellipticine to DNA adducts, while isolated CYP1A1/2 detoxify ellipticine mainly to its 9-hydroxy derivative. Cytochrome b 5 has been shown to modulate CYP catalysis by either transferring the second electron to CYPs, or by allosteric interaction thereby leading to higher CYP enzyme activity (Schenkman, J.B., I. Jansson Pharmacol. Ther. 97(2003) 139-52). Isolated cytochrome b 5 was added to isolated CYP1A1 or 1A2 reconstituted with NADPH-CYP reductase in liposomes at different ratios of the three enzymes. Apo-cytochrome b 5 (containing no heme) was generated in transfected E.coli (Kotrbova V. et al. Protein Expr Purif., 66(2009) 203-209) and used as such or reconstituted with heme or Mn-protoporphyrin IX. Metabolites were analysed by HPLC and UV detection and quantified with standards. DNA adducts were determined by the nuclease P1 version of the 32 P-postlabeling assay. With both CYP1A1 and 1A2 the pattern of ellipticine metabolites in incubations containing cytochrome b 5 shifted from detoxication to activation. This shift depended on the ratio of CYP1A to cytochrome b 5 with optimal yield of 12-hydroxy- and 13-hydroxy ellipticine at a ratio of 1:5, CYP1A to cytochrome b 5 . The shift was more pronounced for CYP1A1 than for 1A2 leading to 6.5 fold higher DNA adduct levels than without cytochrome b 5 correlating with 6 fold higher 13-hydroxy-ellipticine amounts, the metabolite responsible for the major deoxyguanosin adduct. The cytochrome b 5 holo-enzyme is necessary for this effect, since neither the apo-enzyme nor the apo-enzyme reconstituted with Mn-protoporphyrin IX had the same effect. Other heme containing proteins, like myoglobin or cytochrome c had no effect. Ellipticine is, to our knowledge, the first xenobiotic compound where cytochrome b 5 shifts the metabolism by CYP from detoxication to activation and DNA adduct formation. This is important also when analysing the pharmacological effects in vivo, since ellipticine induces both CYP1A and cytochrome b 5 . Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5419. doi:10.1158/1538-7445.AM2011-5419