title: 26-hydroxylation of ecdysteroids is catalyzed by a typical cytochrome P-450-dependent oxidase and related to ecdysteroid resistance in an insect cell line.
abstract: The epithelial cell line from the dipteran Chironomus tentans responds to the insect steroid hormone 20-hydroxyecdysone and the non-steroidal analogue tebufenozide by undergoing a morphogenetic and biochemical differentiation program. Long-term culture in the presence of 20-hydroxyecdysone has resulted in the selection of subclones that are resistant to the steroid but respond normally to the non-steroidal analogue. In the present study, several subclones that were resistant to the steroid hormone have been compared with steroid-sensitive subclones with respect to their capability to metabolize 20-hydroxyecdysone. Homogenates of both types of cells, when incubated with 3H-labelled steroid in the presence of NADPH, producecd 20,26-dihydroxyecdysone, which was further metabolized to two compounds, which behaved less polar than 20-hydroxyecdysone on reverse-phase HPLC. Ecdysone, a less-active hormone precursor, provided 26-hydroxyecdysone as the only product. The metabolites were identified by mass spectrometry coupled to HPLC, chromatography with authentic samples, and formation of acetonides. The structure of 20,26-dihydroxyecydsone was confirmed by 1H-NMR. The enzyme responsible for the synthesis of 20,26-dihydroxyecdysone in the Chironomus cell preparations has been characterized as a typical cytochrome P-450-dependent monooxygenase. It was a strictly microsomal enzyme, sensitive to inhibition by carbon monoxide and imidazole/triazole-based fungicides, and required NADPH for maximal activity. NADH could partly replace NADPH. The Michaelis constant (Km) for 20-hydroxyecdysone was 0.96 microM, and the maximal enzyme velocity (Vmax) was 50 pmol substrate metabolized x mg protein(-1) x min(-1). 26-Hydroxylation of 20-hydroxyecdysone was inhibited by ecdysone, an alternative substrate, and by inokosterone, a product analogue, to 50% at 1.4 microM and 0.73 microM, respectively. When various subclones were compared with respect to their in vitro rate of 20-hydroxyecdysone metabolization, those clones known to be resistant to the steroid were 'high metabolizers' (> 70% relative rate), whereas the sensitive clones were 'poor metabolizers' (< 30% relative rate). Hence, it is tempting to conclude that ecdysteroid resistance of the Chironomus cell clones is due to metabolic inactivation of the steroid hormone.
authors.affiliation: Novartis Crop Protection AG, Research, Biochemistry Insecticides, Basel, Switzerland. hartmut.kayser@cp.novartis.com
authors.forename: H
authors.initials: H
authors.lastname: Kayser
authors.forename: T
authors.initials: T
authors.lastname: Winkler
authors.forename: M
authors.initials: M
authors.lastname: Spindler-Barth
mesh_terms: D000818:Animals; D000935:Antifungal Agents / Q000494:pharmacology; D002458:Cell Fractionation; D002460:Cell Line; D002683:Chironomidae / Q000201:enzymology* / Q000378:metabolism; D053493:Cholestanetriol 26-Monooxygenase; D002851:Chromatography, High Pressure Liquid; D002999:Clone Cells; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004351:Drug Resistance; D004440:Ecdysone / Q000378:metabolism*; D004441:Ecdysterone / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D006900:Hydroxylation; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D015394:Molecular Structure; D009249:NADP / Q000494:pharmacology; D013053:Spectrophotometry; D013250:Steroid Hydroxylases / Q000378:metabolism*
chemical_list: D000935:Antifungal Agents; C021997:20,26-dihydroxyecdysone; D004440:Ecdysone; D004441:Ecdysterone; D009249:NADP; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D053493:Cholestanetriol 26-Monooxygenase