XENOBIOTIC BIOTRANSFORMATION IN UNICELLULAR GREEN-ALGAE - INVOLVEMENTOF CYTOCHROME-P450 IN THE ACTIVATION AND SELECTIVITY OF THE PYRIDAZINONE PRO-HERBICIDE METFLURAZON

The N-demethylation of the pyridazinone pro-herbicide metflurazon into norflurazon implies a toxification in photosynthetic organisms. This is confirmed by quantitative structure activity relationships determin ed for two unicellular green algae, Chlorella sorokiniana and Chlorell a fusca; however, the latter is 25 to 80 times more sensitive to metfl urazon. This sensitivity is linked to differences in the N-demethylase activity of both algae, as determined by an optimized in vivo biotran sformation assay. Apparent K-m values of the metflurazon-N-demethylase indicate a 10-fold higher affinity for this xenobiotic substrate for Chlorella fusca. Furthermore, algal metflurazon-N-demethylation is cha racterized by distinct variations in activity, depending on the stage of cell development within the cell cycle. Several well-established in hibitors of cytochrome P450-mediated reactions, including piperonylbut oxide, 1-aminobenzotriazole, 1-phenoxy-3-(1H-1,2,4-triol-1 yl)-4-hydro xy-5,5-dimethylhexane, and tetcyclacis, as well as cinnamic acid, a po tential endogenous substrate, inhibited the N-demethylation of metflur azon. The results suggest that the N-demethylation of metflurazon by b oth algae is mediated by a cytochrome P450 monooxygenase. The determin ation of antigenic cross-reactivity of algal proteins with heterologou s polyclonal antibodies originally raised against plant P450s anti-cin namic acid 4-hydroxylase (CYP73A1), anti-ethoxycoumarin-O-dealkylase, anti-tulip allene oxidase (CYP74), and an avocado P450 (CYP71A1) or th ose of bacterial origin, CYP105A1 and CYP105B1,suggests the presence o f distinct P450 isoforms in both algae.