Metabolism of a Diphenylether Herbicide to a Volatile Thioanisole and a Polar Sulfonic Acid Metabolite in Spruce (Picea)

Abstract

[14C]Fluorodifen herbicide (2,4′-dinitro-4-trifluoromethyl[CF3-14C[diphenylether) was metabolized to a volatile metabolite in 25% yield in 5 weeks by a Norway spruce (Picea abies L. Karst) cell suspension culture. This metabolite was purified by HPLC and identified by mass spectrometry. The metabolite was 2-nitro-4-trifluoromethylthioanisole. Extracts from Norway spruce callus culture and from the needles of Norway spruce, white spruce (Picea glauca L. Moench), and Colorado spruce (Picea pungens L. Engelm) were assayed for enzymes that could be utilized in the biosynthesis of 2-nitro-4-trifluoromethylthioanisole from fluorodifen via the GSH pathway. Glutathione transferase and cysteine lyase were detected in all of the extracts. S-Adenosylmethionine methyltransferase was detected in extracts from the needles of Norway spruce, white spruce, and Colorado spruce, but not in extracts from Norway spruce callus. It appeared that 2-nitro-4-trifluoromethylthioanisole was synthesized from the GSH pathway through a cysteine conjugate and a thiophenol intermediate. Although additional routes of biosynthesis seemed likely, attempts to determine if several fluorodifen metabolites were precursors of 2-nitro-4-trifluoromethylthioanisole were inconclusive. A polar metabolite, 2-amino-4-nitrobenzenesulfonic acid, was also identified as a metabolite of fluorodifen. This metabolite accounted for 6.7% of the 14C dose after 5 weeks. It was identified by negative ion, fast atom bombardment mass spectrometry, and by chemical and chromatographic comparison to model compounds. This metabolite was also formed by the metabolism of 2-nitro-4-trifluoromethylphenyl-S-glucoside, the most abundant metabolite of fluorodifen in Norway spruce cell suspension cultures after 2 weeks. Ten metabolites of fluorodifen have now been identified from this Norway spruce cell suspension culture. Each of these metabolites appears to have been formed by the metabolism of a GSH conjugate.