Alternate pathways of metribuzin metabolism in soybean: Formation of N-glucoside and homoglutathione conjugates

Abstract

Metribuzin [4-amino-6- tert-butyl-3(methylthio)-1,2,4-triazin-5(4H)-one] metabolism was studied in soybean [ Glycine max (L.) Merr. Tracy]. Pulse treatment studies with seedlings and excised mature leaves showed that [5- 14C]metribuzin was absorbed rapidly and translocated acropetally. In seedlings, >97% of the root-absorbed 14C was present in foliar tissues after 24 hr. In excised leaves, 50–60% of the absorbed 14C remained as metribuzin 48 hr after pulse treatment, 12–20% was present as polar metabolites, and 20–30% was present as an insoluble residue. Metabolites were isolated by solvent partitioning, and were purified by adsorption, ion-exchange, thin-layer, and high-performance liquid chromatography. The major metabolite (I) was identified as a homoglutathione conjugate, 4-amino-6- tert-butyl-3- S-(γ-glutamyl-cysteinyl-β-alanine)-1,2,4-triazin-5(4H)-one. Metabolite identification was confirmed by qualitative analysis of amino acid hydrolysis products, fast atom bombardment (FAB), and chemical ionization (CI) mass spectrometry, and by comparison with a reference glutathione conjugate synthesized in vitro with a hepatic microsomal oxidase system from rat. Minor metabolites were identified as an intermediate N-glucoside conjugate (II), a malonyl N-glucoside conjugate (III), and 4-malonylamido-6- tert-butyl-1,2,4-triazin-3,5(2H,4H)-dione ( N-malonyl DK, IV) by CI and FAB mass spectrometry. Alternative pathways of metribuzin metabolism are proposed.