Integration of metabolomics and in vitro metabolism assays for investigating the stereoselective transformation of triadimefon in rainbow trout.

Triadimefon is a systemic agricultural fungicide of the triazole class whose major metabolite, triadimenol, also a commercial fungicide, provides the majority of the actual fungicidal activity, i.e., inhibition of steroid demethylation. Both chemicals are chiral: triadimefon has one chiral center with two enantiomers while its enzymatic reduction to triadimenol produces a second chiral center and two diastereomers with two enantiomers each. All six stereoisomers of the two fungicides were separated from each other using a chiral BGB-172 column on a GC-MS system so as to follow stereospecificity in metabolism by rainbow trout hepatic microsomes. In these microsomes the S-(+) enantiomer of triadimefon was transformed to triadimenol 27% faster than the R-(-) enantiomer, forming the four triadimenol stereoisomers at rates different from each other. The most fungi-toxic stereoisomer (1S,2R) was produced at the slowest rate; it was detectable after 8 h, but below the level of method quantitation. The triadimenol stereoisomer ratio pattern produced by the trout microsomes was very different from that of the commercial triadimenol standard, in which the most rat-toxic pair of enantiomers (known as "Diastereomer A") is about 85% of the total stereoisomer composition. The trout microsomes produced only about 4% of "Diastereomer A". Complementary metabolomic studies with NMR showed that exposure of the separate triadimefon enantiomers and the racemate to rainbow trout for 48 h resulted in different metabolic profiles in the trout liver extracts, i.e., different endogenous metabolite patterns that indicated differences in effects of the two enantiomers.

Purification and partial characterization of an acid phosphatase from Spirodela oligorrhiza and its affinity for selected organophosphate pesticides.

An acid phosphatase from the aquatic plant Spirodela oligorrhiza (duckweed) was isolated by fast protein liquid chromatography and partially characterized. The enzyme was purified 1871-fold with a total yield of 40%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the pure acid phosphatase resolved a single protein band that migrated to approximately 60 kDa. Nondenaturing SDS-PAGE electrophoresis revealed a single protein band around 120 kDa after staining with Coomassie Brilliant blue. Quantitative gel filtration chromatography estimated a native molecular mass of this enzyme to be 120 kDa. Thus, this acid phosphatase likely functions as a homodimer, consisting of two similar 60 kDa subunits. An electrophoretic technique using the flourogenic substrate 4-methylumbelliferyl phosphate enabled visualization of an acid phosphatase activity that corresponded to the protein band at 120 kDa on a nondenaturing PAGE gel. It was determined that the acid phosphatase had a pH optimum of 6.0 at 25 degrees C. The enzyme activity appeared to be stable over a broad range of temperatures (10-40 degrees C) and in the presence of the metals Zn2+, Mn2+, and Mg2+ as well as the chelating agents ethylenedinitrilotetraacetic acid and ethylene glycol tetraacetic acid. It was shown that this acid phosphatase could hydrolyze a variety of physiological organophosphate compounds including beta-glycerophosphate, phosphoserine, adenosine triphosphate, adenosine diphosphate, adenosine monphosphate, and pyrophosphate. Furthermore, analysis using capillary electrophoresis demonstrated that this hydrolytic enzyme could transform a wide array of organophosphate pesticides including S-2-ethylthioethyl O,O-dimethylphosphorothioate (demeton-S-methyl); S-1,2-bis(ethoxycarbonyl)ethyl O,O-dimethylphosphorodithioate (malathion); O,O-dimethyl O-4-nitrophenyl (paraoxon); O,O,O,O-tetraethyldithiopyrophosphate (sulfatep); O-2-chloro-4-nitrophenyl O,O-dimethylphosphorothioate (dicapthon); and 2,2-dichlorovinyl dimethylphosphate (dichlorvos).