Metabolism of cycloate in radish leaf: Metabolite identification by packed capillary flow fast atom bombardment tandem mass spectrometry

Abstract

The metabolism of cycloate, a thiocarbamate herbicide, was investigated in mature radish leaf. Twelve new metabolites were identified by liquid chromatographic/mass spectrometric analysis using fast atom bombardment and packed capillary liquid chromatography columns. Full-scan and tandem mass spectrometric methods were employed. Application of the on-column focusing technique resulted in identifications with injections of as little as 15 ng of metabolite (20 ppb in radish). This injection technique allows the practical use of packed capillary liquid chromatography/mass spectrometry in sample-limited applications. Cycloate is oxidized to several ring-hydroxylated isomers that are subsequently glucosylated and esterified with malonic acid. Cycloate is also conjugated with glutathione. Metabolic hydrolysis of the glutathione conjugate formed a cysteine conjugate that is further metabolized by amidation with either malonic or acetic acid. Transamination of the cysteine conjugate gave a thiolactic acid derivative. Metabolites were also identified that were the result of both ring-hydroxylation and conjugation with glutathione. One of these, an N-acetylcysteine conjugate, is the first report of a mercapturic acid in plants. The structures of two of the new metabolites were confirmed by chemical synthesis.

Catabolism of Glutathione Conjugates in Arabidopsis thaliana

“…In our studies, this enzyme activity was not detected and to our knowledge has not been described in any other plant. It has been suggested that N-acetylcysteine derivatives of pesticides could instead arise from the decarboxylation of the respective N-malonylated conjugates (18). In support of this hypothesis, we observed that FMC spontaneously decarboxylated to form FAC at a slow but measurable rate of 1.6 mol s Ϫ1 mol Ϫ1 when stored as a methanolic solution.…”

“…Although the formation of such mercapturic acid conjugates is common in drug metabolism in animals (17), it is an unusual reaction in plants (18). In animals, the N-acetylation of cysteine is catalyzed by a specific acetyl-CoA-dependent acetyltransferase (17).…”

“…Based on its parent mass ion of 237 Da, it was clear that this compound had arisen from the extensive processing of the sulfur donor substituent, such that all of its polar functional groups had been removed. Careful scrutiny of the literature (18,19) suggested that compound 10 was the S-methylated derivative of fenclorim, 4-chloro-6-(methylthio)-2-phenylpyrimidine (CMTP; Fig. 3B), and this was confirmed by chemical synthesis of an authentic standard (supplemental materials).…”

“…A review of the literature on the processing of S-glutathionylated drugs suggested that this second sulfur-containing group was N-acetylcysteine. Although the formation of N-acetylcysteine (mercapturic acid) conjugates is very common in drug metabolism in animals (17), such modifications have rarely been determined in pesticide metabolism in plants, notably as residues of the herbicide cycloate in radishes (18). The identity of compound 2 as S-(4-N-acetylcysteine-2-phenylpyrimidin-6-yl)-glutathione (FACG; Fig.…”

Alternate Energy-Dependent Pathways for the Vacuolar Uptake of Glucose and Glutathione Conjugates

“…This is the case for apigenin 7-Oglucoside (the major pigment in parsley), which is conjugated to malonic acid before vacuolar transport (Matern et al, 1986). Although malonic acid hemiesters of glucosylated herbicides have also been found in plants (Onisko et al, 1994;Wink, 1997), it is not known if these auxiliary modifications are required for or contribute to the vacuolar sequestration of xenobiotics of this type.…”