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Locke RK, Chen JY, Damico JN, Dusold LR, Sphon JA: Identification by physical means of organic moieties of conjugates produced from carbaryl by tobacco cells in suspension culture. Arch Environ Contam Toxicol. 1976;4(1):60-100.
Carbaryl (1-naphthyl methylcarbamate), labeled with 14C in the C1-naphthyl, carbonyl, or N-methyl position, was introduced into the culture medium of tobacco cells in suspension culture. Following incubation, cells were homogenized in water, centrifugated, and supernatants hydrolyzed with beta-glucosidase or HCl. Organic moieties (moieties) were characterized by two-dimensional thin-layer chromatography (TLC), and many were subsequently identified by infrared and mass spectrometry. On the basis of the data obtained with 14C1-naphthyl-labeled carbaryl, it appeared that 18.4% of the total characterized metabolites represented unconjugated N-CH2OH- carbaryl [1-naphthyl N-(hydroxymethyl) carbamate], excreted by the cells into the culture medium. The metabolites found in the cells primarily consisted of conjugates of 1-naphthol (73.6% of the total characterized metabolites) and N-CH2OH-carbaryl (2.5%). Conjugates of 7-hydroxycarbaryl (7-hydroxy-1-napthyl methylcarbamate), 4-hydroxycarbaryl (4-hydroxy-1-naphthyl methylcarbamate), and 5-hydroxycarbaryl (5-hydroxy-1-naphthyl methylcarbamate) were also detected in small amounts. Of five unknown 14C1-naphthyl-labeled carbaryl metabolites, three were tentatively characterized as: O-1-naphthylcholesterol (Cholest-5-en-3beta-yl-1-napthol: 3.0%); an unconjugated hydroxylated 1,4-dihydro-1,4-epiperoxynapththalene (1.4%); and an acidlabile, beta-glucosidase-resistant conjugate of a cis-dihydrodiol of 1-naphthol (0.3%; other than the trans-5,6-dihydrodiol). The cholesterol derivative may represent a new "detoxification mechanism" in plants; the epiperoxide may help to elucidate plant oxidation mechanisms. A new TLC procedure was developed which successfully separated the acetate derivative of N-hydroxycarbaryl (1-naphthyl N-hydroxy-N-methylcarbamate) from 12 other common moieties of carbaryl metabolites and their acetate derivatives. A new two-dimensional TLC system was developed for the separation of underivatized N-hydroxycarbaryl from 14 other moieties of carbaryl metabolites; two additional two-dimensional TLC systems were utilized for moiety separations. With these TLC procedures, no conjugated or unconjugated N-hydroxycarbaryl could be detected in any tobacco cell culture fraction after incubation of cells in medium containing radiolabeled carbaryl. Authentic 14C1-naphthyl-labeled N-CH2OH-carbaryl was shown to be converted to desmethylcarbaryl (1-naphthylcarbamate) 97%) and 1-naphthol (3%) by 0.1N HCl hydrolysis. |
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