Gerald G. Still

The metabolism of 3,4-dichloropropionanilide in plants. Partial purification and properties of an aryl acylamidase from rice☆

Abstract An aryl acylamidase (aryl-acylamine amidohydrolase, EC 3.5.1.a) from rice, which hydrolyzes 3,4-dichloropropionanilide, has been partially purified and characterized. The distribution of the enzyme in rice and barnyard grass tissues was determined. The enzyme displayed a broad specificity for chlorinated ring-substituted propionanilide analogs, but was specific for 3,4-dichloropropionanalide when compared with several alkyl substituted analogs. The enzyme was inhibited by sulfhydryl reagents and was strongly inhibited at 1·0 × 10 −6 M by the insecticidal carbamates 1-naphthylmethylcarbamate,4-benzothienyl methylcarbamate, 2-chloro-4,5-xylyl methylcarbamate, 2,4,5-trimethylphenyl methylcarbamate, 3,4,5-trimethylphenyl methylcarbamate and 4-(methylthiol) 3,5-xylyl methylcarbamate. The partially purified enzyme had a broad pH optimum between 7·5 and 7·9, with an apparent K m of 2·93 × 10 −3 M for 3,4-dichloropropionanilide. The K i for 1-naphthyl methylcarbamate was 1·51 × 10 −8 M with 3,4-dichloropropionanilide as the substrate. The significance of the enzyme distribution in the resistant and susceptible species and the inhibition of the rice enzyme by insecticidal carbamates is discussed.

Metabolism of 3,4-Dichloropropionanifide in Plants: The Metabolic Fate of the 3,4-Dichloroaniline Moiety

Studies to elucidate the fate of 3,4-dichloropropionanilide (propanil) in rice (Oryza sativa L. var. Nato) plants have shown that the propanil molecule is cleaved and the propionic acid moiety metabolized. To ascertain the fate of the 3,4-dichloroaniline moiety of propanil, rice plants were exposed to propanil in liquid culture. The roots and shoots of treated rice plants were extracted and quantitatively assayed for four aniline-containing metabolites. One of the four metabolites proved to be 3,4-dichloroaniline, while the remaining three metabolites contained complexed 3,4-dichloroaniline. N-(3,4-dichlorophenyl)-glucosylamine was identified as one of the complexes. A time-course study of the four metabolites indicated the appearance of the 3,4-dichloroaniline-containing metabolites within 6 hours. After 14 days of treatment, the complexed aniline metabolites amounted to only 10 percent of the total propanil administered to the rice plants.

Soybean shoot metabolism of isopropyl-3-chlorocarbanilate: Ortho and para aryl hydroxylation

Abstract This laboratory reported that isopropyl-3-chlorocarbanilate-phenyl-U- 14 C (chlorpropham-phenyl- 14 C) was absorbed, translocated, and metabolized by soybean plants. Both polar metabolites and insoluble residues were found in roots, whereas only polar metabolites were found in shoot tissues. In both roots and shoots the polar metabolites were shown to be the O -glucoside of isopropyl-2-hydroxy-5-chlorocarbanilate (2-hydroxy-chlorpropham). In shoot tissue there were other polar metabolites that were not identified. The experiments with soybeans have been repeated, but with new isolation and purification procedures. The plants were root treated with both chlorpropham-phenyl- 14 C and isopropyl-3-chlorocarbanilate-2-isopropyl- 14 C. The roots and shoots were extracted and separated into the polar, nonpolar, and insoluble metabolic components, using the Bligh-Dyer extraction method. The polar metabolites were separated by gel permeation chromatography. Further purification was accomplished on Amberlite XAD-2. The polar metabolites from the shoot and root tissues were hydrolyzed either by β-glucosidase or hesperidinase. The enzyme liberated aglycones were derivatized and separated by gas-liquid chromatography, and the components were characterized by mass spectrometry or NMR. The results of this study showed that the polar metabolites of soybean shoots were 2-hydroxy-chlorpropham and isopropyl-4-hydroxy-3-chlorocarbanilate (4-hydroxy-chlorpropham). These two hydroxy-chlorpropham metabolites were found in soybean shoots at a ratio of approximately 1:1. The only aglycone found in root tissue was 2-hydroxy-chlorpropham. Using the new procedures, no evidence was obtained for the presence of the unidentified polar metabolites that were previously observed in shoot tissues.

Animal metabolism of propham (isopropyl carbanilate): The fate of residues in alfalfa when consumed by the rat and sheep☆

Abstract Alfalfa was root-treated with [14C]propham (isopropyl carbanilate[14C-phenyl(U)]) for 7 days and then harvested and freeze-dried. Rats and sheep were orally given either 14C-labeled alfalfa roots ([14C]root) or 14C-labeled alfalfa shoots ([14C]shoot). When the [14C]root was given, 6.5–11.0% of the 14C was excreted in the urine and 84.6–89.4% was excreted in the feces within 96 h after treatment. Less than 3% of the 14C remained in the carcass (total body—gastrointestinal tract and contents) 96 h after treatment. When [14C]shoot was given, 53.2–55.2% of the 14C was excreted in the urine, 32.1–43.4% was excreted in the feces, and the carcass contained 0.2–1.1% of the 14C 96 h after treatment. When the insoluble fraction (not extracted by a mixture of CHCl3, CH3OH, and H2O) of both alfalfa roots and shoots was fed to rats, more than 86% of the 14C was excreted in the feces and less than 3% remained in the carcass 96 h after treatment. The major radiolabeled metabolites in the urine of the sheep fed 14C shoot were purified by chromatography and identified as the sulfate ester and the glucuronic acid conjugates of isopropyl 4-hydroxycarbanilate. Metabolites in the urine of the sheep treated with [14C]root were tentatively identified as conjugated forms of isopropyl 4-hydroxycarbanilate, isopropyl 2-hydroxycarbanilate, and 4-hydroxyaniline. The combined urine of rats dosed with [14C]shoot and [14C]root contained metabolites tentatively identified as conjugated forms of isopropyl 4-hydroxycarbanilate, isopropyl 2-hydroxycarbanilate, and 4-hydroxyaniline.

Aryl hydroxylation of isopropyl-3-chlorocarbanilate by soybean plants

Abstract Isopropyl-3-chlorocarbanilate-phenyl UL- 14 C (CIPC- 14 C) is absorbed, translocated and metabolized by soybean plants. Water-soluble metabolites in root and shoot were purified and the root major metabolite characterized. The acetylated aglucones from the β-glucosidase hydrolysis and the esters from the direct acetylation of CIPC- 14 C polar metabolites were purified by GLC and analysed by mass spectrometry. The data showed that the phenyl riong of CIPC- 14 C was hydroxylated by both root and shoot tissues. Isopropyl-5-chloro-2-hydroxycarbanilate (hydroxy-CIPC) was the predominant aglucone liberated by β-glucosidase from polar metabolites in root and shoot. The o -glucoside of hydroxy-CIPC was shown to be present, by direct acetylation and characterization. In shoot tissue the major metabolites were dechlorinated hydroxy-CIPC and were not hydrolysed by β-glucosidase. These data show that soybean root or shoot tissues hydroxylate the phenyl ring of CIPC- 14 C but do not alter either the isopropyl alcohol moiety or the e arbamate bond.

The elimination of (N-[[(4-chlorophenyl)amino]carbonyl]-2,6-difluorobenzamide) by the boll weevil☆

Abstract Boll weevils, Anthonomus grandis Boheman, were either dipped in or injected with a solution of [14C]diflubenzuron (N-[[(4-chlorophenyl)amino]carbonyl]-2,6-difluorobenzamide) or fed on cotton squares that had been treated with the chemical to determine its turnover time and metabolic fate. No significant differences were observed between male and female weevils in their ability to eliminate [14C]diflubenzuron. Only minor differences were observed when immersion and injection treatments were compared. When weevils were treated with 66.3 ng of [14C]deflubenzuron per weevil by injection, the insects contained 13 to 15% of the radiolabel after 6 days and 4 to 6% after 13 days. The remainder of the radiolabel was in the frass. When weevils fed for 66 hr on cotton squares that had been treated with a wettable [14C]diflubenzuron preparation (Dimilin W-25), the insects averaged 120 ng of diflubenzuron per weevil. Forty-four hours after removing insects from the treated squares, 50% of the radiolabel had been excreted. In all cases, the radiolabel found in the frass or in the weevil was unchanged diflubenzuron. There were no data to indicate that the boll weevil could metabolize appreciable amounts of diflubenzuron.

Fate of [14C]diflubenzuron on cotton and in soil

Abstract Aqueous suspensions and oil emulsions of a commercial [14C]diflubenzuron (N-[[(4-chlorophenyl)amino]carbonyl]-2,6-difluorobenzamide) formulation (Dimilin W-25) remained on the leaf surface of greenhouse-treated plant tissues. Absorption, translocation, and metabolism of the [14C]diflubenzuron were not significant. Less than 0.05% of the applied 14C was found in newly developed plant tissues 28 days after spray treatment. [14C]Diflubenzuron was degraded in soil. After 91 days, biometer flask studies showed that 28% of the 14C incorporated into the soil as [14C]diflubenzuron was recovered as 14CO2. Major dichloromethane-soluble soil residues were identified as unreacted [14C]diflubenzuron and [14C]4-chlorophenylurea. A minor unknown degradation product cochromatographed with 2,6-difluorobenzoic acid. Insoluble 14C-residues increased with time and represented 67.8% of the residual 14C in the soil 89 days after treatment. Cotton plants grown for 89 days in [14C]diflubenzuron-treated soil contained only 3% of the 14C applied to the soil. Small quantities of acetonitrile-soluble [14C]4-chlorophenylurea were isolated from the foliar tissues. Root tissues contained small amounts of [14C]diflubenzuron and trace quantities of a minor 14C-product that chromotographed similarly to 2,6-difluorobenzoic acid. Most of the 14C in the plant tissues (84–93%) was associated with an insoluble residue fraction 89 days after treatment.

Partial purification and properties of S-cysteinyl-hydroxychlorpropham transferase from oat (Avena sativa L.)☆

Abstract S -Cysteinyl and glutathione conjugates of isopropyl-3′-chloro-4′-hydroxycarbanilate (4-hydroxychlorpropham) were synthesized directly in the presence of soluble enzyme systems isolated from etiolated shoots of oat seedlings. The enzyme systems responsible for these reactions were partially purified and charaterized. Enzyme A appeared to be a multicomponent system, equally reactive with either cysteine or glutathione. Enzyme B was twice as active as enzyme A in the formation of S -cysteinyl-hydroxychlorpropham. Affinity chromatography of enzyme A produced an enzyme fraction with properties similar to those of enzyme B. Both enzymes (A and B) were significantly inhibited by increased cysteine concentrations. The reaction of glutathione with enzyme B was limited. However, when low concentrations of a nonreacting effector, cysteine ethyl ether, were added, glutathione conjugation increased significantly. At higher concentrations, cysteine ethyl ester formed a conjugate with 4-hydroxychlorpropham. Isopropyl-5′-chloro-2′-hydroxycarbanilate (2-hydroxy-5-chlorpropham) did not conjugate with either cysteine or glutathione but did react with cysteine ethyl ester. Isopropyl-3′-chlorocarbanilate (chlorpropham) was not a substrate for thioether conjugation. These data suggest that para - and/or ortho -hydroxylated carbanilates and cysteine-related substrates may form thioether conjugates when incubated under appropriate conditions with these complex enzyme systems.

S-cysteinyl-hydroxychlorpropham: Formation of the S-cysteinyl conjugate of isopropyl-3′-chloro-4′-hydroxycarbanilate in oat (Avena sativa L.)

Abstract Isopropyl-3′-chlorocarbanilate (chlorpropham) forms phenolic metabolites, isopropyl-3′-chloro-4′-hydroxycarbanilate (I), and isopropyl-5′-chloro-2′-hydroxycarbanilate (II), in several plant species. In oat, which is a chlorpropham-susceptible plant, I was converted to an S-cysteinyl-conjugate (III). The reaction in vitro was catalyzed by a partially purified, soluble enzyme. The formation of III by the enzyme preparation and by oat shoot sections was compared. Mass spectral data indicated the presence of an aryl-thioether bond, and chloro-, hydroxy-, and isopropylcarbanilate groups in III. The results of this investigation indicate that III was isopropyl-[(2-amino-2-carboxyethyl)thio]-chloro-hydroxycarbanilate (S-cysteinyl-hydroxychlorpropham).

Alfalfa metabolism of propham

Abstract Root-treated alfalfa absorbs, translocates, and metabolizes [phenyl- 14 C]isopropyl carbanilate ([ 14 C]propham). After 7 days of root treatment, the distribution of radiolabel was 73% for shoots and 27% for roots. Shoots and roots were extracted and separated into the polar, nonpolar, and solid residual components using a mixture of chloroform, methanol and water. The insoluble residues accounted for approximately 40% of the 14 C found in shoots and roots. The nonpolar fraction (6.1% of the radiolabel in shoots and roots) was not characterized, but was shown to be some component other than parent propham. Propham was not found in either shoots or roots. The polar metabolites were partly purified on Amberlite XAD-2. Cellulase-liberated aglycones were derivatized and separated by high-performance liquid and gas-liquid chromatography. The infrared, nuclear magnetic resonance, and mass spectral data showed that the polar metabolites of alfalfa shoots and roots were glycoside conjugates of isopropyl 2-hydroxycarbanilate (2-hydroxypropham) and isopropyl 4-hydroxycarbanilate (4-hydroxypropham). Conjugated 4-hydroxypropham accounted for 45.9% of the 14 C in the shoots and 3.4% of the 14 C in the roots. Conjugated 2-hydroxypropham accounted for 3.4% of the 14 C in the shoots and 1.4% of the 14 C in the roots.