Donald G. Rusness

Propachlor metabolism in soybean plants, excised soybean tissues, and soil

Abstract Propachlor was rapidly metabolized to a homoglutathione conjugate in the roots and foliage of soybean plants. No other competing reactions were observed. The homoglutathione conjugate was rapidly metabolized to the cysteine conjugate which was slowly converted to a variety of other metabolites, four of which were present up to 72 days after treatment. Those four metabolites were the malonylcysteine, malonylcysteine S -oxide, 3-sulfinyllactic acid, and the O -malonylglucoside conjugates of propachlor. A cysteine S -oxide conjugate was also observed as a transient metabolite. Fast atom bombardment mass spectrometry was used to characterize the polar metabolites. Some metabolites were isolated and identified from both soybean plants and peanut cell suspension cultures. The terminal metabolites in soybean plants were concentrated in the roots and foliage. Less than 1% of the metabolites were present in the beans and pods of the mature plants. In a sandy loam soil, propachlor and the cysteine conjugate of propachlor were both metabolized in approximately the same ratio to the bound residue fraction and three major metabolites: N -isopropyloxanilic acid, 2-sulfo- N -isopropylacetanilide, and 2-(sulfinylmethylenecarboxy)- N -isopropylacetanilide. The methyl sulfoxide and methyl sulfone analogs of propachlor were characterized as minor soil metabolites. The major metabolites present in soybean plants grown in soil treated with propachlor were those produced in the soil and taken up by the plants. It was speculated that propachlor metabolism in the soil involved microbial activity and conjugation with glutathione or cysteine. It was concluded that homoglutathione conjugates are metabolized in soybean in a manner similar to the metabolism of other glutathione conjugates in species such as peanut.

Diphenyl ether herbicide metabolism in a spruce cell suspension culture: The identification of two novel metabolites derived from a glutathione conjugate☆

Abstract [ CF 3 - 14 C]Fluorodifen herbicide (2,4′-dinitro-4-trifluoromethyldiphenyl ether) was rapidly metabolized by a spruce cell suspension culture ( Picea abies L. Karst). The primary route of metabolism involved cleavage of the diphenyl ether bond by glutathione (GSH). The resulting conjugate of fluorodifen ( S -[4-trifluoromethyl-2-nitrophenyl]glutathione) appeared to be metabolized sequentially to the corresponding γ-glutamylcysteine conjugate, the cysteine conjugate, and two novel metabolites. These novel metabolites, identified by fast atom bombardment mass spectrometry and enzyme hydrolysis, were the S -glucoside and the S -(3-thio-2- O -glucosyl) lactic acid conjugate of 4-trifluoromethyl-2-nitrobenzene. This appears to be the first report on the plant metabolism of GSH conjugates to metabolites of these classes. During the metabolism of [ 14 C]fluorodifen, a loss of water-soluble 14 C-labeled metabolites from the spruce cells to the growth medium was observed. After 1 day, 63% of the applied 14 C was present as metabolites in the cells and 30% was present as metabolites in the medium. However, after 10 days only 14% of the applied 14 C was present as metabolites in the cells and 50% was present as metabolites in the medium. During this period, the recovery of 14 C declined from 97% after 1 day to 64% after 10 days. The decrease in recovery may have been due to the loss of volatile metabolites such as 4-trifluoromethyl-2-nitrothiophenol. The major metabolites present after 1 day were the GSH conjugate (36%), the γ-glutamylcysteine conjugate (35%), and the cysteine conjugate (14%). After 10 days, the major metabolites were the S -glucoside (45%) and the S -(2- O -glucosyl)-3-thiolactic acid conjugate (12%).

Synergism of diazinon toxicity and inhibition of diazinon metabolism in the house fly by tridiphane: Inhibition of glutathione S-transferase activity☆

Abstract Diazinon toxicity to a susceptible strain of house fly ( Musca domestica L.) was synergized by tridiphane [2-(3,5-dichlorophenyl)-2-(2,2,2-trichloroethyl)oxirane], a herbicide synergist. Both diazinon and tridiphane were partially metabolized in the house fly by glutathione (GSH) conjugation. Synergism appeared to be due to inhibition of diazinon metabolism/detoxification. Crude glutathione S -transferase (GST) preparations from the house fly catalyzed GSH conjugation of diazinon, tridiphane, 3,4-dichloronitrobenzene (DCNB), and chloro-2,4-dinitrobenzene (CDNB). Tridiphane and the GSH conjugate of tridiphane appeared to inhibit diazinon GSH conjugation, but diazinon did not inhibit tridiphane GSH conjugation. The enzymatic rate of tridiphane GSH conjugation was 22 times the rate of diazinon GSH conjugation; therefore, attempts to assay tridiphane as an inhibitor of diazinon GSH conjugation were inconclusive because of the high concentration of tridiphane GSH conjugate produced during the assay. CDNB underwent enzymatic GSH conjugation at a rate 240 times faster than that of tridiphane and 5000 times faster than that of diazinon. GSH conjugation of CDNB was not inhibited by tridiphane, but was inhibited by the GSH conjugate of tridiphane. In vivo , the GSH conjugate of tridiphane was produced in sufficient concentration to cause the observed inhibition of diazinon metabolism and synergism of diazinon toxicity. However, the possibility that parent tridiphane caused or contributed to the inhibition of diazinon metabolism and synergism of diazinon toxicity could not be excluded. Inhibition of diazinon metabolism did not appear to be due to depletion of either GSH or GST.

The effect of CDAA (N,N,-diallyl-2-chloroacetamide) pretreatments on subsequent CDAA injury to corn (Zea mays L.)

Abstract The effects of CDAA ( N,N -diallyl-2-chloroacetamide) pretreatment on subsequent CDAA injury to corn were examined and compared with the effects of the herbicide protectant R-25788 ( N,N ,-diallyl-2,2-dichloroacetamide). In addition, the effects of CDAA pretreatment on subsequent CDAA metabolism were determined. It was found that 5μ M CDAA protected corn from injury by 200 μ M CDAA when given as a 2.5- or 1-day pretreatment. R-25788 at similar concentrations did not protect corn from subsequent R-25788 injury. Pretreatment with CDAA increased GSH levels of corn roots by 61% within 1 day, and these levels did not increase with a longer 2.5-day pretreatment with CDAA. GSH- S -transferase activity was assayed spectrophotometrically using CDNB (1-chloro-2,4-dinitrobenzene). A 1-day pretreatment with CDAA increased the root GSH- S -transferase activity by 35%, but did not affect shoot GSH- S -transferase activity. A 2.5-day pretreatment resulted in a 50% increase in root GSH- S -transferase activity but no response of the shoot enzyme was observed. Even longer pretreatments with CDAA did not result in any further increases in enzyme activity. When corn roots pretreated with CDAA for 2.5 days were excised and incubated with radiolabeled CDAA, they exhibited greater rates of uptake and metabolism than did nonpretreated roots. With in vitro studies, a fairly high rate of nonenzymatic degradation of CDAA was observed. However, the enzymatic rate was always double that of the nonenzymatic rate under the experimental conditions used. It is concluded that elevations in the GSH levels and GSH- S -transferase activities of corn roots following CDAA pretreatments may be involved in the protection of corn from subsequent CDAA injury.

Glutathione S-transferase activity in spruce needles

Abstract Glutathione S -transferase activity was present in extracts from needles of two different spruce species ( Picea abies and Picea glauca ). In vitro conjugation studies were conducted with three 14 C herbicides and one 14 C fungicide: atrazine (2-chloro-4-ethylamino-6-isopropylamino- s -triazine), fluorodifen (2,4′-dinitro-4-trifluoromethyl diphenyl-ether), propachlor (2-chloro- N -isopropylacetanilide), and pentachloronitrobenzene (PCNB). The enzymes from both P. abies and P. glauca showed the highest rates of enzymatic conjugation for fluorodifen as the substrate while intermediate to low rates of enzymatic conjugation were observed with PCNB and propachlor. Atrazine was not an appreciable substrate for the enzymes of either species. The water-soluble 14 C conjugation products of the enzymatic reactions were assayed by liquid scintillation spectrometry. The [ 14 C]glutathione conjugates from fluorodifen and PCNB were identified by a combination of thinlayer chromatography (TLC), high-performance liquid chromatography (HPLC), and fast atom bombardment mass spectrometry and the [ 14 C]glutathione conjugate of propachlor was identified by TLC and HPLC comparison to an authentic standard. The catalytic properties of glutathione S -transferase from P. abies were analyzed with CDNB as substrate. The apparent K M values were 0.14 m M for GSH and 0.67 m M for CDNB, respectively, the pH optimum was between 7.6 and 8.0, and the temperature optimum was 40–45°C. The activation energy was calculated to be 32.4 kJ mol −1 .

In vitro metabolism of pentachloronitrobenzene to pentachloromethylthiobenzene by onion: Characterization of glutathione S-transferase, cysteine C-S lyase, and S-adenosylmethionine methyl transferase activities☆

Abstract Pentachloromethylthiobenzene (PCTA) was synthesized in vitro from pentachloronitrobenzene (PCNB) at pH 7.9 by an enzyme system from onion root that required dithiothreitol, glutathione, and S -adenosylmethionine. The soluble enzyme system was isolated from onion root by ammonium sulfate fractionation and differential centrifugation. The system contained glutathione S -transferase activity with PCNB, C-S lyase activity with S -(pentachlorophenyl)cysteine, S -adenosylmethionine methyl transferase activity with pentachlorothiophenol (PCTP), and presumably several peptidase activities. All activities were stable when the crude enzyme system was stored at −25°C. Evidence for the following sequence of reactions in PCTA synthesis was presented: PCNB→ 1 S -(pentachlorophenyl)glutathione→ 2 S -(pentachlorophenyl)-γ-glutamylcysteine→ 3 S -(pentachlorophenyl)cysteine→ 4 PCTP→ 5 PCTA. The first reaction was studied with [ 14 C]PCNB. Reactions 2–4 were studied with S -([ 14 C]pentachlorophenyl)glutathione, S -([ 14 C]pentachlorophenyl)cysteine, and peptide inhibitors. Reaction 5 was studied with [ 14 C]PCTP, S -[ 14 C]adenosylmethionine, and inhibitors. The possible use of the enzyme system in the characterization of other glutathione conjugates was discussed.

The mechanism of action of BAS 145 138 as a safener for chlorimuron ethyl in corn: Effect on hydroxylation, glutathione conjugation, glucoside conjugation, and acetolactate synthase☆

Abstract BAS 145 138 (1-dichloroacetylhexahydro-3,3,8α-trimethylpyrrolo[1,2-α]pyrimidin-6-(2 H -one)) protected corn from injury due to low levels of chlorimuron ethyl ( N -[4-chloro-6-methoxy-pyrimidin-2-yl]- N′ -[2-ethoxycarbonylbenzenesulfonyl]urea). Inhibition of root growth was used to monitor injury. BAS 145 138 did not affect uptake, but it caused a ca. two-fold increase in the rate of chlorimuron ethyl metabolism in corn roots and shoots. The increase in chlorimuron ethyl metabolism was correlated positively to growth. Routes of metabolism that were accelerated in response to BAS 145 138 included hydroxylation of chlorimuron ethyl at the 5-position of the pyrimidine ring, the formation of the corresponding glucoside, the formation of two glutathione conjugates, and the formation of two unidentified metabolites. The most dramatic of these effects was on the formation of the glucoside of 5-hydroxychlorimuron ethyl. In the roots, the level of this metabolite was increased ca. six-fold in response to BAS 145 138. Part of this increase was due to an elevation in the in vivo rate of glucosylation of 5-hydroxychlorimuron ethyl. BAS 145 138 did not alter the qualitative routes of chlorimuron ethyl metabolism, nor did it appear to affect the levels or catalytic properties of acetolactate synthase, the target enzyme of chlorimuron ethyl. 5-Hydroxychlorimuron ethyl was 152 times less effective as an inhibitor of acetolactate synthase than chlorimuron ethyl. The glucoside of 5-hydroxychlorimuron ethyl was hydrolyzed during the acetolactate synthase assay; therefore, an I 50 value for this metabolite was not obtained.

MALONYLCYSTEINE CONJUGATES AS END-PRODUCTS OF GLUTATHIONE CONJUGATE METABOLISM IN PLANTS

Malonylcysteine conjugates were formed in peanut cell suspension cultures from seven out of twelve pesticides examined. Each of these pesticides was thought to be initially metabolized to a glutathione conjugate. Malonylcysteine conjugates of PCNB were detected in eight out of eleven plant species examined and a malonylcysteine conjugate was a major metabolite of EPTC in corn, cotton and soybean. The malonylcysteine conjugate of propachlor was a major terminal metabolite in soybean grown to maturity in hydroponic culture, but was less important in soil-grown soybean. The malonylcysteine sulfoxide conjugate of propachlor was also detected as a metabolite in soybean.

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.

Chlorimuron ethyl metabolism in corn

Abstract [14C]Chlorimuron ethyl was readily absorbed by the roots of young intact corn seedlings and through the cut ends of excised leaves, but it was not readily absorbed by intact leaves. Under the conditions employed, [14C]chlorimuron ethyl was metabolized at a moderate rate in both intact roots and excised leaves (ca. 2.4 nmol/g fresh wt tissue/hr). Based upon high-performance liquid chromatography (HPLC) analysis, [14C]chlorimuron ethyl appeared to be metabolized by similar routes in both the roots and leaves. [14C]Chlorimuron ethyl and 10 radioactive metabolites were detected in the roots of corn 7 hr following herbicide treatment. [14C]Chlorimuron ethyl and seven metabolites, listed in approximate order of their abundance, were isolated and characterized: chlorimuron ethyl (N-[4-chloro-6-methoxypyrimidine-2-yl]-N′-[2-ethoxycarbonylbenzenesul-fonyl]urea; (I) N-(4-chloro-5-hydroxy-6-methoxypyrimidine-2-yl)-N′-(2-ethoxycarbonylbenzene-sulfonyl)urea; (II) 2-ethoxycarbonylbenzene sulfonamide, (IV) N-(4-[S-glutathionyl]-6-methoxypyrimidine-2-yl)-N′(2-ethoxycarbonylbenzenesulfonyl)urea, (VI) N-(4-[S-glutathionyl]-5-hydroxy-6-methoxypyrimidine-2-yl)-N′-(2-ethoxycarbonylbenzenesulfonyl)urea, (III) N-(4-chloro-5-[O-β- d -glucosyl]-6-methoxypyrimidine-2-yl)-N′-(2-ethoxycarbonylbenzenesulfonyl)urea, (VII) N-(4-chloro-6-methoxypyrimidine-2-yl)-N′-(2-ethoxy-?-[O-β- d -glucosyl]benzenesulfonyl)urea, and (V) N-(4-[S-cysteinyl]-6-methoxypyrimidine-2-yl)-N′-(2-ethoxycarbonylbenzenesulfonyl)urea. Chlorimuron ethyl and these metabolites were purified by HPLC and were characterized by fast atom bombardment mass spectrometry (FAB MS). In addition to FAB MS, the following methods were used in the characterization of some metabolites: synthesis, hydrolysis with β-glucosidase, analysis of hydrolysis products, electron impact mass spectrometry, and proton nuclear magnetic resonance (400 MH).