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

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.     

Effect of herbicide antidotes on glutathione content and glutathione S-transferase activity of sorghum shoots

The effects of the herbicide antidotes CGA-92194 (α-[(1,3-dioxolan-2-yl-methoxy)-imino]benzeneacetonitrile), flurazole [phenylmethyl 2-chloro-4-(trifluoromethyl)-5-thiazolecarboxylate], dichlormid (2,2-dichloro-N,N-di-2-propenylacetamide), and naphthalic anhydride (1H,3H-naphtho(1,8-cd)-pyran-1,3-dione) on nonprotein thiol content, glutathione content, and glutathione S-transferase (GST) activity in etiolated sorghum (Sorghum bicolor L.) Moench) shoots were examined. CGA-92194 and naphthalic anhydride had no effect on nonprotein thiol or reduced glutathione (GSH) content of sorghum shoots. In contrast, dichlormid and flurazole increased nonprotein thiol content of sorghum shoots by 24 and 48%, respectively. These increases were largely attributable to an increase in GSH. The antidotes increased GST activity less than twofold when using CDNB (1-chloro-2,4-dinitrobenzene) as a substrate. In contrast, when using metolachlor [2-chloro-N-(2-ethyl-6-methylphenyl)-N-(2-methoxy-1-methylethyl)acetamide] as a substrate, the increase in GST activity in response to antidote treatment was much greater: flurazole (30-fold), CGA-92194 (20-fold), naphthalic anhydride (17-fold), dichlormid (5-fold). The degree of protection from metolachlor injury conferred by a particular antidote was strongly correlated (R² = 0.95) with its ability to enhance GST activity, as evaluated with metolachlor as substrate. A comparison of GST activity in untreated and CGA-92194-treated seedlings, over a range of metolachlor concentrations (0.5–500 μM), indicated that the relative enhancement of enzyme activity by CGA-92194 was greater at lower metolachlor concentrations. The rate of nonenzymatic conjugation of metolachlor and GSH in vitro was much less (on a gram fresh weight equivalent basis) than the enzymatic rate. These results are consistent with the hypothesis that the above antidotes protect sorghum by enhancing GST activity which results in accelerated detoxification of metolachlor via GSH conjugation.     

Induction of wheat and maize glutathione S-transferase by some herbicide safeners and their effect on enzyme activity against butachlor and terbuthylazine

The expression of glutathione S-transferase (GST) activity in wheat and maize shoots was investigated in response to treatments with the herbicide safeners benoxacor, cloquintocet-mexyl, fenchlorazole-ethyl, fenclorim, fluxofenim and oxabetrinil. These safeners significantly enhanced the GST activity towards 1-chloro-2,4-dinitrobenzene (CDNB) as a 'standard' substrate, with the exception of oxabetrinil in maize. The enhancements of GST (CDNB) activity were found to be concomitant with increases in V(max) (the reaction rate when the enzyme is fully saturated by the substrate) in wheat following cloquintocet-mexyl and fenchlorazole-ethyl treatments, and in maize following fenchlorazole-ethyl treatment. Otherwise, decreases in V(max) were observed in wheat and maize following fenclorim and fluxofenim treatments. With the exception of oxabetrinil, all the safeners significantly reduced the apparent K(M) (the substrate concentration required for 50% of maximum GST activity) of both wheat and maize GST. The V(max) and K(M) variations following safener treatments are discussed in terms of an increased expression of GST enzymes and an increased affinity for the CDNB substrate. The activity of wheat and maize GST was also assayed towards butachlor and terbuthylazine respectively; the results indicate the ability of cloquintocet-mexyl, fenchlorazole-ethyl and fluxofenim to enhance the enzyme activity in wheat and of benoxacor and fenchlorazole-ethyl to do so in maize.     

Toxic effects of cadmium,acetochlor and bensulfuron-methyl on nitrogen metabolism and plant growth in rice seedlings

The adverse effects of cadmium (Cd), acetochlor (AC), bensulfuron-methyl (BSM) alone and their coexistence on nitrogen metabolism and plant growth in rice seedlings were studied with aquatic culture experiments. The results demonstrated that in all exposure groups there were marked decrease in the fresh weight of rice seedlings and the activities of nitrate reductase (NR), glutamine synthetase (GS) in the roots and shoots, consequently leading to an increase in free amino acids content and decrease in soluble protein content and nitrate content in the roots and shoots. These results suggested that the toxicity of Cd, AC and BSM to the rice seedlings took place partly through the influence on nitrogen metabolism.     

Relationship between safening activity of dymron and fenclorim to pretilachlor and glutathione S-transferase activity in rice

Dymron [1‐(α,α‐dimethybenzyl)‐3‐(p‐tolyl)urea] and fenclorim (4,6‐dichloro‐2‐phenylpyrimidine) were found to exhibit a safening activity on the growth of rice (Oryza sativa L.) seedlings against pretilachlor [2‐chloro‐2′,6′diethyl‐N‐(2‐propoxyethyl)acetanilide] injury. By pretilachlor treatment at 10^–6 and 10^–5 mol L^–1, the elongation of the third leaves of rice seedlings was reduced by approximately 20 and 40%, and that of the fourth leaves was reduced by approximately 40 and 80%, respectively. Upon the treatment of dymron at 3 × 10^–6 and 10^–5 mol L^–1 in combination with pretilachlor, the growth inhibition was half alleviated in the third leaves, and the length of the fourth leaves was almost recovered from 10^–6 mol L^‐1 pretilachlor injury, and was 20–25% recovered from 10^–5 mol L^–1 pretilachlor injury. Upon the treatment of fenclorim at 3 × 10^–6 and 10^–5 mol L^–1 in combination with pretilachlor, the growth inhibition of rice seedlings was almost alleviated in both the third and the fourth leaves. This result indicated that dymron and fenclorim showed almost the same safening effect on the fourth leaf growth against 10^–6 mol L^‐1 pretilachlor injury, although fenclorim showed higher effects at higher concentrations of pretilachlor. Glutathione S‐transferase (GST) activities in rice seedlings were investigated after being treated with a herbicide and safener. By pretilachlor treatment at 10^–6 and 10^–5 mol L^–1, the GST activity was approximately 32 and 72% increased in roots, respectively, and a little increased (7–13%) in shoots of two‐leaf‐stage rice seedlings. By dymron treatment at 3 × 10^–6?10^–5 mol L^–1, the GST activity was 2–30% increased in roots, but was not increased in shoots. By their combination treatment, the GST activity was almost the same or less than that by treatment with pretilachlor alone. In contrast, by fenclorim treatment alone, the GST activity was 43–52 and 33–45% increased in roots and shoots of rice seedlings, respectively. By the combination treatment of pretilachlor and fenclorim, the GST activity was increased 73–126% in shoots and 101–139% in roots, and was much more increased in both shoots and roots compared with treatment of pretilachlor or fenclorim alone. It was found that dymron showed less effect in increasing the GST activity than fenclorim. It is also suggested that dymron did not increase the GST activity in shoots but did increase it slightly in roots, and showed almost no effect on GST increase by pretilachlor in shoots, or rather reduced the increase in roots. From the above results, fenclorim and dymron may have different mechanisms of safening effects on the protection of rice seedlings against pretilachlor injury.     

Dichloroacetamide antidotes enhance thiocarbamate sulfoxide detoxification by elevating corn root glutathione content and glutathione S-transferase activity

Glutathione (GSH) content and GSH S-transferase activity are consistently increased in corn roots on 24-hr exposure of corn seedlings to part per million levels of N,N-diallyl-2,2-dichloroacetamide (R-25788) and related antidotes for thiocarbamate herbicide injury in susceptible corn varieties. This combined enhancement of enzyme activity and cofactor level leads to rapid detoxification of thiocarbamate sulfoxides, which are proposed to be the active herbicidal compounds formed on metabolic sulfoxidation. S-(N,N-Dipropylcarbamyl)-GSH is formed by this enzyme-catalyzed detoxification of EPTC sulfoxide. This hypothesis on antidote mode of action is supported by studies on 32 dichloroacetamides and related compounds and on the concentration- and time-dependent relationships of R-25788 action. The liver GSH content is normal in mice injected with high doses of R-25788, but the content is reduced when EPTC or EPTC sulfoxide is administered. EPTC sulfoxide also carbamoylates the thiol group of coenzyme A in neutral aqueous medium.     

Effect of monooxygenase inhibitors on bentazon uptake and metabolism in maize cell suspension cultures

Maize “black Mexican sweet” (BMS) cell suspension cultures were used to study the effects of various cytochrome P450 monooxygenase inhibitors on the uptake and metabolism of the herbicide bentazon. Maize cells rapidly absorbed bentazon and metabolized it via aryl hydroxylation and glycosylation to a glycosyl conjugate of 6-hydroxybentazon. BMS cells accumulated bentazon to levels approximately 20-fold greater than those in the external medium. When BMS cells were incubated in an external medium containing 25 μM bentazon, the formation of the glycosyl conjugate (ca. 2 nmol/min/g fresh wt) was rate-limited by aryl hydroxylation. Tetcyclacis, a plant growth retardant, phenylhydrazine, a mechanism-based cytochrome P450 inhibitor, and piperonyl butoxide, an insecticide synergist, inhibited bentazon metabolism with I₅₀ values of approximately 0.1, 1.0, and 1.0 μM, respectively. Other mechanism-based cytochrome P450 inhibitors, 3(2,4-dichlorophenoxy)-1-propyne and aminobenzotriazole, also inhibited bentazon metabolism but were less effective. The results obtained with selected inhibitors are consistent with the hypothesis that aryl hydroxylation of bentazon is catalyzed by a cytochrome P450 monooxygenase.     

Propanil activity, uptake and metabolism in resistant Echinochloa spp. biotypes

Field resistance of Echinochloa spp. to propanil has been previously reported in Costa Rica, Colombia and Arkansas (USA). In this study, the mechanism of resistance was investigated in three resistant (R) and three susceptible (S) biotypes. The shoot fresh weight reduction in pot-grown plants from a post-emergence spray of propanil at 2.44 kg a.i. ha⁻¹ on biotypes R/S from Costa Rica, Colombia and Arkansas was 35/98%, 25/79% and 20/82% respectively. In vitro chlorophyll fluorescence data from leaf tissue incubated in propanil showed that photosynthesis was inhibited in all biotypes, indicating that the propanil-binding site and enzyme were not altered. After transfer to herbicide-free solution, photosynthesis recovered only in resistant biotypes, indicating that the mechanism of resistance was caused by enhanced metabolism of the herbicide. Simultaneous treatment with fenitrothion, an aryl acylamidase inhibitor, prevented the recovery of photosynthesis in leaf tissue in two resistant biotypes. In contrast, the cytochrome P450 mono-oxygenase inhibitor, 1-aminobenzotriazole, did not prevent recovery from propanil in leaf tissue. Application of ¹⁴C-propanil to the second leaf of intact Echinochloa plants showed that c . 90% of the radioactivity remained in the treated leaf for up to 72 h after application. No major differences in translocation between R and S biotype plants were found. TLC analysis of tissue extracts from the treated leaves showed substantially less radioactivity associated with propanil, present after 72 h in rice or in the three R biotypes, compared with S biotypes.     

The effect of temperature on glutathione S-transferase activity and glutathione content in Alopecurus myosuroides (black grass) biotypes susceptible and resistant to herbicides

Herbicide‐resistant populations of Alopecurus myosuroides (black grass) have become widespread throughout the UK since the early 1980s. Previous observations in this laboratory have demonstrated that natural climatic fluctuations caused increases in endogenous glutathione S‐transferase (GST) enzyme activity in A. myosuroides plants as they mature, which is thought to be linked to herbicide resistance in this species. The present study has investigated the effects of plant growth at 10°C and 25°C, and reports GST specific activity and glutathione (GSH) pool size in resistant and susceptible A. myosuroides biotypes. Findings demonstrate differences in GST activity between resistant and susceptible populations, which are transient at lower growth temperatures. The GSH pool size was elevated at lower growth temperature in both biotypes. We speculate that these endogenous responses are part of a natural mechanism of acclimation to environmental change in this species and suggest that resistant plants are more able to adapt to environmental stress, as indicated in this instance by temperature change. These observations imply that the control of resistant A. myosuroides by graminicides may be more effective when applied at lower temperatures and at earlier growth stages.     

阔草清与乙草胺混用防除大豆、玉米田杂草药效试验

为了明确阔草清与乙草胺混用的剂量、配伍量、防除效果、安全性及残留对后茬作物的影响，特安排本试验，现将试验结果报告如下：     

扑草净·乙草胺防除玉米田杂草的效果

玉米田用40%扑草净.乙草胺WP 100~150g/667m2,在玉米播后苗前对水喷雾,能有效防除马唐、马齿苋、凹头苋、辣蓼、酢浆草、碎米莎草、粟米草等杂草。可在生产中推广应用。     

New, quick tests for herbicide resistance in black-grass (Alopecurus myosuroides Huds) based on increased glutathione S-transferase activity and abundance

Black-grass (Alopecurus myosuroides Huds) is a major grass weed in winter cereals in Europe. It reduces yields and can act as a secondary host for a range of diseases. Herbicide resistance in this species was first detected in the UK in the early 1980s, and has now been reported in thirty counties. To successfully manage herbicide resistance it is vital that suspect populations are tested so that appropriate action can be taken. Ideally, a test will be quick, cheap and easy to use. Furthermore, it should provide an unequivocal result before post-emergence herbicides are to be applied, allowing alternative strategies to be adopted where necessary. This paper reports the development of new tests for herbicide resistance based on our observation that the resistant black-grass biotype Peldon contains approximately double the activity of the enzyme glutathione S-transferase (GST) compared with susceptible biotypes. Data are presented on the production of a monoclonal antiserum to a novel 30 kDa GST polypeptide purified from the biotype Peldon. An ELISA using this antiserum is described and the utility of this assay to detect resistant black-grass biotypes in plants grown under glass and in the field is presented. In addition, a microtitre assay for GST activity is described, which allows the rapid assessment of GST activities of plants. Both abundance and activity of GSTs are discussed as markers for herbicide resistance in black-grass.     

Pre-treatment environmental effects on the uptake, translocation, metabolism and performance of fluazifop-butyl in Elymus repens

The performance of fluazifop-butyl against Elymus repens (L.) Gould was significantly influenced by the environmental conditions in which the plants had grown prior to treatment as follows: soil moisture deficit (greatest reduction of herbicide performance) > cool temperatures > low light intensity. The level of control under conditions in which none of these factors was reduced (so-called ‘standard’ conditions) was similar to that observed for‘low light’regime plants. Significant effects of environment on spray retention, foliar uptake and amounts of herbicide translocated to the roots and rhizomes were observed. The lowest rates of herbicide uptake were found with plants grown under cool conditions, the greatest amount of basipetal herbicide translocation being associated with low light intensities. Rates of herbicide de-esterification were much lower in plants grown under low light intensities, cool temperatures, or soil moisture deficits than in those plants grown under the ‘standard’ conditions. This result was confirmed by studies of herbicide deesterification using cell-free leaf homogenates.     

The effects of patulin from Penicillium vulpinum on seedling growth,root tip ultrastructure and glutathione content of maize

A culture of Penicillium vulpinum CM1 isolated from a soil sample cultivated with maize was examined for the production of the mycotoxins cyclopiazonic acid, griseofulvin, patulin (PAT), and roquefortine C. The fungal strain was positive for PAT- and roquefortine C–producing ability, while it showed negative producing ability for cyclopiazonic acid, and griseofulvin. Both PAT and roquefortine C toxins were tested for their inhibitory effect on the germination of maize seeds. Roquefortine C showed no phytotoxicity up to a concentration of 100 μg ml^?1. However, a notable reduction in the average values of fresh weight and length of roots and shoots was observed following application of PAT and a culture filtrate of P. vulpinum to maize seedlings. The phytotoxic effect was found to be dose-dependent. To analyse the cellular effects of PAT, maize root tips were treated with PAT at different concentrations. The higher dose of 25 μg ml^?1 exhibited morphological changes in the nature of cytoplasm and cytoplasmic organelles as was shown by transmission electron microscopy. Furthermore, an increase in the vacuolation with localized tonoplast dissolution was observed. At this concentration, there was evidence of a disruption of lipid metabolism. On the metabolic level, the phytotoxicity of PAT and the culture filtrate of P. vulpinum led to alterations in the total reduced glutathione (GSH) concentration in maize seedlings. The GSH concentrations examined in roots and shoots after PAT, fungal filtrate or fungal crude extract treatments were enhanced compared with control treatments. Findings further showed higher total GSH levels in shoots than in roots following treatments either with PAT or the fungal crude extract. The results are discussed in the context of known herbicide and metal effects on GSH synthesis and transport mechanisms.     

Differential glutathione S-transferase isozyme activities in rice and early watergrass seedlings

Glutathione S -transferase (GST) isozymes were investigated in one-leaf-stage rice ( Oryza sativa L. cv. Nipponbare) and early watergrass ( Echinochloa oryzicola Vasing) shoots after being induced by treatment with a combination of pretilachlor [2-chloro-2',6'-diethyl-N-(2-propoxyethyl)acetanilide] and fenclorim (4,6-dichloro-2-phenylpyrimidine) using DEAE-Sephacel anion exchange chromatography. Non-treated plants contained GST isozymes that had activity to the following substrates: three isozymes for l-chloro-2,4-dinitrobenzene (CDNB), six isozymes for pretilachlor and three isozymes for fenclorim in rice shoots; and four isozymes for CDNB, three or four isozymes for pretilachlor and two or three isozymes for fenclorim in early watergrass shoots. Glutathione S -transferase isozyme activities of non-treated plants were higher in rice than in early watergrass, especially in the case of GST activity with fenclorim as a substrate. Pretreatment of rice roots with a combination of pretilachlor and fenclorim increased the activity of the constitutively expressed isozymes that exhibited activity with CDNB, pretilachlor and fenclorim. This pretreatment also caused the appearance of one new GST(fenclorim) isozyme. Pretreatment of early watergrass roots with a combination of pretilachlor and fenclorim produced almost no increase in activity of some constitutively expressed isozymes that exhibited activity to CDNB and fenclorim, although it partly increased the peaks to corresponding to pretilachlor. The induction of GST was higher in rice than that in early watergrass. These results indicated that the isozyme pattern and substrate specificity of GST isozymes in rice were different from those in early watergrass. Furthermore, the selectivity of pretilachlor between rice and early watergrass may be related to different constitutively expressed GST(pretilachlor) isozyme activities and the induction of GST(pretilachlor) isozyme activities in the combination treatment.     

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

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→¹S-(pentachlorophenyl)glutathione→²S-(pentachlorophenyl)-γ-glutamylcysteine→³S-(pentachlorophenyl)cysteine→⁴ PCTP→⁵ PCTA. The first reaction was studied with [¹⁴C]PCNB. Reactions 2–4 were studied with S-([¹⁴C]pentachlorophenyl)glutathione, S-([¹⁴C]pentachlorophenyl)cysteine, and peptide inhibitors. Reaction 5 was studied with [¹⁴C]PCTP, S-[¹⁴C]adenosylmethionine, and inhibitors. The possible use of the enzyme system in the characterization of other glutathione conjugates was discussed.     

Rapid effects of a thiocarbamate herbicide and its dichloroacetamide protectant on macromolecular syntheses and glutathione levels in maize cell cultures

The rapid effects of the thiocarbamate herbicide S-ethyl dipropyl thiocarbamate (EPTC) and the herbicide protectant N,N-diallyl-2,2-dichloroacetamide (DDCA) on macromolecular syntheses and glutathione (GSH) levels in maize cell cultures were studied to determine whether stimulation of GSH could be the primary mechanism of action of DDCA. EPTC (0.5 and 1 mM) reduced incorporation of radioactive precursors within 1 hr after treatment, and affected incorporation of [³H]acetate into lipids more than incorporation of [³H]adenosine into acid-precipitable nucleic acids, or [¹⁴C]protein hydrolysate into protein. [¹⁴C]EPTC was rapidly biotransformed within 8 hr by maize cell suspensions. Measureable decreases in GSH levels following treatment with 1 mM EPTC occurred after 15 hr. DDCA stimulated incorporation of [³H]acetate into lipids within 4 hr but did not affect incorporation of [¹⁴C]protein hydrolysate into protein or [³H]adenosine incorporation into nucleic acids. Measureable increases in GSH following DDCA treatment began after 12 hr. Treatment with EPTC and DDCA in combination inhibited incorporation of [³H]acetate into lipids less than EPTC given alone. Increases in GSH levels could be observed following pretreatments with glutathione precursors, but no protectant activity could be detected, in contrast to treatments with DDCA. It is suggested that DDCA has an initial rapid effect on lipid metabolism followed by a slower effect involving increases in cellular GSH.     

Effect of E-cinnamaldehyde against Sclerotinia sclerotiorum on potato and induction of glutathione S-transferase genes

This research was carried out to assess the effect of E-cinnamaldehyde (EC) against Sclerotinia sclerotiorum, causal agent of potato white mold, under invivo and invitro conditions. Based on the results, EC was able to completely inhibit (P < 0.05) mycelial growth of four isolates in both volatile and contact phases after five days of inoculation at the concentrations 500 μl and 5 μl/ml, respectively. Moreover EC at concentrations 30 and 50 μl EC/ml sterile distilled water significantly reduced white mold on six potato cultivars including Pashandi, Istambouli, Agria, Marfauna, Alpha and Spartaan in greenhouse trials. In next step, induction of glutathione S-transferase (GST)-like genes identified from the pathogen (isolate 3) genome was assessed. After 2 h of exposure to both EC and cinnamom extract, four genes with locus numbers SS1G_07319.1, SS1G_07195.1, SS1G_00703.1 and SS1G_01918.1 were up-regulated. Furthermore, EC and cinnamon extract appeared to have no effect on gene expression in SS1G_10295.1. In this study, enzyme activity of GST was determined with 1-chloro-2, 4-dinitrobenzene as substrate. While enzymatic activity was 42.3 and 45.6 units mg⁻¹ protein in sterilized distilled water and ethyl acetate, respectively, it was recorded as 78.9 and 86.4 units mg⁻¹ protein for cinnamon extract and EC, respectively.