Inhibition of the hill reaction of isolated chloroplasts by herbicidal phenylureas

I₅₀ values have been determined for inhibition of the Hill reaction of lettuce chloroplasts by thirteen phenylurea herbicides, all of which are potent inhibitors. The compounds fall into two groups on the basis of the relationship between pI₅₀ and log (water solubility).     

Inhibitory effects of pesticides on growth and respiration of cultured cells

Effects of various drugs including pesticides on the growth and respiration of cultured cells were evaluated comparatively using cell lines derived from mosquito ovary and subcutaneous mouse tissues. The concentration producing 50% inhibition of cell growth, I₅₀ (M), was determined for each of 42 drugs. Inhibitors of respiration and nucleic acid and protein biosyntheses such as rotenone, piericidin A₁, actinomycin D, and puromycin had very high pI₅₀ values of approximately 8. Except for the compounds known to be uncouplers of oxidative phosphorylation, the drugs suppressed the respiration rate of the cultured cells to various degrees. The pI₅₀ value (and the pEC₁₅₀, 150% enhancement of the control, value for uncouplers) was determined for each compound. By examining the relation of pI₅₀ (and pEC₁₅₀) values between cell growth and respiration, the compounds could be classified into two groups according to their modes of inhibitory action against the cultured cells. One mode relates to the inhibition of energy synthesis and the other, perhaps, to interference with the biosynthesis of biomacromolecules.     

Effect of some herbicides on CO2 fixation,intermediates pattern,and ruDP-carboxylase and FDPase activities of spinach chloroplasts

We have studied the inhibitory effect of the herbicides phenmediphan, chloroxuron, dinoseb, dichlobenil, dicamba, 2,4-D, 2,4-DB, and 2,4-DP on photosynthetic CO₂ fixation and on the level of intermediates of the CO₂ assimilation cycle by isolated chloroplasts, as well as their in vitro activities on the enzymatic systems ribulose-1,5-diphosphate carboxylase and fructose-1,6-bisphosphatase. Phenmedipham showed the strongest inhibition of CO₂ assimilation, with an I₅₀ of 0.05 μM, followed by chloroxuron and dinoseb, with a 50% inhibition in the range of 0.5–1 μM. A weaker inhibitory effect, with an I₅₀ of 50 μM, is promoted by 2,4-DB, whereas dicamba and 2,4-DP showed this inhibition at 100 μM; dichlobenil and 2,4-D were completely ineffective. In the presence of phenmedipham and chloroxuron, the $\text{trioses-}\text{P}\text{P}\text{-glycerate}$ ratio showed a sharp decrease, which means an inhibition of the P-glycerate reduction step by a low NADPH synthesis; a low ratio is also promoted by 2,4-D, but it may be a consequence of induced collateral metabolic pathways of P-glycerate. Dinoseb showed a 25% inhibition of ribulose-1,5-diphosphate carboxylase activity in the concentration range of 10–100 μM and an I₅₀ of 50 μM of the fructose-1,6-bisphosphatase. Thus these effects could contribute, in addition to the photochemical ones, to an explanation of the dinoseb inhibition of CO₂ assimilation by isolated chloroplasts. The other herbicides tested showed a weak or no effect on these enzyme systems.     

Binding characteristics of methyl parathion to photosynthetic membranes of Chlorella

When methyl parathion is added at a given concentration to exponentially growing Chlorella cultures containing different numbers of cells, the inhibition of cell number, packed cell volume, pigment content, or photosynthesis has been found to be a function of the cell number, the inhibition being decreased with increased number of cells. Inhibition of photosynthesis has been studied further with a view to characterizing the mechanism of inhibition with a simple assumption that methyl parathion binds to a specific component in Chlorella cells to form an inhibitory complex. The concentration of methyl parathion causing 50% inhibition (I₅₀) of photosynthetic O₂ evolution increases linearly with increasing concentration of chlorophyll in the culture medium. With whole cells the inhibition constant (K_i) is 15 times greater than that with cell-free photosynthetic membranes. This shows that the cell wall acts as a permeability barrier. The relation between the I₅₀ and K_i values and the analyses of the Hill plot of the inhibition curves reveal one binding site per 0.5 chlorophyll molecule and a cooperative binding of methyl parathion with at least three binding sites per binding molecule. Mild treatment of the photosynthetic membranes with trypsin makes the photosynthetic electron transport insensitive to the insecticide, suggesting that the binding component is proteinaceous in nature and the binding sites are located on the external surface of the membrane. The reversal of methyl parathion inhibition is parallel to that of 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (diuron) inhibition during trypsin treatment suggesting that the binding proteins for these two inhibitors are similar.     

Diphenyl ether-chloroplast interactions

The diphenyl ethers acifluorfen (sodium-5-[2-chloro-4-(trifluoromethyl)-phenoxyl]-2-nitrobenzoate), acifluorfen-methyl (methyl-5-[2-chloro-4-(trifluoromethyl)-phenoxyl]-2-nitrobenzoate), and oxyfluorfen (2-chloro-1-[3-ethoxy-4-nitrophenoxy]-4-(trifluoromethyl)benzene have an absolute light requirement for herbicidal activity. CO₂-dependent O₂ evolution was inhibited in leaf disks obtained from 5-week-old spinach plants as a result of incubation in the light in the presence of each of the three diphenyl ethers. I₅₀'s were determined for inhibition by the diphenyl ethers of CO₂-dependent O₂ evolution in intact chloroplasts obtained from three species of varying susceptibilities (spinach, coffeeweed, and pea). Rankings obtained correlated well with relative susceptibilities and with relative effectiveness of the three compounds tested. Coupled and uncoupled photosynthetic electron transport in susceptible species were unaffected by the three compounds at concentrations in the I₅₀ range. Exposure to herbicidally inactive isomeric analogs of oxyfluorfen and acifluorfen did not affect photosynthesis in leaf disks but was effective in inhibiting photosynthesis in isolated chloroplasts. Photosynthetic abilities of intact tissue were not affected by herbicide treatment in red light. Red light was, however, as effective as white light in mediating the inhibition of photosynthesis in isolated intact chloroplasts by diphenyl ethers. The existence of two photoreceptors for diphenyl ether action, one located at the chloroplast envelope and a second outside of the chloroplast, is suggested as a possible basis for these findings.     

In vitro and in vivo analyses of the mechanism of action of SWEP

In vitro experiments with intact chloroplasts from hydroponically grown spinach (Spinacia oleracea L. var. Winter Giant) plants, have shown an I₅₀ value for SWEP (methyl N-3,4-dichlorophenyl) carbamate) of 0.1 μM in PS I and II-linked electron transport H₂0 → NADP⁺. With thylakoid membranes the I₅₀ values for PS II-linked Hill reactions H₂O → [Fe(CN)₆]³⁻ and H₂O → dichlorophenolindophenol are in the range 0.05-0.1 μM, whereas the I₅₀ shifts to 0.45 μM in short PS II-linked transport chain diphenylcarbazide → dichlorophenolindophenol. Trypsination of PS II-enriched particles produces a negligible increase of the I₅₀ value in diphenylcarbazide → dichlorophenolindophenol electron transport, a much smaller increase than occurs with diuron- or atrazine-type inhibitors. All these data show SWEP as a strong inhibitor of electron transport in the Q-B region of the PS II-reducing side. However, it appears to have a different binding site than that of urea and triazine herbicides, either on a trypsin resistant or on a non-surface cluster. As a consequence of the NADPH shortage, SWEP brings about a strong inhibition of CO₂ assimilation, with an I₅₀ of 0.04 μM, and a lower percentage of trioses-P among the intermediates of the Calvin cycle. In vivo experiments have shown a three to five times higher inhibition of PS II-linked electron transport, when SWEP was supplied through the roots than when it was applied to the leaves. We have found I₅₀ values of CO₂ assimilation by isolated chloroplasts of foliar disks of 3 and 5 μM, respectively, when the herbicide was root supplied, as opposed to 10 and 25 μM after leaf application.     

Synthesis and characterization of a group of dihydropyrimido-benzimidazole photosystem II herbicides

A group of 28 dihydropyrimido-benzimidazole analogs and derivatives was tested for herbicidal activities. The 1:1 mixture of 7-fluoro- and 8-fluorodihydropyrimido-benzimidazole had the greatest biological activity. Activity is increased by methylation of nitrogen 2. None of the active compounds was inhibitory to nonphotosynthetic tissue-culture cells in vitro. The I₅₀ of inhibition of growth of green duckweed plants and that of in vitro photosynthesis activity matched the I₅₀ of inhibition of photosystem II activity. Fluorescence analyses of photosystem II activity indicated inhibition at the same step as did many triazine, phenylurea, pyridazinone, and uracil herbicides. As the compounds showed insufficient activity on recently evolved triazine-resistant weed biotypes, their further development was terminated, despite their novel chemistry.     

Inhibition of acetyl-coenzyme a carboxylase by the novel grass-selective herbicide 3-(2,4-dichlorophenyl)-perhydroindolizine-2,4-dione

The novel herbicidal compound 3-(2,4-dichlorophenyl)-perhydroindolizine-2,4-dione which is active as a pre- and post-emergence grass-selective herbicide is a new type of inhibitor of plant acetyl-coenzyme A carboxylase (ACCase; E.C. 6.4.1.2). This has been demonstrated by in-vitro experiments using isolated corn enzyme. Besides an I₅₀ value of 4.7 μM under the assay conditions used, a mixed-type inhibitory activity on the enzyme is established. The compound is inactive on rat liver A CCase enzyme and shows decreased activity (pI₅₀ less than 3.0) on the spinach enzyme.     

Structure-activity relationships of triazinone herbicides on resistant weeds and resistant Chlamydomonas reinhardtii

Weeds resistant to the s-triazine herbicide atrazine also show resistance to the triazinone herbicide metribuzin. However, with highly lipophilic triazinones, thylakoids isolated from atrazine-resistant Amaranthus retroflexus (mutation at position Ser₂₆₄ of the photosystem II D-1 reaction centre protein) in general show a higher pI₅₀ value in photosystem II electron transport than those from the wild type (i.e. negative cross-resistance; ‘supersensitivity’). A quantitative structure–activity relationship (QSAR) can be established, wherein the lipophilicity of the compound plays a major role. In in-vivo experiments, it was found that the triazinone DRW2698 killed resistant Amaranthus retroflexus and Chenopodium album whereas the wild type was almost unaffected. Triazinones were further investigated in five different mutants of Chlamydomonas rheinhardtii (mutations in the D-1 protein at positions Ser₂₆₄, Ala₂₅₁, Leu₂₇₅, Phe₂₅₅, and Val²¹⁹). Inhibitory activity of all triazinones was generally enhanced in the Phe₂₅₅ mutant but decreased in the Val₂₁₉ mutant. In the other mutants, biological activity was decreased when position 3 of the triazinone was substituted by CH₃, OCH₃, SCH₃, NHCH₃ or N(CH₃)₂. However, negative cross-resistance was again observed when this position was occupied by free thiol. It is therefore suggested that these two groups of triazinones orient themselves differently within the herbicide binding niche of the photosystem II D-1 protein.     

Growth, photosynthetic pigments and photosynthetic activity during seedling stage of cowpea (Vigna unguiculata) in response to UV-B and dimethoate

UV-B (0.4 W m⁻²) irradiation and dimethoate (100 and 200 ppm) treatments, singly and in combination, declined the growth, photosynthetic pigment contents and photosynthesis (O₂ evolution and CO₂-fixation) of cowpea (Vigna unguiculata). Contrary to this, low concentration of dimethoate (50 ppm) caused stimulation on these parameters, while together with UV-B it showed inhibitory effects. Carotenoids (Car) showed varied responses. It was found that carbon-fixation (¹⁴CO₂) was more sensitive to both the stresses than photosynthetic oxygen evolution. Photosynthetic electron transport activity was reduced by both the stresses, however, 50 ppm dimethoate besides inhibiting photosystem II (PSII) and whole chain activity, showed slight stimulation in photosystem I (PSI) activity. The individual effect of two stresses on PSII activity was probably due to interruption of electron flow at oxidation side of PSII which extended to its reaction center following simultaneous exposure. A similar trend was also noticed in case of CO₂ liberation (measured as ¹⁴CO₂ release) in light and dark. Results suggest that dimethoate (100 and 200 ppm) and UV-B alone caused heavy damage on pigments and photosynthetic activity of cowpea, leading to the significant inhibition in growth. Further, the interactive effects of both the stresses got intensified. However, low concentration (50 ppm) of dimethoate showed stimulation, but in combination, it slightly recovered from the damaging effect, caused by UV-B.     

Properties of acetolactate synthase from sulfonylurea-resistant Scirpus juncoides Roxb. var. ohwianus T. Koyama

Properties of acetolactate synthase (EC 4.1.3.18; ALS) from sulfonylurea-resistant (SUR) Scirpus juncoides Roxb. var. ohwianus T. Koyama were studied biochemically and physiologically in comparison with those from sulfonylurea-susceptible weed (SUS). GR₅₀ values for growth inhibition and I₅₀ values for ALS inhibition by imazosulfuron were determined for both SUR and SUS. Imazosulfuron controlled the SUS above 80% at the dosage more than 10 g a.i./ha but did not control the SUR at the even great dosage of 1000 g a.i./ha. The rates required for 50% growth inhibition of the SUR relative to the SUS (R/S ratio) were 271-fold. The I₅₀ value for inhibition of ALS from the SUS was 15 nM, compared to I₅₀ of >3000 nM for inhibition of ALS from the SUR. These results suggest that a resistance may due to an altered ALS that is insensitive to imazosulfuron. The K_m (pyruvate) value of ALS from the SUR was similar to the K_m for ALS from the SUS, suggesting that a mutation resulting in resistance does not change the affinity of the enzyme for pyruvate. The specific activity of the SUR ALS was similar to that of the SUS ALS, which indicates that resistance is not an over-expression of the enzyme. ALS activity from both biotypes was inhibited by isoleucine, valine, and leucine in this order. However, the SUR ALS was less sensitive to inhibition by valine than the SUS ALS.     

药剂对小菜蛾抗性及敏感品系乙酰胆碱酯酶抑制作用比较

采用浸叶法测定了云南通海、元谋和澜沧的小菜蛾plutella xylostella田间种群对常用杀虫剂的抗药性。结果表明,云南上述地区小菜蛾田间种群对各类杀虫剂均产生了不同程度的抗性。对有机磷类药剂的抗药性为1.74~31.1倍;对菊酯类药剂的抗药性为7.41~764倍;对阿维菌素类药剂则产生了 5.60~4.06×104倍的抗性。通过离体和活体试验测定了药剂对小菜蛾头部乙酰胆碱酯酶(AChE)的抑制作用。敌敌畏和灭多威对通海抗性品系AChE离体和活体内的抑制中浓度(I50)分别是敏感品系的209、26.5倍和2.21、2.16倍;敌敌畏对通海小菜蛾种群的离体和活体内抑制中时间(IT50)小于敏感品系,分别是敏感品系的0.32和0.17倍;而灭多威对通海小菜蛾种群的离体和活体内抑制中时间(IT50)则大于敏感品系,分别是敏感品系的1.37和1.74倍。     

Co-stressors chilling and high light increase photooxidative stress in diuron-treated red alga Kappaphycus alvarezii but with lower involvement of H2O2

Diuron is one of the most commonly found N-phenylurea herbicides in marine/estuarine waters that promotes toxic effects by inhibiting photosynthesis and affecting the production of reactive oxygen species (ROS) in autotrophs. Since photo- and thermoacclimation are also ROS-mediated processes, this work evaluates a hypothetical additive effect of high light (HL) and chilling (12 °C) on 50 nM diuron toxicity to the highly-photosynthetically active apices of the red alga Kappaphycus alvarezii. Additive inhibition of photosynthesis was mainly evidenced by significant decreases of quantum yield of photosystem II and electron transfer rates upon co-stressors exposure to diuron-treated algae. Under extreme 12 °C/HL/diuron conditions, unexpected lower correlations between H₂O₂ concentrations in seawater and radical-sensitive protein thiols were concomitantly measured with the highest indexes of photoinhibition (parameter β). Altogether, these data support the hypothesis that co-stressors chilling/HL additively inhibit photosynthesis in diuron-exposed K. alvarezii but with less involvement of H₂O₂ in injury effects than with only chilling or HL.     

Effects of herbicidal carbamates on mitochondria and chloroplasts

At concentrations near 2 × 10^?4M, barban, chlorpropham, and phenmedipham are inhibitors of the electron transfer in potato and mung bean mitochondria. The inhibition seems to be localized in the flavoprotein region. It affects preferentially the exogenous NADH dehydrogenation, in potato mitochondria (I₅₀, 10^?4M). Succinate dehydrogenation is less inhibited. At noninhibiting concentrations, the studied carbamates cannot uncouple the oxidative phosphorylations. Photosynthesis is completely inhibited by 2.10^?7M phenmedipham, 5 × 10^?5M barban, and 2 × 10^?4M chlorpropham. The inhibition takes place at the PS II level. Moreover, barban and chlorpropham are uncouplers of the photophosphorylations for concentrations between 5 × 10^?5 and 5 × 10^?4M. The effects observed on mitochondrial respiration can also be found on respiration of Acer cultured cells. The effects on isolated chloroplast photosynthesis are also observed for slightly higher concentrations on cultured Chlorella and on pea and oat leaf fragments.     

Assessment of Cyfluthrin commercial formulation on growth, photosynthesis and catalase activity of green algae

Aquatic environments of the pampasic region of Argentina are severely affected by agricultural contamination due to an increase in a glyphosate tolerant transgenic variety of soybean crops. The present study is aimed to determine the effects of a commonly used Cyfluthrin commercial formulation (CCF) on growth, some physiological and biochemical parameter of four species of green algae. Significant inhibition of algal growth was observed from 0.1 mg Cyf/l. 96 h IC₅₀ were between 0.92 and 4.85 mg Cyf/l. CCF caused algicidal effects. Photosynthesis was stimulated by 50% in Scenedesmus quadricauda cultures exposed to the lowest concentration (hormesis). Algal photosynthesis inhibition was observed at higher concentrations with IC₅₀ values between 1.7 and 8.9 mg Cyf/l. Similar toxicity endpoints were found as a consequence of applying the traditional methodology of short-term chronic toxicity test of 96 h of exposition and the methodology developed using the Clark type photosynthetic oxygen evolution method. CAT activity was significantly increased between 23% and 33% considering the four species, at a lower concentration than those affecting algal growth and photosynthesis, indicating a potential biomarker. Taking into account that the extent of the soybean crops in the region is about fourteen million hectares, the improvement and extension of environmental tools for early detection of the action of pesticides on this essential group of organisms are discussed.     

Structural Effects of N-Arylcarbamoylpyrazolines on Calcium Uptake in Rat Brain Synaptosomes

N-Arylcarbamoylpyrazolines with various substituents at the para position of the carbamoyl benzene ring inhibited ATP-dependent Ca²⁺-uptake in synaptosomes prepared from the rat brain. The activity of these compounds was evaluated as log(1/I₅₀), the reciprocal logarithm of half inhibitory concentration, I₅₀ (m ), from the concentration–response curve for the inhibition of Ca²⁺-uptake. Among the compounds tested, methyl 3-(4-chlorophenyl)-4-methyl-1-[N-(4-trifluoromethylphenyl)carbamoyl]-2-pyrazoline-4-carboxylate was the most potent, the I₅₀ value of which as 9·12×10⁻⁷ m . Variations in the activity in terms of log(1/I₅₀) were quantitatively analysed using a substituent parameter, showing that the higher the electron-withdrawing effect of the substituent, the higher was the activity. The substituent effects were similar to those on insecticidal activity against the Americal cockroach. The higher the inhibitory activity against Ca²⁺ uptake, the higher seemed to be the insecticidal activity. Methyl(4S) - 3 - (4 - chlorophenyl) - 4 - methyl - 1 - [N - (4 - chlorophenyl)carbamoyl] - 2 - pyrazoline -4-carboxylate had higher inhibitory activity against Ca²⁺-uptake and higher in-secticidal activity than the R-isomer, but the difference was greater in theCa²⁺-uptake system.     

Effects of the herbicides benzoylpropethyl and flampropisopropyl on rat liver mitochondria: An alteration in membrane fluidity?

The action of the herbicides benzoylpropethyl and flampropisopropyl, and the corresponding unesterified acids was studied in rat liver mitochondria. The herbicides were found to (a) inhibit the mitochondrial electron transfer in complex III or at the level of ubiquinone (I₅₀ of 4 nmol mg protein^?1 for flampropisopropyl and 18 for benzoylpropethyl with succinate as a substrate); (b) have an additional (however less sensitive) site of inhibition near succinate dehydrogenase; and (c) interfere with energy transfer. Sensitivity was increased 2- (benzoylpropethyl) and 3.5-fold (flampropisopropyl) as the rats age increased from 12–13 weeks to 23–26 weeks. The free acids were far less effective. Since the herbicides benzoylpropethyl and flampropisopropyl are readily hydrolyzed by animals and since the free acids are less effective than the herbicides, it explains why these potentially harmful compounds have a low acute toxicity. Swelling studies in isoosmotic salts suggested that the two herbicides decrease membrane fluidity, an action which was assumed to be responsible for the electron transfer inhibition and, via inhibition of phosphate transport, the interference with energy transfer.     

Long-term effects of herbicides on the photosynthetic apparatus II. Investigations on bentazone inhibition

Non-cyclic electron transport by isolated chloroplasts from the alga Bumilleriopsis is inhibited by bentazone, while the activity of photosystem I is not affected. Chloroplast material isolated from cells grown for several days in the presence of bentazone also shows inhibition of photosystem II activity, similar to the decreased cellular photosynthesis. A slow partial recovery is possible, whereas it is fast and complete after exposure of cells or isolated chloroplasts to the herbicide for some hours only. Irreversibility of longterm inhibition could be a metabolic process which brings about the binding of bentazone (or an active derivative) to the thylakoids in vivo, but not in vitro.     

Potential involvement of alkoxyl and hydroxyl radicals in the peroxidative action of oxyfluorfen

The potential involvement of hydroxyl and alkoxyl radicals in the peroxidative action of the p-nitro diphenyl ether herbicides acifluorfen (5-[2-chloro-4-(trifluoromethyl)phenoxyl]-2-nitrobenzoic acid), acifluorfen-methyl (methyl ester of acifluorfen), nitrofen [2,4-dichloro-1-(4-nitrophenoxy)benzene], nitrofluorfen [2-chloro-1-(4-nitrophenoxy)-4-(trifluoromethyl)benzene], and oxyfluorfen [2-chloro-1-(3-ethoxy-4-nitrophenoxy)-4-(trifluoromethyl)benzene] was evaluated under laboratory conditions. Methional was added to illuminated thylakoids from peas (Pisum sativum L., cv Little Marvel) and its oxidation to ethylene was used as an indicator of hydroxyl and alkoxyl radical production. Oxyfluorfen stimulated the rate of methional oxidation by 138% at 10 μM and 175% at 1 mM. This oxyfluorfen-induced stimulation of the rate of methional oxidation was dependent on light, photosynthetic electron transport, and hydrogen peroxide since it was not observed under dark conditions or in the presence of DCMU and catalase. Addition of Fe-EDTA, a catalyst of the Fenton reaction, stimulated the oxyfluorfen-induced enhancement of methional oxidation sixfold, suggesting that hydroxyl radicals are synthesized through a Fenton reaction. Acifluorfen, nitrofen, and nitrofluorfen inhibited the rate of methional oxidation whereas acifluorfen-methyl had no effect on the rate of methional oxidation, even at high concentrations (1 mM). Nitrofluorfen at 1 mM was the only p-nitro diphenyl ether herbicide tested to inhibit photosynthetic electron transport of pea thylakoids. In experiments with pea leaf disks, acifluorfen at low concentrations stimulated the rate of methional oxidation, whereas acifluorfen-methyl, nitrofen, and nitrofluorfen had no effect. These data indicate that hydroxyl and alkoxyl radicals could be involved in the mechanism of cellular damage caused by oxyfluorfen but they are not important for the activity of the diphenyl ether herbicides acifluorfen, acifluorfen-methyl, nitrofen, and nitrofluorfen.