Phorate sulfoxidation by plant root extracts

Soybean root homogenates were found to oxidize phorate to phorate sulfoxide. Neither PO nor sulfone derivatives of phorate were detected. Fractionation of soybean root homogenate indicates that the enzyme(s) which oxidize phorate to the sulfoxide are largely (72%) in the 25,000g pellet. In vitro studies of the 25,000g pellet indicate a linear reaction rate at 34°C for periods of up to 2 h at the pH optimum of 5.5. Soybean and bean root 25,000g pellets were twice as active on a mg protein basis as those from barley, corn, wheat, sorghum and over ten times more active than tomato.In vitro studies indicate that Counter, disulfoton, fenthion, aldrin, heptachlor, piperonyl butoxide, sulfoxide, atrazine, SKF525A and p-chlorothioanisole when present at a 10:1 ratio with phorate significantly inhibited soybean root phorate sulfoxidation.     

Effect of hepatic enzyme inducers on the in vivo and in vitro metabolism of dicrotophos,dimethoate, and phosphamidon in mice

Daily 75 mg/kg phenobarbital ip injections for 3 days or 25 ppm dieldrin in the diet of mice for 14 days caused an increase in liver cytochrome P-450 and blood B-esterase. Liver A-esterase was not significantly increased. Under in vitro conditions, phenobarbital and dieldrin induced the oxidative as well as hydrolytic metabolism of dicrotophos, dimethoate, and phosphamidon by liver homogenates or combined microsomes plus 105,000g supernatant fractions. The concentration of dimethoxon was increased more than fourfold by the pretreatments after incubation for 4 hr at 37.5°C with NADPH added. The organophosphorus insecticides used in this study were not metabolized as well by the liver microsomes alone or 105,000g supernatant alone, as by the combination of microsomes and 105,000g supernatant. Under in vivo conditions in mice, phenobarbital and dieldrin treatments increased the urinary recovery of metabolites in the initial 6 hr after [¹⁴C]carbonyl-dimethoate or [¹⁴C]N-ethyl-phosphamidon administration. Analysis of urine showed that the inducers caused a more than sixfold increase in dimethoxon recovered and twofold increase in water-soluble nontoxic metabolites within 6 hr after dimethoate administration. With phosphamidon both inducers increased the rate of metabolism, and the total recovery in aqueous and chloroform fractions was decreased. These results suggest that increased dimethoate toxicity after phenobarbital and dieldrin treatments in whole animals results from stimulation of the activation of dimethoate to dimethoxon, while the increase in hydrolytic products after both pretreatments results in decreased toxicity of the direct acetylcholinesterase inhibitors, dicrotophos and phosphamidon.     

Acetylcholinesterase of Aphis citricola: Properties and significance in determining toxicity of systemic organophosphorus and carbamate compounds

In apterous adults of the spirea aphid, Aphis citricola van der Goot, the optimum conditions for determining acetylcholinesterase (AChE) activity consist of reaction mixture of 0.1 M phosphate buffer (pH 7.5), 10^?3M acetylthiocholine (ASCh), and enzyme extract equivalent to 80 ± 3 μg protein incubated for 15 min at 30°C. The K_m value for ASCh (6.7 × 10^?5M) was much lower than that of butyrylthiocholine (BuSCh) (1.25 × 10^?2M). The enzyme activity was almost completely inhibited by 10^?6M paraoxon or 10^?5M eserine and was 84% inhibited by 10^?5M BW284C51 (a specific AChE inhibitor). DTNB was found to inhibit the enzyme activity and was therefore added at the end of the reaction. AChE activity of A. citricola was inhibited in vitro and in vivo by dimethoxon > dimethoate, and aldicarb sulfoxide > aldicarb > aldicarb sulfone. The in vivo effect correlates well with the toxicity level of the various toxicants. A neurotoxicity index which combines both mortality and in vivo inhibition of the aphid AChE activity is suggested as a measure for determining the toxicity of organophosphorus and carbamate compounds toward aphids.     

Accumulation pattern and insecticidal effect of aldicarb in cotton following soil treatment for the control of the tobacco whitefly (Bemisia tabaci)

The accumulation pattern of the pesticide aldicarb [2-methyl-2-(methylthio) propionaldehyde O-(methylcarbamoyl)-oxime] and of its sulfoxide and sulfone metabolites was studied in field-grown cotton, following soil treatments at various intervals from planting. Control of the tobacco whitefly(Bemisia tabaci) was determined and correlated with the concentration of aldicarb and of its metabolites in cotton leaves. The main constituent found in the leaves was aldicarb sulfoxide, which reached its maximum concentration there at about 22 days post-treatment. Late application of the insecticide (mid-July) resulted in higher concentrations toward the end of the growing season and so gave improved control of the pest. Results are presented for residues in young and mature leaves and in the seeds.     

In vitro metabolism of EPN and EPNO by mouse liver

The in vitro metabolism of EPN (O-ethyl O-p-nitrophenyl phenylphosphonothionate) and EPNO (O-ethyl O-p-nitrophenyl phenylphosphonate) in mouse liver was studied. EPNO was metabolized faster than EPN, and the highest metabolic activity was found in the 10,000g supernatant in the presence of both NADPH and glutathione. Liver microsomes in the presence of NADPH metabolize EPN to its oxygen analog, EPNO and p-nitrophenol. With the 100,000g supernatant only slight metabolism of EPN occurred in the presence of GSH. Metabolism of EPNO by liver microsomes increased upon the addition of NADPH. p-Nitrophenol was the only metabolite isolated in the presence of microsomes, whereas, with the addition of NADPH, both p-nitrophenol and desethyl EPNO were formed. Quantitative studies showed that there was little, if any, oxidative dearylation of EPNO by liver microsomes. The 100,000g supernatant was found to actively degrade EPNO, and this increased upon addition of glutathione. The initial rate of p-nitrophenol formation as a result of incubation of EPN and EPNO with liver microsomes was found to be higher with EPN than EPNO.     

In vitro inhibition of α-amylase and protease by nematocides in Cicer arietinum L

The in vitro effects of four systemic nematocides, i.e., aldicarb, carbofuran, oxamyl, and phorate, on the α-amylase and protease activities in Cicer arietinum has been revealed. All four nematocides markedly inhibited the activities of both the enzymes, with a general tendency of increased inhibition with corresponding increase in the concentrations of the nematocides. There was complete inhibition of α-amylase activity by the highest concentration (500 μM) of aldicarb and carbofuran, while oxamyl at the same concentration showed the same effects on protease activity. The lowest concentration (10 μM) was almost ineffective.     

The metabolism of [14C]aldicarb in the root of sugar beet plants

Sugar beet plants were grown in the field, after in-furrow application of [¹⁴C]- aldicarb (3 kg of aldicarb ha^?1) at planting. The ripe sugar beet plants were harvested, and the roots were analysed. The roots were fractionated according to a procedure similar to the normal beet-sugar manufacturing process. Expressed as a proportion of the total radioactivity incorporated into the root, the pulp contained 29.7%, the lime cake 9.7%, the crystallised sugar 17.7% (which gave, with the radioactivity found in the sugar in the molasses, a total of 20.7% of the radioactivity in the total sugar), and the molasses, 42.9%. A part of the labelled carbon from the radio- active aldicarb and its metabolites had thus been metabolised and incorporated into sugar molecules. Except for the radioactivity in the sugar and in the lime cake from the processing, the proportion of radioactive non-conjugated organosoluble compounds was very low (2.6%), and perhaps partially corresponded to the very low amount of aldoxycarb (aldicarb sulphone) in the root (less than 0.001 mg of [¹⁴C]-aldicarb equivalents kg^?1 fresh weight). Hydrolysis of the molasses yielded free radioactive 2-methyl-2-(methylsulphinyl)propan-1-ol (3.1%), 2-mesyl-2-methyl-propan-I-ol (8.9%) and 2-mesyl-2-methylpropionic acid (12.0%) which had been conjugated to plant constituents in the root. The corresponding concentrations (expressed as mg of [¹⁴C]aldicarb equivalents kg^?1 fresh weight of root) were 0.004, 0.011, and 0.016, respectively. No aldicarb, aldicarb sulphoxide or aldoxycarb (nor the corresponding nitrile, generated from aldicarb during the fractionation procedure) was liberated by the hydrolysis, indicating the absence of conjugates of these compounds in the root.     

Metabolism of [14C]vernolate in soybeans

Penetration and metabolism of [¹⁴C]vernolate in soybean [Glycine max (L.) Merr. var Ransom] pods and seeds were measured 0, 1, 4, 24, 48, or 72 hr after treatment which occurred at 40 days after flowering. Total ¹⁴C recovery decreased ca. 50% within 4 hr and the loss of ¹⁴C was considered to be a measure of volatility. Total nonpolar extractants decreased in a logarithmic pattern which approached 10% of total ¹⁴C recovered within 24–48 hr. Total polar extractants increased in a logarithmic pattern to a maximum of 90% of total ¹⁴C recovered within 24 hr. Seed nonpolar extractants never exceeded 2% of the total ¹⁴C recovered while pod nonpolar extractants consisted of vernolate plus an unidentified component that did not thin-layer chromatograph (TLC) as the sulfone or sulfoxide. Pod polar extractants increased with time to ca. 75% of the total ¹⁴C recovered (24–48 hr) and decreased to ca. 58% at 72 hr after treatment. Seed polar extractants averaged ca. 10% of total ¹⁴C recovered for the first 48 hr after treatment and then increased to 30% of total ¹⁴C recovered. Thus, [¹⁴C]vernolate per se concentration decreased to <1% of applied material within 72 hr through volatilization and degradation of nonpolar extractants to polar products. Polar metabolites showed two major patterns of vernolate detoxification. One detoxification system produced ¹⁴C-metabolites whose R_f's were equivalent to that reported in corn (Zea mays L.) [J. P. Hubbell and J. E. Casida, [J. Agric. Food Chem. 25, 404 (1977)] and accounted for <30% of the pod polar extractants. A second detoxification system was most prevalent in soybean pod and seed tissues and resulted in very rapid modification of vernolate with an unidentified product that was 85% of the extracted ¹⁴C within 4 hr after treatment and which decreased in concentration with time. Therefore, unexplained vernolate detoxification system(s) exist in soybean pod and seed.     

Reductive metabolism of heptachlor,parathion, 4,4′-dichlorobenzophenone,and carbophenothion by rat liver systems

An attempt was made to survey systems which carry out reduction of four pesticidal chemicals, heptachlor, parathion, 4,4-dichlorobenzophenone, and carbophenothion sulfoxide, in rat liver microsomes and in the supernatant in vitro. On the basis of the stimulatory effects of nicotinamide cofactors (NADPH and NADH), a flavin cofactor (FAD), localization of the systems, and their sensitivities to heat-inactivation treatment, four different reductive systems were recognized. They are (a) the mixed-function oxidase-coupled reductive systems, (b) an independent NADH-stimulated (instead of NADPH as above) microsomal system for parathion reduction, (c) FAD-stimulated system for reduction of carbophenothion sulfoxide and parathion, present specifically in the supernatant, and (d) a heat stable, nonenzymatic reduction system in the microsomes operated by flavoproteins and a flavin cofactor.     

Sulfonylurea herbicides: Growth inhibition in soybean cell suspension cultures and in bacteria correlated with block in biosynthesis of valine,leucine, or isoleucine

The sulfonylurea herbicides chlorsulfuron (CS) and sulfometuron methyl (SM) inhibit the growth of soybean cells (Glycine max L. var. Amsoy 71) in suspension culture with 50% inhibition at 170 and 62 ppb, respectively, relative to the initial cell dry weight, and CS is not rapidly metabolized in these cultures. In Glycine max L. cv Merrill var. Mandarin, CS inhibits the growth by 50% at 4 ppm on the basis of initial cell dry weight. This inhibition is partially reversed by valine, leucine, or 2-ketoisovalerate, but not by pyruvate, isoleucine, or any other single amino acid. CS drastically reduces the content of free valine and leucine in soybean cells without significant effect on the amount of other free amino acids. Deoxyribonucleosides alleviate a portion of the CS growth inhibition in soybean cells in vivo, though CS and SM do not inhibit ribonucleoside diphosphate reductase in vitro. CS and SM are bacteriostats for Escherichia coli and Salmonella typhimurium in minimal growth medium. E. coli growth is retarded at CS concentrations (100–300 μg/ml) that inhibit RNA and protein synthesis but not DNA synthesis. CS growth inhibition in E. coli is enhanced by cysteine and valine and partially alleviated by isoleucine and the aromatic amino acids, but not by leucine. The sulfonylureas appear to act in soybean by blocking the synthesis of valine and leucine between pyruvate and 2-ketoisovalerate and in E. coli by inhibiting isoleucine biosynthesis.     

Differential effect of diclofop-methyl on fatty acid biosynthesis in leaves of sensitive and tolerant plant species

The herbicide diclofop-methyl caused an early and pronounced inhibition of the incorporation of [¹⁴C]acetate into leaf lipids of the sensitive plant species maize (Zea may L.), wild oat (Avena fatua L.), and barnyardgrass (Echinochloa crus-galli L.). With an EC₅₀ value of approximately 10^?7M inhibition was already apparent 0.5–4 hr after herbicide application. The fatty acid biosynthesis of tolerant bean (Phaseolus vulgaris L.), sugar beet (Beta vulgaris L.), and soybean (Glycine max L.) was not affected, with one exception [wheat (Triticum aestivum L.) belongs to the more tolerant species]; the inhibition of fatty acid biosynthesis, however, was in the same order of magnitude as in sensitive plants. More detailed studies showed that in wheat a recovery from inhibition of fatty acid biosynthesis occurred. Four days after herbicide application (0.18 kg diclofop-methyl/ha) in wheat normal fatty acid biosynthesis was restored, whereas in sensitive maize a 60% inhibition was maintained over the whole experimental period (8 days). The results support the view that tolerance of wheat to diclofop-methyl is based on its inactivation in leaves, whereas the tolerance of dicotyledonous species may probably lie at the level of the site of action of diclofop-methyl. In experiments with intact leaves, the inhibition of fatty acid biosynthesis resulted in an enhanced flow of [¹⁴C]acetate into organic acids and amino acids. This effect, however, was not always reproducible in experiments with leaf pieces or isolated root tips.     

Transformation of aldicarb sulfoxide and aldicarb sulfone in four water-saturated sandy subsoils

The transformation of aldicarb sulfoxide and aldicarb sulfone was studied in incubations with water-saturated subsoils under simulated field conditions at 10°C. The subsoils were collected at four locations from beneath the water table at a depth of 2.5 to 3.5 m. In three of the subsoils, the half-life of sulfoxide, incubated at concentrations of 0.14-0.17 mg litre^?1, ranged from 0.7 to 2.8 years. At higher concentrations (8-13 mg litre^?1), its half-life ranged from 3.4 to 6.4 years. At the lower concentration, a large fraction of sulfoxide was transformed into sulfone. The rates of transformation of the sulfone at the lower concentration in the three subsoils corresponded to half-lives of 3.3 to 8.1 years, but in only one subsoil was a significant transformation rate (half-life 6.7 years) measured at the higher concentration during the 2.3-year incubation period. The half-lives at the lower concentrations were more like those in field studies, and perhaps would still underestimate transformation rates under field conditions. After a year, 2.5-15% of the higher sulfoxide and sulfone doses had been trapped as [¹⁴C] carbon dioxide. In the fourth subsoil, with more anaerobic conditions, the half-life of sulfoxide at both concentrations was less than 0.02 year and that of sulfone was about 0.04 year. Four or five radio-labelled transformation products could be traced in this subsoil and about half of the dose of both compounds was trapped as [¹⁴C] carbon dioxide.     

Effects of picloram on the synthesis of soluble plant proteins as determined by gel electrophoresis

Changes in soluble proteins synthesized in soybean (Glycine max L.), safflower (Carthamus tinctorius L.), radish (Raphanus sativus L.), and barley (Hordeum vulgare L.) treated with either growth promotive or inhibitory concentrations of picloram (4-amino-3,5,6-trichloropicolinic acid) were determined by polyacrylamide gel electrophoresis and isoelectric focusing. A special gel mixture was developed which provided resolution of protein bands superior to that obtained by standard gel electrophoresis. Growth promotive concentrations of picloram caused both qualitative and quantitative alterations in the band patterns of soluble proteins of safflower, radish, and barley roots and shoots. Isoelectric focusing was applied for the separation and identification of soluble protein fractions from soybean and barley roots and shoots treated primarily with growth inhibitory concentrations of picloram (except for barley shoot tissues). More than 35 clear bands were distinguishable in a typical gel electrophoretogram for either soybean or barley tissue (4-day-old plants). Approximate pI values of the bands from barley root protein were determined from a pH gradient diagram. Protein band patterns of picloram-treated samples were changed qualitatively and quantitatively, in comparison with controls, mostly in the range above pI 6, and predominantly in the neutral and basic protein regions. Band patterns for 96-hr root samples treated with growth inhibitory concentrations of picloram were more similar to those from 48-hr (soybean) or 55-hr (barley) than 96-hr control seedlings. A quantitative decrease in intensity of a band which had the same pI value as that of RNase was noticed in both the treated samples and 2- or 3-day-old control seedlings.     

Assessment of leaching potential of aldicarb and its metabolites using laboratory studies

The metabolites of pesticides can contaminate groundwater and pose a risk to human health when this water is used for drinking. This paper reports the results of a laboratory study on aldicarb and its main metabolites, aldicarb sulfone and aldicarb sulfoxide. Aldicarb and its metabolites showed K_oc values (6–31) which were lower than that of atrazine (55), indicating that they are very mobile in soil. They are less persistent than atrazine (DT₅₀ = 25 days), with DT₅₀ values from less than 1 day and up to 12 days. Aldicarb behaved as a non‐leacher, whereas its metabolites clearly showed the characteristics of leachers. Aged residue leaching experiments showed that aldicarb can occur at high concentrations in the leachate, together with its two metabolites. The leachate composition depends on the incubation time of the parent compound. Aldicarb and its metabolites can form various mixtures in groundwater on the basis of the time elapsing between the application of the insecticide and the first significant rainfall. This study confirms the characteristics of contaminants of aldicarb and especially its metabolites, as reported in the literature. © 2001 Society of Chemical Industry     

固相萃取液相色谱串联质谱法测定生姜中涕灭威及其代谢产物

建立了固相萃取-液相色谱串联质谱法测定生姜基质中涕灭威及其代谢产物涕灭威亚砜、涕灭威砜的分析方法。试样经乙腈提取,氨基SPE小柱净化后,采用多反应监测（MRM）正离子模式检测,外标法定量。实验结果表明：3种目标物在10ng/mL到50ng/mL质量浓度范围内呈现良好的线性,方法的检出限均为2μg/kg,3个加标水平下平均回收率在72～94%之间。该方法准确、灵敏、重现性好,可用于生姜样品中涕灭威及其代谢产物涕灭威亚砜、涕灭威砜的实际检测。     

A comparison of the potency of some fungicides as inhibitors of sterol 14-demethylation

An enzymatic assay system has been developed to measure the relative potency of fungicides such as triadimefon, triarimol, triforine, and buthiobate as inhibitors of sterol 14-demethylation. The enzyme preparation used is the 8000g supernatant derived from a homogenate of an aerobically adapted, anaerobically grown, high sterol strain of Saccharomyces cerevisiae. After incubation of the enzyme with [2-¹⁴C]mevalonic acid and the fungicide the ratio, radioactivity in 4,4-dimethyl sterols/radioactivity in 4-demethyl sterols is determined. The higher this ratio is, the more efficient is the fungicide as an inhibitor of fungal sterol 14-demethylation. The ratio has been determined for a number of commercial fungicides and two series of triazole compounds. A similar assay system based on the 10,000g supernatant from a rat liver homogenate was also tested but gave an inaccurate assessment of the relative potency of fungicides as inhibitors of fungal sterol 14-demethylation.     

吲哚乙酸引导下三唑醇在大豆植株中的传导与积累研究初报

将吲哚乙酸与三唑醇发生酯化反应后生成的吲哚乙酸三唑醇酯 ,在0.5 mmol/L浓度下水培和喷雾处理6~8叶期大豆植株,同时以相同浓度的三唑醇及其与吲哚乙酸的混合物为对照,色谱法测定不同时间植株不同部位吲哚乙酸三唑醇酯和三唑醇的含量。结果表明,与对照相比,吲哚乙酸三唑醇酯在大豆中具有明显的双向传导和向根部积累的特点。喷雾处理后,在12~60 h之间内吸量和根部积累量均出现最大值。     

The activity of amino acids produced by <Emphasis Type="Italic">Paenibacillus macerans</Emphasis> and from commercial sources against the root-knot nematode <Emphasis Type="Italic">Meloidogyne exigua</Emphasis>

The rhizobacterium Paenibacillus macerans was grown in tryptic soy broth and after separating the cells by centrifugation the activity of fractions of the supernatant was tested against Meloidogyne exigua juveniles. From HPLC analyses and spectral data, the most active fractions were found to contain alanine, glutamic acid, glycine, histidine, threonine and valine, which were probably produced by bacterial hydrolysis of proteic nutrients. Amino acids from commercial sources were then assayed to confirm these results and to evaluate their potential for the control of nematodes. LC₅₀ of 26 and 283 μg ml⁻¹ were shown for the nematicide aldicarb and L-cysteine respectively when tested on M. exigua juveniles. At a concentration 38.4 times>LC₅₀, the amino acid diminished the nematode population on coffee plants to values statistically equal to those obtained with aldicarb at a concentration 19.2 times>LC₅₀.     

Seed treatments associated with resistance inducers for management of <Emphasis Type="Italic">Pratylenchus brachyurus</Emphasis> in soybean

Pratylenchus brachyurus is one of the main limiting factors of soybean yield in Brazil, particularly because of inefficiency of the control methods when used individually. The present study aimed to assess the effect of associated methods, using seed treatment with nematicides (ST) and resistance inducers (RI), on nematode control in soybean plant in both field and greenhouse conditions. A field assay was conducted in an infested field and nematode population was assessed at sowing, 45, 75, and 100 days after sowing and the yield measured at end of crop cycle. The experiment was repeated in greenhouse. In another experiment, that was conducted in two different periods in a greenhouse, seed treatments and resistance inducers, alone or combined, were assessed under two initial populations of P. brachyurus (low IP = 500 specimens and high IP = 2000 specimens). The treatments did not reduce the number of nematodes g^?1 of root in field assay, but all seed treatments effectively controlled nematode population in the greenhouse assay. Most treatments reduced the number of nematodes g^?1 of root when nematode initial population was low (IP = 500) but when initial nematode population was high (IP = 2000) combinations of treatments which includes abamectin inhibited P. brachyurus reproduction. Chemical products did not affect yield but acibenzolar-S-methyl, alone or associated with other products, generally inhibit plant growth.     

Alternate pathways of metribuzin metabolism in soybean: Formation of N-glucoside and homoglutathione conjugates

Metribuzin [4-amino-6-tert-butyl-3(methylthio)-1,2,4-triazin-5(4H)-one] metabolism was studied in soybean [Glycine max (L.) Merr. Tracy]. Pulse treatment studies with seedlings and excised mature leaves showed that [5-¹⁴C]metribuzin was absorbed rapidly and translocated acropetally. In seedlings, >97% of the root-absorbed ¹⁴C was present in foliar tissues after 24 hr. In excised leaves, 50–60% of the absorbed ¹⁴C remained as metribuzin 48 hr after pulse treatment, 12–20% was present as polar metabolites, and 20–30% was present as an insoluble residue. Metabolites were isolated by solvent partitioning, and were purified by adsorption, ion-exchange, thin-layer, and high-performance liquid chromatography. The major metabolite (I) was identified as a homoglutathione conjugate, 4-amino-6-tert-butyl-3-S-(γ-glutamyl-cysteinyl-β-alanine)-1,2,4-triazin-5(4H)-one. Metabolite identification was confirmed by qualitative analysis of amino acid hydrolysis products, fast atom bombardment (FAB), and chemical ionization (CI) mass spectrometry, and by comparison with a reference glutathione conjugate synthesized in vitro with a hepatic microsomal oxidase system from rat. Minor metabolites were identified as an intermediate N-glucoside conjugate (II), a malonyl N-glucoside conjugate (III), and 4-malonylamido-6-tert-butyl-1,2,4-triazin-3,5(2H,4H)-dione (N-malonyl DK, IV) by CI and FAB mass spectrometry. Alternative pathways of metribuzin metabolism are proposed.