[Effect of substrate-dependent microbialy produced ethylene on plant growth].

Various compounds have been identified as precursors/substrates for the synthesis of ethylene (C2H4) in soil. This study was designed to compare the efficiency of four substrates, namely L-methionine (L-MET), 2-keto-4-methylthiobutyric acid (KMBA), 1-aminocyclopropane-1-carboxylic acid (ACC), and calcium carbide (CaC2) for ethylene biosynthesis in a sandy clay loam soil by gas chromatography. The classic "triple" response in etiolated pea seedling was employed as a bioassay to demonstrate the effect of substrate-dependent microbialy produced ethylene on plant growth. Results revealed that an amendment with L-MET, KMBA, ACC (up to 0.10 g/kg soil) and CaC2 (0.20 g/kg soil) significantly stimulated ethylene biosynthesis in soil. Overall, ACC proved to be the most effective substrate for ethylene production (1434 nmol/kg soil), followed by KMBA, L-MET, and CaC2 in descending order. Results further revealed that ethylene accumulation in soil released from these substrates created a classic "triple" response in etiolated pea seedlings with different degrees of efficacy. A more obvious classic "triple" response was observed at 0.15, 0.10, and 0.20 g/kg soil of L-MET, KMBA/ACC, and CaC2, respectively. Similarly, direct exposure of etiolated pea seedlings to commercial ethylene gas also modified the growth pattern in the same way. A significant direct correlation (r = 0.86 to 0.97) between substrate-derived [C2H4] and the classic triple response in etiolated pea seedlings was observed. This study demonstrated that the presence of substrate(s) in soil may lead to increased ethylene concentration in the air of the soil, which may affect plant growth in a desired direction.

In vitro inhibition of human cytochrome P450-mediated metabolism of marker substrates by natural products.

Spices, herbal and black teas, and soybean products were analyzed for their capacity to inhibit in vitro metabolism of drug marker substrates by human cytochrome P-450 (CYP) isoforms. Inhibition of drug metabolism was determined using aliquots or infusions from these products in a fluorescence-detection assay. Aliquots and infusions of all natural product categories inhibited 3A4 metabolism to some extent. Of the 26 aliquots from teas and spices further tested with 2C9, 2C19 and 2D6, many demonstrated significant inhibitory activity on the metabolism mediated by these isoforms. Black teas and herbal tea mixtures were generally more inhibitory than single-entity herbal teas. Spices and single-entity herbal teas showed species-specific isoform inhibition with sage, thyme, cloves, St John's Wort and goldenseal having the highest activity against several isoforms. Seven soybean varieties tested, as well as daidzein and genistein isolated from soybean, were found to inhibit 3A4-mediated metabolism. Genistein was found to inhibit 3A7- but not 3A5-mediated metabolism of the marker substrate. Assessment of the in vitro CYP inhibition potential for these natural products has important implications for predicting the likelihood of natural product-drug interactions if these products are taken concomitantly.

Extraction of incurred sulphamethazine in swine tissue by microwave assisted extraction and quantification without clean up by high performance liquid chromatography following derivatization with dimethylaminobenzaldehyde.

A rapid and reliable method using microwave energy is described for the extraction of spiked and incurred (freeze-dried and fresh) sulphamethazine residues from swine tissues/organs (muscle, liver and kidney). Incurred tissues were obtained from an abattoir and freeze-dried pig tissue reference materials were produced as part of a reference material study for the Community Bureau of References, European Communities. The extraction was achieved by irradiating the sample in methanol for 25 s in a household microwave oven, commonly referred to as microwave-assisted extraction (MAE). The extracts were analysed without clean-up by high performance liquid chromatography (HPLC) using a C18 column and detected at 450 nm after derivatization with 4-dimethylaminobenzaldehyde (DMABA) in a heated rector at 40 degrees C. The limit of quantitation was 2.5 micrograms kg-1 of wet tissues. A comparison of MAE with an homogenization technique indicated that MAE worked extremely well for freeze-dried samples, while it showed significant variation for wet tissues. No sulphamethazine was detected in retail pork meat and liver samples when analysed by the MAE technique.

Fate and residues of 14C-chloramphenicol in laying chickens.

Studies were conducted to determine the metabolic fate of chloramphenicol (CAP) in White Leghorn using the 14C-labelled compound. In one experiment birds were administered orally via intra-crop, a single dose of 100 mg (equivalent to 66 mg kg-1 body weight) of CAP containing 14 microCi 14C-CAP, and its absorption, elimination and distribution in plasma were recorded. Orally dosed 14C-compound was rapidly absorbed, efficiently distributed in plasma and eliminated in excreta (> 70% in 5 hr). After 5 h, CAP equivalent residues in issues were lower than 15 micrograms g-1 for this treatment. In a second experiment birds were given intra-crop dose of either 0.5 or 5 mg of CAP (each dose contained 2.5 microCi 14C-CAP) daily for five consecutive days followed by a seven day withdrawal period and elimination of 14C in excreta and eggs was monitored. More than 95% of the administered 14C was eliminated within the first 24 h after dosing. Radiocarbon (14C) was deposited preferentially in yolks compared to albumen or other tissues. Residues declined when feeding was stopped. Various metabolites were isolated and identified by a combination of TLC, LC, and LC-MS. The main metabolic route of CAP in laying hens appears to be the glucuronidation. Cleavage of the dichloroacetate moiety was only a minor route.

Microwave extraction of incurred salinomycin from chicken tissues.

A rapid, accurate, environmentally friendly and cost-effective microwave extraction technique was developed for the extraction of spiked and incurred salinomycin from chicken tissues (kidney, liver, muscle, ovarian yolk and fat). Extraction of salinomycin from various tissues was achieved by irradiating the sample in absolute ethanol and 2-propanol (15 + 2) for 9 sec. in a common household microwave oven. The extract was analysed without further cleanup by HPLC on a C18 column (5 microns) and detected at 592 nm via post-column reaction with 4-dimethylaminobenzaldehyde (DMABA) in a heated reactor coil at 86 degrees C. Recoveries of salinomycin from spiked tissues at 30 ng/g level ranged between 87 and 100%. The limit of quantitation was found to be 10 ng/g. The developed method was applied for the analysis of incurred tissues and ovarian yolk of laying chickens given sodium salinomycin in feed at different levels for 14 consecutive days followed by withdrawal periods. Residues were detected in all tissues and ovarian yolk at 0 withdrawal time but declined during the withdrawal period. Highest residue were found in fat and ovarian yolk.

Bioavailability of bound pesticide residues and potential toxicologic consequences - an update.

The growing demand for enhanced food productivity to meet the needs of the global population has led farmers to use sophisticated agricultural technology in which pesticides play a crucial role. Pesticide use has a positive and dramatic impact on agricultural production through protection of crops against insects, pests, and disease, but every effort must be made to ensure that application is safe and, more importantly, to assure safety for human and environmental health. Since our initial review (1), global usage of pesticides has increased and knowledge regarding the biological significance of bound pesticide residues has expanded. The fact that more reports are appearing in the literature signifies an increased awareness of the presence of bound pesticide residues and indicates that a greater number of scientists are attempting to establish the potential toxicologic consequences of this pesticides residue fraction. Rico (2), in a review in 1990, concluded that covalently bound residues in edible animal tissues were not carcinogenic, were not readily bioavailable, and hence produced little, if any, adverse effects. Whether this conclusion is applicable to the presence and consequences of bound pesticide residues in plants and food commodities remains unanswered. Thus, the aim of this updated review is not simply to list studies on bioavailability of bound pesticide residues in grains subsequent to the review in 1992 (1) but also to establish the toxicological impact of this chemical fraction on health.

Fate of wheat bound malathion residues in rats during gestation.

Malathion [S-1,2-di(ethoxycarbonyl) ethyl 0,0-dimethyl phosphorodithioate], treated wheat when stored for 28 months at 20 degrees C with or without food grade white mineral oil on grains contained about 62 and 79% of the applied insecticide as bound residues, respectively. These bound residues were present mainly in the form of the parent compound. The stored wheat containing bound malathion residues, as well as wheat material freshly spiked with malathion were fed to rats during gestation. No residues of malathion and/or metabolites were detected in urine, feces and body tissues. Further no significant effect on body weight, serum chemistry and cytochrome P450 levels were observed in the mothers. There was no evidence for the histopathological alteration or teratogenic anomalies in the fetuses. However, placental transfer of malathion was indicated by the presence of the insecticide residues in fetuses from rats fed wheat material containing bound residues.

Comparative metabolism of deltamethrin and 3-phenoxybenzoic acid in chickens.

A comparative metabolism of [benzyl 14C]deltamethrin and 3-phenoxybenzoic acid (3-PBacid) was carried out in chickens. The effect of oral and intravenous route of administration was also investigated. There was no difference in the metabolic profile of [14C] portion of deltamethrin and 3-PBacid. Similarly, no effect on the nature of metabolites excreted due to mode of administration was observed. Biotransformation of orally dosed compounds did not occur in the intestine, but were absorbed and metabolized by liver (deltamethrin) and kidney (3-PBacid). 3-PBacid, the primary metabolite of deltamethrin, was readily metabolized into 3-hydroxy benzoic acid (3-HO-Bacid) which is conjugated with a variety of endogenous substances to form sulphates, glucuronides, butyl ester and peptides. HPLC and LC-Ms methods were used to ascertain the structures of metabolites.

Cleavage of 3-phenoxybenzoic acid by chicken microsomal preparations.

3-Phenoxybenzoic acid, labelled at either the 3-phenoxy[U-ring C-14] or carbonyl C-14, produced the corresponding labelled transitory metabolite 4'-hydroxy-3-phenoxybenzoic acid when incubated with chicken liver microsomal preparation. The other radioactive reaction product identified from the metabolism of the carbonyl-labelled 3-phenoxybenzoic acid was labelled 3-hydroxybenzoic acid, whereas metabolism of the [U-ring] labelled 3-phenoxybenzoic acid produced labelled hydroquinone in addition to 4'-hydroxy-3-phenoxybenzoic acid. Formation of both 4'-hydroxy-3-phenoxybenzoic acid, and its subsequent conversion to 3-hydroxybenzoic andhydroquinone were NADPH and oxygen dependent. The reaction was inhibited by carbon monoxide, but not by piperonyl butoxide or metyrapone. The inhibition by carbon monoxide suggests that the enzyme(s) involved in the cleavage is a form of P450. The enzyme(s) is a monooxygenase and has requirement for NADPH and oxygen.

Deltamethrin residues in milk and tissues of lactating dairy cows.

Lactating dairy cows were fed deltamethrin (2 or 10 mg kg-1 feed) for 28 consecutive days and deltamethrin residues measured in milk and tissues. Deltamethrin residues were higher relative to dose administered. The order of relative concentrations of deltamethrin in tissues, measured 1, 4, and 9 days after the last dose was: renal fat greater than subcutaneous fat greater than forequarter muscle greater than hindquarter muscle greater than liver greater than kidney. Depletion of deltamethrin residues in milk was very rapid indicating the half-life of the insectide of about 1 day. Trace amounts of deltamethrin metabolites 3-(2,2-dibromovinyl)-2,2-dimethylcyclopropane carboxylic acid (less than 0.0235 ppm) and 3-phenoxybenzoic acid (less than 0.034 ppm) were also detected in milk and tissues of treated cows.

Diphenyl ether cleavage of 3-phenoxybenzoic acid by chicken kidney microsomal preparations.

Incubation of 3-phenoxybenzoic acid[carbonyl-14C] with chicken kidney microsomal fraction produced two major radioactive compounds identified as 3-hydroxybenzoic acid and 4'-hydroxy-3-phenoxybenzoic acid. It was found that 4'-hydroxy-3-phenoxybenzoic acid was transitory in nature and was rapidly converted into 3-hydroxybenzoic acid. The conversion of 3-phenoxybenzoic acid to the products was NADPH dependent. This is a first example of metabolism of 4'-hydroxy-3-phenoxybenzoic acid to 3-hydroxybenzoic acid. Cytosol did not promote any cleavage. Similar activities were present in liver microsomes, but the activity was lower than in kidney. Intestinal contents, homogenates, or microsomal fractions did not metabolize 3-phenoxybenzoic acid.

Identification and characterization of a pseudomonas strain capable of metabolizing phenoxybenzoates.

3-Phenoxybenzoate is a transient metabolite from the breakdown of a number of pyrethroid insecticides in soil. In this study, we identified and characterized a bacterium which could grow on 3-phenoxybenzoate, converting it to phenol. On the basis of morphological and biochemical features, the 3-phenoxybenzoatedegrading isolate was determined to be a Pseudomonas species, probably a strain of Pseudomonas delafieldii, now designated Pseudomonas strain ET1. Pseudomonas strain ET1 grew on 3-phenoxybenzoate with a generation time of 3 h and a specific rate of metabolism of (2.6 +/- 0.9) x 10 g of 3-phenoxybenzoate consumed cell h. The K(m) for 3-phenoxybenzoate metabolism was 1.4 +/- 0.8 muM. The metabolism of 3-phenoxybenzoate was constitutive and not subject to catabolite repression. The metabolism of a variety of substituted diaryl ether compounds was examined. 3- and 4-Phenoxybenzoates were metabolized, but 2-phenoxybenzoate was not. Phenoxy-substituted benzyl aldehyde was metabolized, but phenoxy-substituted benzyl alcohol, benzene, phenol, and aniline were not. Derivatives of 3-phenoxybenzoate substituted in the 4' position with hydroxyl, methyl, or chlorine were metabolized, yielding the corresponding 4-substituted phenol. 3-(2-Hydroxyphenoxy)benzoate was not metabolized, but 3-phenoxy-4-fluorobenzoate was. These results indicate that the metabolism of the tested diaryl ether compounds was restricted to 4-phenoxybenzoate, 3-phenoxybenzyl aldehyde, and 3-phenoxybenzoate derivatives without a substitution in the 2' position.

Gas chromatographic determination of deltamethrin in biological samples.

A gas chromatographic method is described for the detection and estimation of deltamethrin in spiked samples of urine, feces, milk, liver, kidney, muscle and subcutaneous fat. It involved extraction with acetone, with the exception of milk and urine samples, liquid-liquid partition with acetonitrile, followed by clean-up on a micro-column using benzene-hexane (1:1) solvent system. Milk and urine samples were extracted with hexane followed by liquid-liquid partition with acetonitrile. Under the gas chromatographic conditions employed, deltamethrin eluted in less than 4 min.