Semiautomated preparation of 3,5,6-trichloro-2-pyridinol in human urine using a Zymate XP laboratory robot with quantitative determination by gas chromatography-negative-ion chemical ionization mass spectrometry.
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A rapid and sensitive semiautomated method was developed for quantitation of the chlorpyrifos metabolite 3,5,6-trichloro-2-pyridinol (TCP) in human urine. A Zymark Zymate XP laboratory robotics system was used to mix urine samples, transfer aliquots, add the stable-isotope-labeled TCP internal standard (13C2- or 13C2,15N-), and liberate conjugates of TCP from urine via acid hydrolysis. Samples were manually extracted into toluene, derivatized, and analyzed by gas chromatography-negative-ion chemical ionization mass spectrometry. Determination of the metabolic TCP was performed by selected ion monitoring of the dichloropyridinol fragment ions: m/z 161 for TCP and m/z 165 for 13C2-TCP or m/z 168 for 13C2,15N-TCP. Interday precision and accuracy were demonstrated over 3 years of analyses using the 13C2-TCP internal standard, with an average recovery from fortified urine samples of 93+/-12% (N = 54, concentration range 1-140 ng/mL). The method was found to be linear over the range of 0.5 to 200 ng/mL, and the limit of detection for TCP in urine was estimated to be 0.2 ng/mL with a limit of quantitation of 1 ng/mL. The effect of solids distribution on the concentration of TCP in the thawed urine samples was examined, and the results indicated that homogeneous distribution is critical for quantitation. The precision and accuracy of the automated method with respect to the transfer of homgeneous urine aliquots and delivery of internal standard yielded equivalent or improved results over the manual techniques. Overall, this method is more simple than existing methodologies, and it yields results with improved precision, accuracy, and sensitivity over previously developed methods. (+info)
Effect of phenylurea herbicides on soil microbial communities estimated by analysis of 16S rRNA gene fingerprints and community-level physiological profiles.
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The effect of three phenyl urea herbicides (diuron, linuron, and chlorotoluron) on soil microbial communities was studied by using soil samples with a 10-year history of treatment. Denaturing gradient gel electrophoresis (DGGE) was used for the analysis of 16S rRNA genes (16S rDNA). The degree of similarity between the 16S rDNA profiles of the communities was quantified by numerically analysing the DGGE band patterns. Similarity dendrograms showed that the microbial community structures of the herbicide-treated and nontreated soils were significantly different. Moreover, the bacterial diversity seemed to decrease in soils treated with urea herbicides, and sequence determination of several DGGE fragments showed that the most affected species in the soils treated with diuron and linuron belonged to an uncultivated bacterial group. As well as the 16S rDNA fingerprints, the substrate utilization patterns of the microbial communities were compared. Principal-component analysis performed on BIOLOG data showed that the functional abilities of the soil microbial communities were altered by the application of the herbicides. In addition, enrichment cultures of the different soils in medium with the urea herbicides as the sole carbon and nitrogen source showed that there was no difference between treated and nontreated soil in the rate of transformation of diuron and chlorotoluron but that there was a strong difference in the case of linuron. In the enrichment cultures with linuron-treated soil, linuron disappeared completely after 1 week whereas no significant transformation was observed in cultures inoculated with nontreated soil even after 4 weeks. In conclusion, this study showed that both the structure and metabolic potential of soil microbial communities were clearly affected by a long-term application of urea herbicides. (+info)
Cytotoxic effect of paraquat on rat C6 glioma cells: evidence for the possibility of non-oxidative damage to the cells.
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Although paraquat has been shown to cause oxidative damage to neuronal cells, little is known about its effect on glial cells. Thus the effect of paraquat on glial cells was examined using rat C6 glioma cells as a model system. Paraquat reduced cell viability in a concentration- and time-dependent manner, and this toxic effect was not significantly attenuated by various kinds of antioxidants. Furthermore, paraquat failed to increase 8-hydroxy-deoxyguanosine formation in the cells. These results indicate that paraquat can be toxic to glial cells and suggest that this cytotoxic effect may not be associated with the oxidative damage to the cells. (+info)
High-performance liquid chromatography column switching applied to the trace determination of herbicides in environmental and drinking water samples.
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A selective and sensitive coupled-column high-performance liquid chromatographic method is developed for the simultaneous determination of 5 phenylurea herbicides (monuron, linuron, isoproturon, monolinuron, and diuron) in environmental and drinking water samples. Sample clean-up is performed automatically by means of a column switching technique. Using 2 octadecyl silica columns connected via two programmable 6-port valves and ultraviolet detection at 244 nm, the aforementioned compounds can be determined at the low concentration levels required for pesticide residue analysis in water samples. A mobile phase consisting of a mixture of methanol-water (55:45, v/v) is pumped at 1 mL/min. For the 5 phenylureas, high recoveries ranging from 94.9 to 101.6%, good reproducibility with relative standard deviations lower than 5%, and wide linear ranges up to 20 micrograms/L are observed with determination limits of 0.05 microgram/L. The method is successfully applied to the screening of different environmental water samples such as surface, ground, rain, and drinking water. (+info)
Construction and characterization of a functional mutant of Synechocystis 6803 harbouring a eukaryotic PSII-H subunit.
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A Synechocystis 6803 mutant carrying a chimaeric photosystem II (PSII), in which the Zea mays PsbH subunit (7.7 kDa calculated molecular mass) replaces the cyanobacterial copy (7.0 kDa), was constructed. With the exception of the N-terminal 12 amino acid extension, which has a phosphorylatable threonine, the eukaryotic polypeptide is 78% homologous to its bacterial counterpart. Biochemical characterization of this mutant shows that it expresses the engineered gene correctly and is competent for photoautotrophic growth. Fluorescence analysis and oxygen evolution measurements in the presence of exogenous acceptors indicate that the observed phenotype results from a chimaeric PSII rather than from the absence of function associated with PsbH, suggesting that the heterologous protein is assembled into a functional PSII. Inhibition of oxygen evolution by herbicides belonging to different classes shows that the sensitivity of the mutant PSII is changed only towards phenolic compounds. This result indicates slight conformational modification of the QB/herbicide binding pocket of the D1 polypeptide caused by the bulky PsbH protein in the mutant, and also suggests close structural interaction of the D1 and PsbH subunits in the topological arrangement of PSII. (+info)
A comparison of electron-capture GLC, electrolytic-conductivity GLC and UV-absorption HPLC for the analysis of some herbicides in foods.
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A comparison of gas chromatography with electron-capture or electrolytic-conductivity (nitrogen mode) detection, and high-pressure liquid chromatography (HPLC) with UV-absorption detection (254 nm) was carried out for the analysis of several herbicides in foods. Linuron, propanil, terbacil, benzoylprop-ethyl, and the fungicide DCNA in samples of cabbage, corn, potato, and wheat spiked at 2 and 0.2 ppm were examined. The pesticides were extracted with acetone, partitioned into petroleum ether-methylene chloride, and cleaned up on a 2% deactivated Florisil column before direct chromatographic analysis. Electron-capture gas-liquid chromatography (GLC) was most suitable for DCNA and benzoylprop-ethyl while UV-absorption HPLC was best for terbacil analysis. Linuron and propanil gave similar results for both electron-capture GLC and HPLC. Electrolytic-conductivity GLC could detect all pesticides at the 0.2 ppm level and exhibited the least number of extraneous peaks in the chromatograms. (+info)
Selected phenolic compounds in cultivated plants: ecologic functions, health implications, and modulation by pesticides.
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Phenolic compounds are widely distributed in the plant kingdom. Plant tissues may contain up to several grams per kilogram. External stimuli such as microbial infections, ultraviolet radiation, and chemical stressors induce their synthesis. The phenolic compounds resveratrol, flavonoids, and furanocoumarins have many ecologic functions and affect human health. Ecologic functions include defense against microbial pathogens and herbivorous animals. Phenolic compounds may have both beneficial and toxic effects on human health. Effects on low-density lipoproteins and aggregation of platelets are beneficial because they reduce the risk of coronary heart disease. Mutagenic, cancerogenic, and phototoxic effects are risk factors of human health. The synthesis of phenolic compounds in plants can be modulated by the application of herbicides and, to a lesser extent, insecticides and fungicides. The effects on ecosystem functioning and human health are complex and cannot be predicted with great certainty. The consequences of the combined natural and pesticide-induced modulating effects for ecologic functions and human health should be further evaluated. (+info)
Induction of the multispecific organic anion transporter (cMoat/mrp2) gene and biliary glutathione secretion by the herbicide 2,4,5-trichlorophenoxyacetic acid in the mouse liver.
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The canalicular multispecific organic anion transporter, cMoat, is an ATP-binding-cassette protein expressed in the canalicular domain of hepatocytes. In addition to the transport of endo- and xenobiotics, cMoat has also been proposed to transport GSH into bile, the major driving force of bile-acid-independent bile flow. We have shown previously that the herbicide 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), a peroxisome-proliferator agent, significantly increases bile-acid-independent bile flow in mice. On this basis, the effect of the herbicide on cMoat gene expression was studied. A 3.6-fold increase in cMoat mRNA levels and a 2.5-fold increase in cMoat protein content were observed in the liver of mice fed on a diet supplemented with 0.125% 2,4,5-T. These effects were due to an increased rate of gene transcription (3.9-fold) and were not associated with peroxisome proliferation. Significant increases in bile flow (2.23+/-0.39 versus 1.13+/-0.15 microl/min per g of liver; P<0.05) and biliary GSH output (7.40+/-3.30 versus 2.65+/-0.34 nmol/min per g of liver; P<0.05) were observed in treated animals. The hepatocellular concentration of total glutathione also increased in hepatocytes of treated mice (10.95+/-0.84 versus 5.12+/-0.47 mM; P<0.05), because of the induction (2.4-fold) of the heavy subunit of the gamma-glutamylcysteine synthetase (GCS-HS) gene. This is the first model of co-induction of cMoat and GCS-HS genes in vivo in the mouse liver, associated with increased glutathione synthesis and biliary glutathione output. Our observations are consistent with the hypothesis that the cMoat transporter plays a crucial role in the secretion of biliary GSH. (+info)