Activities of the enzymes participating in purine and free-radical metabolism in cancerous human colorectal tissues

Liquid chromatography-pneumatically assisted electrospray mass spectrometry with negative ionization has been used for the determination of acidic herbicides in ground water. Eighteen pesticides or pesticide degradation products belonging to several different groups of acidic herbicides (phenoxy acids, sulfonylureas, phenols, etc.) were covered in the study. Optimization of electrospray inlet conditions is described as well as results from investigations of the linearity of the detector response. Conditions for tandem mass spectrometry (MS-MS) detection of characteristic daughter ions formed by collision-induced dissociation (CID) of the parent ion are described and a comparison of obtainable instrument detection limits by single MS and MS-MS was made. Detection limits using MS in the selected ion monitoring (SIM) mode were generally in the order of 1 microgram/l or below, whereas detection limits were three-four times higher using MS-MS detection. A principle of analysis is proposed based on single quadrupole MS as a method for quantitative determination followed by verification of positive findings by CID MS-MS. Application of the method for detecting acidic herbicides residues in a "real-world" ground water sample is demonstrated.     

Use of solid phase extraction disks for the GC-MS analysis of acidic and neutral herbicides in drinking water

An analytical procedure was developed in which thirteen herbicides (10 acidic and 3 neutral compounds) may be extracted from drinking water samples using solid phase extraction disks and subsequently analyzed by gas chromatography-mass spectrometry. Using 8 replicate samples consisting of reagent water fortified with nanogram quantities of each herbicide, the average percent recoveries and method detection limits were determined for each analyte. The method was found to be suitable for the determination of individual herbicides in drinking water at concentrations of approximately 10 ng/L.     

Monitoring impact of a pesticide treatment on bacterial soil communities by metabolic and genetic fingerprinting in addition to conventional testing procedures

Herbogil (dinoterb), a reference herbicide, the mineral oil Oleo (paraffin oil used as an additive to herbicides), and Goltix (metamitron) were taken as model compounds for the study of impacts on microbial soil communities. After the treatment of soil samples, effects on metabolic sum parameters were determined by monitoring substrate-induced respiration (SIR) and dehydrogenase activity, as well as carbon and nitrogen mineralization. These conventional ecotoxicological testing procedures are used in pesticide registration. Inhibition of biomass-related activities and stimulation of nitrogen mineralization were the most significant effects caused by the application of Herbogil. Even though Goltix and Oleo were used at a higher dosage (10 times higher), the application of Goltix resulted in smaller effects and the additive Oleo was the least-active compound, with minor stimulation of test parameters at later observation times. The results served as a background for investigation of the power of "fingerprinting" methods in microbial ecology. Changes in catabolic activities induced by treatments were analyzed by using the 95 carbon sources provided by the BIOLOG system. Variations in the complex metabolic fingerprints demonstrated inhibition of many catabolic pathways after the application of Herbogil. Again, the effects of the other compounds were expressed at much lower levels and comprised stimulations as well as inhibitions. Testing for significance by a multivariate t test indicated that the sensitivity of this method was similar to the sensitivities of the conventional testing procedures. The variation of sensitive carbon sources, as determined by factor weights at different observation times, indicated the dynamics of the community shift induced by the Herbogil treatment in more detail. DNA extractions from soil resulted in a collection of molecules representing the genetic composition of total bacterial communities. Distinct and highly reproducible community patterns, or genetic fingerprints, resulting from application of the different herbicides were obtained by the sequence-specific separation of partial 16S rDNA amplification products in temperature gradient gel electrophoresis. Significant pattern variations were quantified. For detailed analysis, application-responsive bands from the Herbogil and Oleo treatments were sequenced and their tentative phylogenetic positions were identified. Data interpretation and the potentials and biases of the additional observation windows on microbial communities are discussed.     

Synthesis and Study on Physical Properties for Magnetic Superconductor RESr2RuCu2O8（RE = Gd and Eu）

Single-phase magnetic superconductors RESrzRuCuzOs(RE = C,d and Eu) were successfully synthesized and investigations on their physical properties were carried out. It is found that the structure of the compounds is similar to that of YBa2Cu307 and its superconductivity coexists with weak ferromagnetic order in both compounds. The compounds exhibit magnetic order below TM = 130 and 136 K and superconducting below TC^onset = 35 and 46 K, respectively, for RE = Eu and Gd samples. There is slight difference in the magnetic properties between RE = Euand C,d compounds owing to various magnetic moments for C,d3 and Eu3 .     

Structures of herbicides in complex with their detoxifying enzyme glutathione S-transferase - explanations for the selectivity of the enzyme in plants

BACKGROUND: Glutathione S-transferases (GSTs) are detoxifying enzymes present in all aerobic organisms. These enzymes catalyse the conjugation of glutathione with a variety of electrophilic compounds. In plants, GSTs catalyse the first step in the degradation of several herbicides, such as triazines and acetamides, thus playing an important role in herbicide tolerance. RESULTS: We have solved the structures of GST-I from maize in complex with an atrazine-glutathione conjugate (at 2.8 A resolution) and GST from Arabidopsis thaliana (araGST) in complex with an FOE-4053-glutathione conjugate (at 2.6 A resolution). These ligands are products of the detoxifying reaction and are well defined in the electron density. The herbicide-binding site (H site) is different in the two structures. The architecture of the glutathione-binding site (G site) of araGST is different to that of the previously described structure of GST in complex with two S-hexylglutathione molecules, but is homologous to that of GST-I. CONCLUSIONS: Three features are responsible for the differences in the H site of the two GSTs described here: the exchange of hydrophobic residues of different degrees of bulkiness; a slight difference in the location of the H site; and a difference in the degree of flexibility of the upper side of the H site, which is built up by the loop between helices alpha4 and alpha5. Taking these two structures as a model, the different substrate specificities of other plant GSTs may be explained. The structures reported here provide a basis for the design of new, more selective herbicides.     

Multiresidue method for the chromatographic determination of triazine herbicides and their metabolites in raw agricultural products

A method is described for the determination of 19 triazine herbicides and 4 metabolites in 6 agricultural products that represent diverse matrixes. In addition, a modification of this method to determine the more water-soluble metabolite, desethyldesisopropylatrazine, is described. In both these procedures, residues are extracted with methanol, and the product coextractives are removed using solvent partition and cation-exchange solid-phase extraction chromatography. A nitrogen phosphorus detector and DB-17 megabore column are used for the temperature-programmed chromatographic determination of samples fortified at 0.02-1.0 ppm levels. Average recoveries ranged from 81.1 to 106.2% for the parent herbicides and from 59.6 to 87.5% for the metabolites on all crops. An average recovery of 101.1% was obtained for desethyldesisopropylatrazine.     

Analysis of pesticide residues by chemical derivatization. V. Multiresidue analysis of eight phenoxyalkanoic acid herbicides in natural waters

A multiresidue method is presented for the gas-liquid chromatographic determination of phenoxyalkanoic acid herbicides in natural waters. Extraction efficiencies of different organic solvents are considered in developing a solvent extraction scheme for these herbicides from water. Reactions for derivatizing these compounds by using pentafluorobenzyl bromide, boron trichloride-2-chloroethanol, and dicyclohexylcarbodiimide-2-chloroethanol were studied in order to obtain extracts with low blanks to provide the lowest detection limits. Advantages and disadvantages of the 3 methods are discussed. Retention times are twice as long for the pentafluorobenzyl (PFB) esters as for the 2-chloroethyl (2-Cl) esters under the same conditions, although electron capture sensitivity to the former was greater. The PFB esters are easier to form, but the 2-Cl reaction is more specific for these herbicides. Solutions from the boron trichloride reaction gave the cleanest blanks.     

Biomimetic metabolism of artelinic acid by chemical cytochrome P-450 model systems

PURPOSE: To study the reaction of artelinic acid with chemical model systems of cytochrome P-450 as a means of obtaining authentic samples of the putative metabolites necessary for identification of the mammalian metabolites of artelinic acid. METHODS: Artelinic acid was reacted with different organic complexes of iron(II). The reaction products were isolated and characterized by NMR and thermospray mass spectroscopy. RESULTS: Five compounds which are putative metabolites of artelinic acid were isolated from these reactions and unambiguously identified, while the identity of two other compounds await final confirmation. CONCLUSIONS: Standards of possible metabolites of artelinic acid can be produced by the reaction of the compound with ferrous complexes that may simulate cytochrome P-450 catalyzed metabolism of xenobiotics. This approach may provide a simple and versatile method for the formation of metabolites of artemisinin compounds which is more advantageous than previous approaches with fungal-based systems.     

Plutonium in fish, algae, and sediments in the Barents, Petshora and Kara Seas

This work contributed to a joint research programme between the Finnish Centre for Radiation and Nuclear Safety and the Murmansk Marine Biological Institute in the Arctic. Radioanalyses for plutonium isotopes were performed on more than 50 sediment samples, 12 algae samples and 19 fish samples. Plutonium concentrations in algae and fish samples, including fish meat, bone and liver, were low or in many cases below detection limits. Some differences in plutonium concentrations of sediments were found between different sampling areas. However, the concentrations were low. The Pu isotopic ratios were similar to those found in environmental samples generally when Pu is derived from global fallout or discharges from reprocessing plants. No local enhancement of plutonium contamination was found in the marine areas studied. However, the sampling locations represent only areas of free access; prohibited military areas of North-west Russia with potential pollution sources were not included.     

Reactivity of aliphatic peptides toward hydroxyl radicals in aqueous solution

Dextrostix is now widely used as a method of screening for hypoglycemia of the newborn. There has always been some anxiety about its accuracy for estimating very low blood sugars; this is important, since symptomatic hypoglycemia of the newborn does not usually occur until the blood glucose falls below 20 mg/dl. In 1970 a reflectance meter was introduced which would measure the colour of the strip electronically. The aim of this study was to assess the accuracy of the meter and its reliability in estimating hypoglycemia. The study was continued to investigate a new model of the reflectance meter which superseded the original one. In the pilot study using the original model, blood samples were taken from 46 babies. Readings of the Dextrostix were made by two independent observers and compared with a reading taken on the reflectance meter. These estimations were later compared with blood glucose measured by the glucose oxidase method. the meter showed a marked tendency to overestimate the blood glucose: 44 out of 46 samples were overestimated (Fig. 1, 2). In the second series 180 cord blood samples were collected. Because the intention was to study very low levels of blood glucose, the samples were allowed to stand at room temperature for several hours to allow glycolysis to occur. Again, readings were taken by two independent observers and compared with the readings taken on the new Dextrostix-Eyetone meter. The blood glucose was measured on each of the samples. There was a strong correlation (r = 0.8877, p less than 0.00005) between the blood glucose values and the readings taken from the meter, with no tendency towards overestimation (Fig. 3). A similar correlation (r = 0.8533, p less than 0.00005) was seen for the observers' readings and the chemical method, although there was a tendency to underestimate blood glucose (Fig. 4). When the meter gave an estimate of more than 20 ml/dl, in no case was the actual blood glucose in the profoundly hypoglycemic group of less than 10 mg/dl. When estimated by eye there was one case in which the blood glucose was only 7 mg/dl but the observer had estimated the result of 30 mg/dl. In order to avoid any possibility that a blood glucose was less than 20 mg/dl, it is necessary to take action on any estimate below 40 mg/dl on a Dextrostix. Despite this, Dextrostix remains a very useful method of screening for neonatal hypoglycemia, whether assessed by eye or with the new Dextrostix-Eyetone meter. the meter does give better results than estimating Dextrostix by eye, but the difference was never statistically significant.     

The kinetics of bioaccumulation of zinc, copper, lead and cadmium by oysters (Crassostrea iredalei and C. belcheri) under tropical field conditions

Cyanobacterial neurotoxins have been implicated in animal deaths resulting from drinking contaminated water. Anatoxin-a (AN) and homoanatoxin-a (HMAN) have previously been analysed using high-performance liquid chromatography (HPLC) with UV detection, but this procedure is insufficiently sensitive and is subject to interferences. A sensitive fluorimetric (FL) method for determining AN was recently developed using derivatisation with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) and this has been applied to the simultaneous determination of AN, HMAN and their epoxy and dihydro degradation products. Microscale syntheses were used to prepare the dihydro and epoxy derivatives from AN and HMAN. These compounds were produced in high yields, as confirmed by electrospray MS and HPLC-FL of their benzoxadiazole derivatives. All six NBD derivatives were readily separated using isocratic reversed-phase HPLC. The recoveries of these compounds from spiked water samples, using weak cation-exchange (WCX) solid-phase extraction (SPE), were 83.2-84.9% at concentrations of 10 micrograms/l. The R.S.D. values were 1.7-3.9% (n = 8) and the limits of detection were better than 10 ng/l for all six compounds, illustrating the high sensitivity of the method. This methodology was successfully applied to the analysis toxin degradation products in natural samples. Dihydroanatoxin-a (0.8 mg/g) was isolated from a benthic Oscillatoria bloom from Caragh Lake, Ireland, and was found to contain two isomers but their ratio was different from that found in the synthetic material.     

A study of the degradation of phenoxyacid herbicides at different sites in a limestone aquifer

The biodegradation of three phenoxyalkanoic acid herbicides, viz. MCPA, dichlorprop and mecoprop, under aerobic and anaerobic conditions was investigated using microcosm techniques. The field studies were conducted in a limestone aquifer that had suffered contamination from leaking landfill sites in which phenoxyalkanoic acid herbicides (mostly mecoprop) had undergone disposal. The results from in situ field and laboratory microcosms indicated that under microbially active aerobic conditions the biotransformation of all three herbicides was rapid and that lag phases were short. Under fully aerobic conditions the concentration of each of the three herbicides was reduced from 2,000 micrograms/l to below the detection limit (approx. 10 micrograms/l) of the HPLC system, used for their analysis, within 14 days. However, under microbially active anaerobic conditions no degradation of the herbicides could be discerned over the 100-200 day duration of the experiments. This finding has significant implications for the disposal of phenoxyalkanoic acid herbicides particularly in situations where any resulting leachate may find its way into underlying water resources.     

Oxide Inclusions at Different Steps of Steel Production

 The formation of oxide inclusions in one of the carbon steel productions of Mobarakeh Steel Complex of Isfahan has been evaluated. Several samples from different steps of steel production were taken, from arc furnace, ladle furnace, tundish, and continuous casting mold. Moreover, samples of slab and hot rolling products were prepared. The samples were investigated by optical and scanning electron microscopes equipped with the EDS system. According to the results, the number, composition, and kind of inclusions were directly influenced by the production variables. It was found that when the amount of dissolved oxygen was high (say more than 0002 5%), the dissolved aluminum was able to reduce silicon oxide and react with the dissolved oxygen simultaneously, whilst, the dissolved aluminum could reduce the magnesium oxide only when the oxygen content was below 0000 5%. Based on this research, a mechanism for forming the complex inclusions was suggested. It was also found that if the aluminum is added to the melt as late as possible, a cleaner melt with fewer inclusions is prepared; this method will be more effective, especially in the case of complex inclusions.     

Immobilized liposome chromatography for studies of protein-membrane interactions and refolding of denatured bovine carbonic anhydrase

Seasonal variations of pesticide residues in surface waters and ground waters of the Imathia area of Central Mecedonia (N. Greece) were determined for the period from May 1996 to April 1997. The sampling cruises included eight sites in rivers Aliakmon, Loudias, Tripotamos, Arapitsa and Canal-66, seven water springs in the mountain Vermion, seven rainfall water collection stations and one hundred underground points. Solid-phase extraction disks followed by gas chromatographic techniques with flame thermionic detection, electron capture detection and mass-selective detection were used for the monitoring of various pesticides their transformation products in environmental waters. The most commonly encountered pesticides in underground waters, were alachlor, atrazine, desethylatrazine (DEA), metolachlor, molinate, propanil, simazine, carbofuran, diazinon and parathion methyl. The above compounds including propazine, trifluralin, malathion, parathion ethyl, lindane, alpha-benzene hexachloride (alpha-BHC), beta-BHC, 4,4'-DDE and heptachlor were determined in river waters. The higher concentrations in underground waters were measured during the period from May to August, 1996, following seasonal application and diminished significantly during the autumn and winter. Water pollution by triazine and chloroacetanilides was highest in the estuarine areas; showing that many of these compounds are transported significant distances from their application sites. The major inputs of atrazine, alachlor, simazine and metolachlor occurred in May and June just after their application. Atrazine, DEA, diazinon and metolachlor were also detected in spring waters at concentration levels below 0.006 microgram/l. Finally, atrazine, DEA, carbofuran, simazine, diazinon, parathion ethyl and parathion methyl were detected in rainfall water samples collected in the agricultural area of Imathia (central part of the plain).     

Urinary excretion of cyclophosphamide in humans, determined by phosphorus-31 nuclear magnetic resonance spectroscopy

Phosphorus-31 NMR spectroscopy was used to analyze urine samples from patients treated with cyclophosphamide (CP) on 2 consecutive days. CP and all of its known phosphorylated metabolites except the tautomeric pair 4-hydroxycyclophosphamide/aldophosphamide, i.e. carboxycyclophosphamide (CXCP), dechloroethylcyclophosphamide (DCCP), alcophosphamide, ketophosphamide, and phosphoramide mustard (PM), were determined. Several other signals corresponding to unknown CP-related compounds were observed. Seven of them were identified; all were hydrolysis products of CP or its metabolites (one from CP, two from CXCP, three from DCCP, and one from PM). Twenty-four-hour urinary excretion of unmetabolized CP was not significantly different on the first (17% of the daily administered dose) and second (16%) days of treatment. The amounts of phosphorylated metabolites excreted in 24-hr urine samples were much higher after the second CP dose (37%) than after the first (20%), suggesting autoinduction of CP metabolism. CXCP and its two degradation products (accounting for 7-10% of CXCP) were by far the major metabolites (11.5 and 23% after the first and second doses, respectively). DCCP plus its degradation products and alcophosphamide each represented 2-3% on the first day of treatment and 5% on the second day of treatment. Levels of PM and its degradation products were extremely low (0.3 and 0.6% after the first and second CP doses, respectively), as were those of ketophosphamide (0.4 and 0.6% on the first and second days of treatment, respectively). We noted only modest interpatient variation in excreted levels of CP and all of its metabolites.     

Dialkylquinoneimine metabolites of chloroacetanilide herbicides induce sister chromatid exchanges in cultured human lymphocytes

Some of the most widely-used herbicides are the chloroacetanilides exemplified by alachlor and butachlor (derived from 2,6-diethylaniline) and metolachlor and acetochlor (synthesized from 2-ethyl-6-methylaniline). This investigation tests the hypothesis that the previously-observed oncogenicity of these herbicides is due to genotoxic intermediates such as diethylbenzoquinoneimine, a purported alachlor metabolite. Syntheses are reported here for the corresponding 2,6-dialkylbenzoquinoneimines, selected chloroacetyldialkylbenzoquinoneimines and several other candidate or known metabolites. The possible mutagenicity of diethylbenzoquinoneimine was tested in Salmonella typhimurium strains TA98 and TA100 with a weakly-positive response in the TA100 strain indicating induction of base-pair substitution mutations. The frequency of sister chromatid exchange (SCE) in Chinese hamster ovary cells was increased by alachlor at 10 microM and diethylaniline but not ethylmethylaniline at 30 and 3 microM. Isolated and cultured peripheral lymphocytes (mostly T cells) were used from two human donors to study the effects of the chloroacetanilides and their metabolites on primary human cells. In tests at 10 microM, the SCE frequency was increased by alachlor and possibly acetochlor but not by butachlor, metolachlor, dimethachlor (a 2,6-dimethyl analog) and dimethenamid (an analog based on 2,4-dimethyl-3-thienylamine). At 0.3 microM in cultured human lymphocytes, alachlor, the corresponding chloroacetanilide (N-dealkyl-alachlor) and aniline metabolites (and their 4-hydroxy derivatives), and diethylbenzoquinone were inactive or active in only one of the two donors whereas at 0.1-0.3 microM the SCE ratio for treated cells divided by the controls was always higher for diethylbenzoquinoneimine than for ethylmethyl- and dimethylbenzoquinoneimines. All the tested compounds were toxic to lymphocytes, but the depression of the mitotic index and increased duration of the cell cycle were not directly linked with SCE induction. Previous investigations have suggested that chloroacetanilide herbicides such as alachlor derived from 2,6-dialkylanilines are metabolized to 2,6-dialkylbenzoquinoneimines and the present study provides the first direct evidence that these metabolites are genotoxic in human lymphocytes.     

Evaluation and confirmation of an alkylation-gas-liquid chromatographic method for the determination of carbamate and urea herbicides in foods

A method is described for the direct analysis of the urea herbicides linuron, monuron, diuron, chlorbromuron, fluometuron, chloroxuron, and fenuron in cabbage, corn, potatoes, turnip, and wheat at 0.01-1.0 ppm. The samples are extracted with acetone; the filtrate is partitioned with hexane-methylene chloride (1+1) followed by 2 other extractions with methylene chloride. The organic phases are dried and concentrated for column chromatographic cleanup on 2% deactivated Florisil. The column fractions are evaporated just to dryness and redissolved in 10 ml isooctane for high-pressure liquid chromatography on a 25 cm silica gel (5 mum) column. Isopropanol in isooctane is the mobile phase, and compounds are measured by ultraviolet absorption at 254 nm. Recoveries are greater than 80% in most cases. These results are confirmed by alkylation with sodium hydride-methyl iodide in dimethyl sulfoxide to form the methyl products which are analyzed by the same chromatographic system or by gas-liquid chromatography.     

Liquid Sodium Ferrate and Fenton’s Reagent for Treatment of Mature Landfill Leachate

As landfills mature, biodegradable matter in leachate is consumed and remaining compounds are increasingly recalcitrant. In this work, ferrate was compared to Fenton’s reagent for the purpose of removing nonbiodegradable organic compounds from mature leachate. Oxidation conditions (time, pH, and dose) were optimized to yield maximum organic removal using two leachate samples from 20- and 12-year-old solid waste cells. Results from this study demonstrated that Ferrate and Fenton’s reagent had similar optimum pH ranges (3–5), but different organic removal capacities, ranging from 54 to 79% of initial leachate organic contents. An advantage of ferrate was that it was effective over a wide pH range. Advantages associated with Fenton’s reagent include that it had higher organic removal capacity, produced more oxidized organic compounds (measured as chemical oxygen demand/dissolved organic carbon), and produced more biodegradable byproducts (measured as chemical oxygen demand/5-day biochemical oxygen demand). Finally, both treatments were found to attack larger molecules (>1,000?Dalton), as indicated by an increase in smaller molecule contribution to organic carbon.     

Acid leaching studies of neutron-irradiated chyrsotile asbestos

Samples of chrysotile from Quebec (UICC B and Jeffrey 4T-30) and from the Coalinga region of California (Calidria RG-144) were irradiated with thermal neutrons in a reactor. The main activation products induced were 46Sc, 51Cr, 59Fe and 60Co. Accurately weighed samples of the irradiated materials were dispersed in N HCl by hand shaking for 10 s. After leaching for predetermined periods at 25 degrees C, the samples were filtered and the concentrations of Mg determined in the filtrates by inductively-coupled plasma atomic emission spectrometry (IPC-AES) and the activities of the four radionuclides by high resolution gamma-ray spectrometry. Similar measurements were made on solutions obtained by refluxing samples of irradiated chrysotile with 2N HCl for 2 h. The specific activities of each of the four activation products were calculated in arbitrary units and, as the concentrations of Sc, Cr, Fe and Co in the UICC B sample had already been determined, it was possible to estimate the concentrations of these elements in the other two samples. Similarities in the leaching patterns of magnesium and of the activation products showed that with all samples, high proportions of the parent trace elements were present in the form of isomorphous substitutes for magnesium in structural brucite. Agreement was closest with the Calidria chrysotile in which all the radionuclides had a similar pattern. With the Jeffrey and UICC B samples, the presence of a high proportion of the iron as relatively insoluble magnetite accounted for the observed discrepancy in behaviour between 59Fe and Mg. More detailed calculation of leaching rates over specific time intervals showed that, initially, 51Cr and 60Co dissolved more rapidly than Mg but that this was followed by a period in which the opposite was the case. It was concluded that the Calidria RG-144 sample is an ideal candidate for studies of magnesium dissolution in vivo.