Priority pesticides in sediments of European coastal lagoons: A review.

This review summarizes the legislation applied to pesticides and highlights the physicochemical properties of the past and recently listed PPs under Water Framework Directive (WFD). It reports important information regarding the analysis, occurrence and long-term screening of PPs in sediments of European coastal lagoons. Among the entire list of PPs, those analyzed have been the organochloride insecticides, such as lindane, hexachlorobenzene and DDT. Although flood events pointed to the possible redistribution of pesticides, which may increase under climate change conditions, few studies are available concerning PPs screening in sediments of European coastal lagoons. Data is scarce not only in terms of the number of listed PPs that have been analyzed but also in terms of their long-term screening. This lack of data on PP concentrations is probably a consequence of the fact that less importance is given to the sediments contamination/quality.

Pathways of priority pesticides in sediments of coastal lagoons: The case study of Óbidos Lagoon, Portugal.

This study reports the concentrations of the priority pesticides (PPs) in 14 surface sediments and 21 layers of a sediment core from Óbidos Lagoon, a shallow Portuguese coastal lagoon. Results show that the PPs are confined to the upper part of the lagoon that receives most of the inputs from surface runoff of the surrounding agricultural fields and from small tributaries. Past and recent applied PPs were registered in sediments, aluminum normalized concentrations varying between 0.05×10(-7) and 6.85×10(-7). The PP risk assessment based on sediment quality guidelines like the "Probable Effect Level" (PEL) shows no biological effects in either sediments or aquatic organisms of Óbidos Lagoon, except for dieldrin, lindane, DDT, heptachlor epoxide and its parent compound heptachlor.

Screening of priority pesticides in Ulva sp. seaweeds by selective pressurized solvent extraction before gas chromatography with electron capture detector analysis.

This work reports a fast and reliable analytical method for the screening of priority pesticides (PPs) in Ulva sp. seaweeds by gas chromatography with electron capture detection. Extraction and sample clean-up were performed in one single step by selective pressurized liquid extraction (SPLE). Several parameters affecting SPLE performance were optimized. Method performance was compared with standard Soxhlet extraction. Significant decrease of the time of analysis with better recoveries for a greater number of PPs was achieved by SPLE. Average recoveries ranged from 71 to 103% with RSD < 10%. Field application showed the presence of PP in the range of 3-11 ng g(-1) in seaweeds collected in a coastal lagoon after a long period of heavy rains. These results suggest that Ulva sp. seaweeds tend to accumulate PPs and have the potential to be used as early alert signals of aquatic pollution especially after rains and storm events.

Detoxification of patulin and ochratoxin A, two abundant mycotoxins, by lactic acid bacteria.

Aim of the present study was to investigate the detoxification of two abundant mycotoxins, namely ochratoxin A (OTA) and patulin (PAT) which are frequently found in human foods, by lactic acid bacteria. The removal of the two mycotoxins from liquid medium by thirty different LAB strains was analyzed in a screening trial by the use of HPLC coupled with UV- or fluorescence detection. Two highly effective strains were identified; Lactobacillus acidophilus VM 20 caused a decrease of OTA by > or = 95% and Bifidobacterium animalis VM 12 reduced PAT levels by 80%. Subsequently experiments showed that the binding of these compounds depends on different parameters, i.e. the concentration of toxins, the cell density, the pH-value and on the viability of the bacteria. To proof that the decrease of the toxins by LAB from liquid medium results in a reduction of their toxic properties, micronucleus (MCN) assays were conducted with a human derived hepatoma cell line (HepG2). Indeed, a substantial decrease (39-59%) of OTA and PAT induced MCN formation was observed with the most effective strains detected in the chemical analyses. Furthermore, also the inhibition of the cell division rates by the toxins was significantly reduced. These findings indicate that certain LAB strains are able to detoxify the two toxins and may be useful to protect humans and/or animals against the adverse health effects of these compounds.

Determination of zearalenone and its metabolites in urine, plasma and faeces of horses by HPLC-APCI-MS.

The paper describes a method for the sensitive and selective determination of zearalenone and its metabolites in urine, plasma and faeces of horses by high performance liquid chromatography and atmospheric pressure chemical ionisation (APCI) mass spectrometry (MS). While only one step sample clean-up by an immunoaffinity column (IAC) was sufficient for plasma samples, urine and faeces samples had to be prepared by a combination of a solid-phase extraction (SPE) and an immunoaffinity column. The method allows the simultaneous determination of zearalenone and all of its metabolites; alpha-zearalenol, beta-zearalenol, alpha-zearalanol, beta-zearalanol and zearalanone. Dideuterated zearalanone was used as internal standard for quantification and the study of the matrix effect. Recovery rates between 56 and slightly above 100% were achieved in urine samples, and more than 80% in plasma and faeces samples. The limits of detection ranged from 0.1-0.5 microg/l or microg/kg, the limits of quantification from 0.5-1.0 microg/l or microg/kg. The practical use of the method is demonstrated by the analysis of spiked and naturally contaminated urine, plasma and faeces of horses.

Determination of less polar heterocyclic aromatic amines in standardised beef extracts and cooked meat consumed in Austria by liquid chromatography and fluorescence detection.

An analysis method was developed for the determination of trace levels of less polar heterocyclic aromatic amines (HAs) in food samples. The development started from a frequently used sample pre-treatment scheme which was slightly improved to make it applicable with high-performance liquid chromatography (HPLC) with fluorescence detection. The method was applied for the analysis of a standardised beef extract containing 5-15 ng/g of HAs and the results are compared with those of the other participants in the same European project. In addition, the method was used for the analysis of less polar HAs in cooked meat consumed in Austria.

Determination of heterocyclic aromatic amines in beef extract, cooked meat and rat urine by liquid chromatography with coulometric electrode array detection.

This paper describes a method for the determination of heterocyclic aromatic amines (HAs; DMIP, IQ, MeIQ, MeIQx, 4,8-DiMeIQx, 7,8-DiMeIQx, AalphaC, PhIP) by high-performance liquid chromatography (HPLC) with coulometric electrode array detection. The compounds are separated on reversed phase columns (LiChroCart Superspher 60 RP-select B, 250 mm x 2 mm, 4 microm and LiChrospher 60 RP-select B, 250 mm x 4 mm, 5 microm) using mobile phases consisting of acetonitrile/buffer/distilled water and detected at eight working electrodes at potentials between +190 and +680 mV against modified palladium electrodes. In the context of an EU-interlaboratory exercise, the method was applied to analyse a standardised test solution and--after isolation of the analytes by several clean-up steps--for the analysis of standardised beef extract and grilled meat. Further, the method could be applied for the analysis of HAs in suspensions of bacteria and rat urine without any sample preparation step beyond sample dilution. The data obtained show that HPLC with coulometric electrode array detection gives accurate results.

Heterocyclic amines: human carcinogens in cooked food?

During the frying of meat and fish, genotoxic heterocyclic amines (HCAs) are formed. The dietary exposure to HCAs may be implicated in the aetiology of human cancer, but there may be other factors in our diet that prevent the genotoxic effects of these compounds. Within the project described here, we plan to identify regional and individual cooking habits that affect HCA-levels in our food. These are determined with a validated analytical method and the exposure to HCAs is estimated by dietary assessment. Biomarker analysis will be employed to estimate recent or long-term exposure to HCAs. In order to identify genetically determined risk factors in humans, cell lines are genetically engineered expressing allelic variants of acetyl- and sulfotransferases implicated in HCA metabolism. Species differences of metabolism and toxicity of HCAs are assessed and the influence of the intestinal microflora on HCA-induced toxicity is evaluated. Dietary constituents that may reduce the genotoxicity of HCAs are screened for potential protective effects in in vitro and in vivo model systems. Finally, we will aim at human intervention studies to investigate if these protective factors are relevant for man. The objectives of this project are to estimate and possibly reduce the exposure levels to HCAs in Europe, to identify populations highly susceptible to HCA toxicity, and to reduce the toxic effects of HCAs by protective factors.

Determination of 1-nitropyrene in herbs after selective enrichment by a sol-gel-generated immunoaffinity column.

Using the determination of 1-nitropyrene as an example the paper demonstrates the advantages of including a highly selective sol-gel-generated immunoaffinity column in the sequence of clean-up steps necessary to determine haptens in complex matrices. The sol-gel method to immobilise antibodies enlarges the variety of immunoaffinity columns available and leads to mechanically stable columns with constant retention characteristics. The sample preparation scheme proposed combines acetonitrile extraction, size-exclusion and immunoaffinity chromatography. 1-Nitropyrene is then separated by reversed-phase HPLC from interfering compounds and determined after catalytic on-line reduction to the corresponding amine by spectrofluorimetry. Concentrations in the range from 0.1 to 1.4 microg/kg 1-nitropyrene were detected in herbs.

Early highly active antiretroviral therapy for acute HIV-1 infection preserves immune function of CD8+ and CD4+ T lymphocytes.

Highly active antiretroviral therapy (HAART) has been advocated for the management of primary HIV-1 infection without clear understanding of its immunological effects. Here, we demonstrate that early use of HAART during primary infection preserves HIV-specific CD8(+) T cells physically and functionally while HIV-specific T cell help is sustained. We also show that even transient administration of HAART at seroconversion can preserve HIV-specific immunity. In contrast, delayed initiation of HAART is associated with a progressive loss of HIV-specific CD8(+) T cells and absent HIV-specific T cell help. These results imply that HIV-specific T help is damaged during primary HIV-1 infection. Early drug treatment, which preserves this immunity, also preserves HIV-specific CD8(+) T cells. These results have implications for understanding the early pathogenesis of HIV-1 infection and suggest that acute HIV infection should be treated aggressively and as early as possible.

Evaluation of sperm washing as a potential method of reducing HIV transmission in HIV-discordant couples wishing to have children.

OBJECTIVE: A number of discordant couples, in whom the man is HIV positive and the woman is HIV negative, wish to have children. To conceive they must abandon protected sex, posing a risk of HIV transmission to the woman and so to the child. In such circumstances purification of spermatozoa ('sperm-washing') to inseminate the woman artificially has been proposed as a method of reducing the risk of transmission. Here we evaluate whether this does represent a true risk reduction. METHODS: Semen samples from HIV-positive patients were separated into spermatozoa, non-sperm cells (NSCs) and plasma fractions. The amount of viral RNA present in each fraction was measured and compared with the level in the peripheral blood. Each fraction was also assessed for the presence of proviral DNA. The ability of spermatozoa to be infected was assessed by evaluating for the presence of HIV receptors, i.e. CD4, CCR5 and CXCR4 on the surface of the sperm, by flow cytometry. RESULTS: A poor correlation was found between the levels of HIV in blood and semen. Within the semen the virus was restricted to the plasma and/or NSCs. All spermatozoa were negative for viral RNA or proviral DNA. Spermatozoa did not express significant levels of CD4, CCR5 or CXCR4, suggesting that they are unlikely to be major targets for HIV infection. CONCLUSIONS: These data suggest that spermatozoa are not major targets of HIV infection. Purifying spermatozoa reduced the level of HIV RNA and proviral DNA to below the detection limit of the assays irrespective of the amount of virus present in the unfractionated semen. On the basis of these data we would recommend 'sperm-washing' followed by insemination as a safer alternative to natural conception for HIV-discordant couples wishing to have children.

Poor reduction of HIV-1 RNA titres in nucleoside reverse transcriptase inhibitor experienced patients treated with indinavir combination therapy.

OBJECTIVES: The long term effectiveness of combination therapy at reducing viral loads in seminal fluid and blood plasma obtained from HIV-1 infected men who had undergone previous antiretroviral therapy was assessed. METHODS: Samples of semen and blood were obtained from a cohort of 12 nucleoside reverse transcriptase inhibitor experienced men before and during 25-68 weeks of combination therapy, which included the protease inhibitor indinavir. HIV-1 RNA titres present in the cell free blood and seminal plasma samples were determined using the nucleic acid sequence based amplification (NASBA)/Nuclisens assay system. RESULTS: Viral RNA was detected in 9/12 and 7/12 baseline blood plasma and seminal plasma samples, with median viral titres of 10(4.81) and 10(4.56) per ml, respectively. By the end of the study period the detection rates of HIV RNA in the blood and seminal plasma samples were 5/12 and 2/12, respectively, with the median viral titres below the assay cut off level for both sample types. Of the nine patients who had detectable viral RNA in the baseline sample, only three cleared virus from both compartments by the end of the study. CONCLUSIONS: These data show that stable reduction of blood and seminal fluid viral titres is not achievable in a significant proportion of nucleoside reverse transcriptase inhibitor experienced men.

Effect of anthraquinone-laxatives on the proliferation and urokinase secretion of normal, premalignant and malignant colonic epithelial cells.

Even though 1,8-dihydroxyanthraquinone (DHA)-laxatives have been implicated in colon carcinogenesis, the available information is still inconclusive. The aim of this study was to demonstrate the effect of the DHA-laxatives, danthrone, rhein, aloe-emodin and sennidine, on colorectal tumour cells. In SW480 carcinoma cultures, dose-dependent induction of urokinase secretion into the medium was the predominant effect. Simultaneously, cell numbers were decreased by DHA-aglycones, but not by sennoside or the biphenylic laxative bisacodyl. DNA synthesis was not similarly reduced: 0.4-4 microM danthrone and sennidine even stimulated 5-bromo-2'-desoxyuridine (BrdU) uptake into DNA. When uptake was normalised to cell number, danthrone and sennidine doubled BrdU uptake/10(6) cells, 18 microM rhein and 0.7 microM aloe-emodin induced increases of 37 and 50%, respectively. This may at least partially be due to selective resistance of S-phase cells to DHA-caused cell loss. In VACO235 adenoma cells, sennidine and aloe-emodin did not affect urokinase secretion, but stimulated growth. Both cell numbers and DNA synthesis were increased. In contrast to SW480 carcinoma cells, VACO235 cells were also sensitive to sennoside and bisacodyl. No effects of DHA were observed in normal colorectal epithelial cells. The biological effects were preceeded by specific phosphorylation of cellular proteins with molecular weights of 110, 78, 63, 57 kDa, indicating the specific induction of a cellular signalling cascade by the laxatives.

Genotoxic effects of crude juices from Brassica vegetables and juices and extracts from phytopharmaceutical preparations and spices of cruciferous plants origin in bacterial and mammalian cells.

Crude juices of eight Brassica vegetables as well as juices and extracts of spices and phytopharmaceutical preparations from cruciferous vegetables were tested for induction of point mutations in Salmonella TA98 and TA100, repairable DNA damage in E.coli K-12 cells and clastogenic effects in mammalian cells. In bacterial assays, all juices caused genotoxic effects in the absence of metabolic activation, the ranking order being: Brussels sprouts > white cabbage > cauliflower > green cabbage > kohlrabi > broccoli > turnip > black radish. In experiments with mammalian cells, six juices induced structural chromosome aberrations. Brussels sprouts, white and green cabbage caused the strongest effects (800 microliters of juice induced a 5-fold increase over the background). In sister chromatid exchange assays, positive results were measured as well, but the effects were less pronounced. With all juices the genotoxic effects seen in mammalian cells were paralleled by a pronounced decrease in cell viability. Column fractionation experiments showed that 70-80% of the total genotoxic activity of the juices is found in the fraction which contains isothiocyanates and other breakdown products of glucosinolates, whereas phenolics and flavonoids contributed to a lesser extent to the overall effects. On the basis of these findings, and considering the negative results obtained with non-cruciferous vegetables (tomato, carrot and green pepper), it seems likely that the genotoxic effects of the juices are due to specific constituents of cruciferous plants such as glucosinolates and/or their breakdown products, in particular, isothiocyanates, which we found previously to be potent genotoxins in bacterial and mammalian cells. Finally, spices (mustards and horse radish paste) and phytopharmaceutical preparations were tested in bacterial assays. Mustards and horse radish caused very weak effects while most of the pharmaceutical preparations gave negative results, except cabbage tablets, which caused a strong and dose dependent induction of his revertants in Salmonella TA100. The present findings clearly indicate that cruciferous vegetables contain DNA damaging constituents. These observations are in contrast to earlier findings, which emphasized the antimutagenic effects of vegetable juices and also raise the question whether greatly increased consumption of Brassica vegetables or their concentrated constituents as a means for cancer prevention is indeed recommendable.

Determination of salicylate in beverages and cosmetics by use of an amperometric biosensor.

A fast and selective enzymatic method for the determination of salicylate in beverages and cosmetics has been developed. The enzyme salicylate hydroxylase was immobilised covalently onto a glassy carbon working electrode of a wall-jet cell coupled with a flow-injection analysis system. The salicylate is enzymatically converted to catechol, which can be detected amperometrically on the glassy carbon electrode at +0.45 V. The response of the biosensor is linearly proportional to the concentration of salicylate between 725 nmol/l and 700 micromol/l. A high sample throughput (60 h(-1)) is possible, and the biosensor is stable for more than three months. Sample pretreatment for beverages and hair lotions is easy and fast. For creams, an extraction of salicylate is necessary. Relative standard deviations are less than 5.5% and the recoveries are between 95 and 105%.

Multichannel coulometric detection coupled with liquid chromatography for determination of phenolic esters in honey.

A method for the determination of phenolic esters in different varieties of honey was developed. The substances were separated by RP-HPLC. A coulometric electrode-array system with sixteen electrodes arranged in series and set at increasing potentials (300-900 mV) was used for electrochemical detection of the compounds. Chromatographic peaks for methyl 4-hydroxybenzoate, methyl vanillate, methyl syringate, trans-p-methyl coumarate and trans-methyl ferulate were identified. The content of the esters varied between 1.3 and 5044 micrograms per kg of honey with detection limits of 0.1-1.0 microgram per kg of honey (S/N = 3). The method described is a sensitive assay to differentiate between rape honey and other varieties.

[Determination of phenolic compounds in alcoholic beverages by HPLC with an electrochemical detector]. / Bestimmung phenolischer Verbindungen in alkoholischen Getränke durch HPLC mit elektrochemischem Detektor.

A method has been developed for the quantitative determination of phenolic lignin degradation products in alcoholic distillates. After diluting the samples with the eluent (1:5-1:10, v/v) the phenolic compounds are separated by high-pressure liquid chromatography and detected by an electrochemical detector at +1.3 V. To identify the compounds the k'-values and the hydrodynamic voltammograms of the samples are compared with those of the standard solution. Several distilled alcoholic beverages were analysed. The content of phenolic compounds was found to vary between 0.15 and 7.7 mg/l. The detection limits were 0.3-0.9 ng.