Carbon nanotube-based electrochemical sensor for assay of salivary cholinesterase enzyme activity: an exposure biomarker of organophosphate pesticides and nerve agents

Certain saliva enzymes may be useful biomarkers for detecting exposures to organophosphate pesticides and chemical nerve agents. In this regard, saliva biomonitoring offers a simple and noninvasive approach for rapidly evaluating those exposures in real time. An electrochemical sensor coupled with a microflow injection system was developed for a simple, rapid, and sensitive characterization of cholinesterase (ChE) enzyme activities in rat saliva. The electrochemical sensor is based on a carbon nanotube (CNT)-modified screen-printed carbon electrode (SPE), which is integrated into a flow cell. Because of the excellent electrocatalytic activity of the CNTs, the sensor can detect electroactive species that are produced from enzymatic reactions with extremely high sensitivity and at low potentials. The electrochemical properties of acetylcholinesterase (AChE) enzymatic products were studied using a CNT-modified SPE, and the operation parameters such as the applied potential and substrate concentration were optimized to achieve the best performance. The AChE enzyme activity was further investigated using the CNT-based electrochemical sensor with commercially available purified AChE and ChE in saliva obtained from nave rats. It is found that the calibration curve is linear over a wide range of AChE concentrations from 5 pM to 0.5 nM, and the sensor is very sensitive with the detection limit down to 2 pM. The dynamics of the ChE enzyme activity in saliva with organophosphate pesticides was further studied using this sensor. The results showthatthe senor can be used to characterize salivary enzyme activity and to detect the exposure to organophosphate compounds. This new CNT-based electrochemical sensor thus provides a sensitive and quantitative tool for noninvasive biomonitoring of the exposure to organophosphate pesticides and nerve agents.     

Highly sensitive and selective amperometric microbial biosensor for direct determination of p-nitrophenyl-substituted organophosphate nerve agents

We report herein a whole cell-based amperometric biosensor for highly selective, highly sensitive, direct, single-step, rapid, and cost-effective determination of organophosphate pesticides with a p-nitrophenyl substituent. The biosensor was comprised of a p-nitrophenol degrader, Pseudomonas putida JS444, genetically engineered to express organophosphorus hydrolase (OPH) on the cell surface immobilized on the carbon paste electrode. Surface-expressed OPH catalyzed hydrolysis of the p-nitrophenyl substituent organophosphorus pesticides such as paraoxon, parathion, and methyl parathion to release p-nitrophenol, which was subsequently degraded by the enzymatic machinery of P. putida JS444. The electrooxidization current of the intermediates was measured and correlated to the concentration of organophosphates. The best sensitivity and response time were obtained using a sensor constructed with 0.086 mg dry weight of cells operating at 600 mV applied potential (vs Ag/AgCl reference) in 50 mM citrate--phosphate pH 7.5 buffer with 50 microM CoCl2 at room temperature. Under optimum operating conditions the biosensor measured as low as 0.28 ppb of paraoxon, 0.26 ppb of methyl parathion, and 0.29 ppb parathion. These detection limits are comparable to cholinesterase inhibition-based biosensors. Unlike the inhibition-based format, this biosensor manifests a selective response to organophosphate pesticides with a p-nitrophenyl substituent only, has a simplified single-step protocol with short response time, and can be used for repetitive/multiple and on-line analysis. The service life of the microbial amperometric biosensor was 5 days when stored in the operating buffer at 4 degrees C. The new biosensor offers great promise for rapid environmental monitoring of OP pesticides with nitrophenyl substituent.     

Merging models and biomonitoring data to characterize sources and pathways of human exposure to organophosphorus pesticides in the Salinas Valley of California

We characterize cumulative intakes of organophosphorus (OP) pesticides in an agricultural region of California by drawing on human biomonitoring data, California pesticide use reporting (PUR) data, and limited environmental samples together with outputs from the CalTOX multimedia, multipathway, source-to-dose model. The study population is the CHAMACOS cohort of almost 600 pregnant Latina women in the Salinas Valley region. We use model estimates of OP intake and urinary dialkylphosphate (DAP) metabolite excretion to develop premises about relative contributions from different exposure sources and pathways. We evaluate these premises by comparing the magnitude and variation of DAPs in the CHAMACOS cohort with those of the whole U.S. population using data from the National Health and Nutrition Examination Survey (NHANES). This comparison supports the premise that diet is the common and dominant exposure pathway in both populations. Biomarker comparisons and model results support the observation that, relative to NHANES, the CHAMACOS population has a statistically significant (p < 0.001) added intake of OP pesticides with low inter-individual variability. We attribute the magnitude and small variance of this intake to residential nondietary exposures from local agricultural OP uses. These results show that mass-balance models can estimate exposures for OP pesticides within the range measured by biological monitoring.     

Highly sensitive and specific detection of neonicotinoid insecticide imidacloprid in environmental and food samples by a polyclonal antibody-based enzyme-linked immunosorbent assay

BACKGROUND: Imidacloprid is one of the main neonicotinoid insecticides widely used in agriculture owing its broad spectrum of activity and low bioaccumulation. However, imidacloprid is toxic to honey bees and other beneficial organisms, and its residues may occur in environmental and food samples, posing a potential hazard to consumers. In this study the imidacloprid derivative bearing a three‐atom length spacer was synthesized and coupled to carrier proteins. Highly sensitive and specific polyclonal antibodies against imidacloprid were successfully produced and the polyclonal antibody‐based enzyme‐linked immunosorbent assay (pAb‐ELISA) was developed. RESULTS: The ELISA standard curve was constructed within the concentration range 0.1–100 ng mL^?1. The IC₅₀ value for nine standard curves was in the range 1.2–3.0 ng mL^?1 and the limit of detection was 0.03–0.16 ng mL^?1. The sensitivity of the assay was one order of magnitude higher than that in most published papers. There was almost no cross‐reactivity of the antibody with four structurally related compounds (acetamiprid, nicotine, clothianidin and nitenpyram) and six other compounds, indicating that the assay displays not only high sensitivity but also high specificity. No detectable imidacloprid was found in 11 collected environmental and food samples by the assay. For imidacloprid‐spiked samples, acceptable recoveries of 73.4–94.4% and intra‐assay coefficients of variation of 2.2–12.8% were obtained. The assay was also validated with high‐performance liquid chromatography (HPLC) and a good correlation of ELISA with HPLC was achieved. CONCLUSION: The proposed ELISA provides a sensitive, specific, simple and cost‐effective quantitative/screening method for detecting imidacloprid in environmental and food samples. Copyright © 2011 Society of Chemical Industry     

The cellular antioxidant activity in red blood cells (CAA-RBC): A new approach to bioavailability and synergy of phytochemicals and botanical extracts

In the present work, human red blood cells (RBC) were used to determine cellular antioxidant activity (CAA-RBC) of pure phytochemicals and botanical extracts, with the aim to predict their bioavailability.     

Development of a highly sensitive and specific assay to detect Staphylococcus aureus in bovine mastitic milk

Diagnosis of udder infections with Staphylococcus aureus by bacteriological milk testing of quarter milk samples is often not satisfactory. To get reliable results, repeated sampling is necessary, which is normally too expensive. Therefore, we developed a test that allows the highly specific detection of Staph. aureus in bovine milk samples at very low concentrations. It is based on a fast procedure to prepare bacteria from milk, followed by DNA extraction and quantitative PCR. The whole analysis is done within 5 h. For clinical milk samples, the analytical sensitivity of the assay was 50.7 times and 507 times higher than conventional bacteriology with 100 and 10 μL, respectively. The diagnostic specificity was 100%. The test is further characterized by a low intra- and interassay variability as well as by a good recovery of Staph. aureus from raw milk. Furthermore, a high correlation (R = 0.925) between the agar plate counts and the quantitative PCR methodology over the whole range of measurement was found. In addition, our test revealed considerably more positive results than bacteriology. Due to its favorable properties, the assay might become an important diagnostic tool in the context of bovine mastitis caused by Staph. aureus.     

Microbial transformation of triadimefon to triadimenol in soils: selective production rates of triadimenol stereoisomers affect exposure and risk

The microbial transformation of triadimefon, an agricultural fungicide of the 1,2,4-triazole class, was followed at a nominal concentration of 50 μg/mL over 4 months under aerobic conditions in three different soil types. Rates and products of transformation were measured, as well as enantiomer fractions of parent and products. The transformation was biotic and enantioselective, and in each soil the S-(+)-enantiomer reacted faster than the R-(-) one. Rates of the first-order reactions were 0.047, 0.057, and 0.107 d(-1) for the three soils. The transformation involves reduction of the prochiral ketone moiety of triadimefon to an alcohol, resulting in triadimenol, which has two chiral centers and four stereoisomers. The abundances of the four product stereoisomers were different from each other, but abundance ratios were similar for all three soil types. Triadimenol is also a fungicide; the commercial product is composed of two diastereomers of unequal amounts (ratio of about 4.3:1), each having two enantiomers of equal amounts. However, the triadimenol formed by soil transformation of triadimefon exhibited no such stereoisomer profile. Instead, different production rates were observed for each of the four triadimenol stereoisomers, resulting in all stereoisomer concentrations being different from each other and very different from concentration/abundance patterns of the commercial standard. This result is important in risk assessment if the toxicity of the environmental transformation product were to be compared to that of the commercial triadimenol. Because triadimenol stereoisomers differ in their toxicities, at least to fungi and rats, the biological activity of the triadimenol formed by microbes or other biota in soils depends on the relative abundances of its four stereoisomers. This is an exposure and risk assessment issue that, in principle, applies to any chiral pesticide and its metabolites.     

Effect of organic complexation on copper accumulation and toxicity to the estuarine red macroalga Ceramium tenuicorne: a test of the free ion activity model

Current water quality criteria (WQC) regulations on copper toxicity to biota are still based on total dissolved (<0.4 μm membrane filter) copper concentrations with a hardness modification for freshwaters. There are however ongoing efforts to incorporate metal speciation in WQC and toxicity regulations (such as the biotic ligand model-BLM) for copper and other metals. Here, we show that copper accumulation and growth inhibition of the Baltic macroalga Ceramium tenuicorne exposed to copper in artificial seawater at typical coastal and estuarine DOC concentrations (similar to 2-4 mg/L-C as fulvic acid) are better correlated to weakly complexed and total dissolved copper concentrations rather than the free copper concentration [Cu2+]. Our results using a combination of competitive ligand exchange-adsorptive cathodic stripping voltammetry (CLE-ACSV) measurements and model calculations (using visual MINTEQ incorporating the Stockholm Humic Model) show that copper accumulation in C. tenuicorne only correlates linearly well to [Cu2+] at relatively high [Cu2+] and in the absence of fulvic acid. Thus the FIAM fails to describe copper accumulation in C. tenuicorne at copper and DOC concentrations typical of most marine waters. These results seem to indicate that at ambient total dissolved copper concentration in coastal and estuarine waters, C. tenuicorne might be able to access a sizable fraction of organically complexed copper when free copper concentration to the cell membrane is diffusion limited.     

Supercritical-CO2 drying of foodstuffs in packed beds: Experimental validation of a mathematical model and sensitive analysis

In this contribution, a mathematical model is built to predict the changes in water concentration in both a solid food matrix and a fluid carrier during supercritical carbon dioxide (SC–CO₂) drying. The mass balance equations of the model involve five dimensionless parameters: Peclet number modified Sherwood number, Fourier number, mass ratio and equilibrium constant. The differential equations were discretized using the finite explicit difference method. The resulting model was implemented and solved in Matlab/Simulink using an explicit Runge–Kutta solver. A very good agreement (ARD = 7.2%) between experimental data, obtained by an independent group, and the present model was observed. The axial dispersion diffusion coefficient seems not to play a significant role during the drying process. A sensitivity analysis revealed that the predictions are relatively more sensitive to the equilibrium constant and the mass ratio than to Peclet and modified Sherwood numbers. Furthermore, in the case of Peclet and modified Sherwood numbers, the sensitivity and the uncertainty of the output are function of the final moisture content. The present model could be used as an optimization tool for kinetic studies to investigate the effects of different operation conditions on the performance and design of the supercritical drying technology.     

Qualitative detection of tetracycline residues in milk with a luminescence-based microbial method: the effect of milk composition and assay performance in relation to an immunoassay and a microbial inhibition assay

Performance of Tet-Lux, a newly developed microbiological test for the detection of tetracycline residues in raw milk, based on tetracycline-controlled luminescence activation of the test bacteria, was evaluated in bovine milks with variable amounts of somatic cells, bacteria, fat, protein, and natural inhibitory compounds. The sensitivity of Tet-Lux was also compared to a commercially available tetracycline immunoassay (Snap, Idexx Laboratories Inc.) and to a microbial inhibition test (Delvotest SP, Gist-Brogades). There were slight differences in the luminescence signals between different milk samples, but no single factor could be pointed out to be responsible for them. There appeared to be a modest inverse relationship between luminescence and increasing fat and protein content. The amount of somatic cells, bacteria, and the natural inhibitors lysozyme and lactoferrin did not affect the luminescence response. The test fulfilled the sensitivity requirement specified by the European Union (maximum residue limit 100 ng/ml for tetracyclines). The Tet-Lux test was clearly more sensitive to all tetracyclines tested (oxytetracycline, tetracycline, chlortetracycline, doxycycline, demeclocycline, methacycline, minocycline) than Delvotest SP, and for five tetracyclines out of seven more sensitive than Snap. The test provides a fast, simple, and robust microbial method for the qualitative detection of tetracycline residues in milk.     

Sequential exposure of porcine cumulus cells to FSH and/or LH is critical for appropriate expression of steroidogenic and ovulation-related genes that impact oocyte maturation in vivo and in vitro

In this study, we collected follicular fluid, granulosa cells, and cumulus cells from antral follicles at specific time intervals following equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG) treatment of gilts. The treatment with eCG increased the production of estrogen coordinately with up-regulated proliferation of granulosa and cumulus cells. eCG also induced the expression of LHCGR and PGR in cumulus cells and progesterone accumulation was detected in follicular fluid prior to the LH/hCG surge. Moreover, progesterone and progesterone receptor (PGR) were critical for FSH-induced LHCGR expression in cumulus cells in culture. The expression of LHCGR mRNA in cumulus cells was associated with the ability of LH to induce prostaglandin production, release of epidermal growth factor (EGF)-like factors, and a disintegrin and metalloprotease with thrombospondin-like repeats 1 expression, promoting cumulus cell oocyte complexes (COCs) expansion and oocyte maturation. Based on the unique expression and regulation of PGR and LHCGR in cumulus cells, we designed a novel porcine COCs culture system in which hormones were added sequentially to mimic changes observed in vivo. Specifically, COCs from small antral follicles were pre-cultured with FSH and estradiol for 10 h at which time progesterone was added for another 10 h. After 20 h, COCs were moved to fresh medium containing LH, EGF, and progesterone. The oocytes matured in this revised COC culture system exhibited greater developmental competence to blastocyst stage. From these results, we conclude that to achieve optimal COC expansion and oocyte maturation in culture the unique gene expression patterns in cumulus cells of each species need to be characterized and used to increase the effectiveness of hormone stimulation.     

Dietary exposure assessment of Danish consumers to dithiocarbamate residues in food: a comparison of the deterministic and probabilistic approach

Probabilistic and deterministic estimates of the acute and chronic exposure of the Danish populations to dithiocarbamate residues were performed. The Monte Carlo Risk Assessment programme (MCRA 4.0) was used for the probabilistic risk assessment. Food consumption data were obtained from the nationwide dietary survey conducted in 2000--02. Residue data for 5721 samples from the monitoring programme conducted in the period 1998--2003 were used for dithiocarbamates, which had been determined as carbon disulphide. Contributions from 26 commodities were included in the calculations. Using the probabilistic approach, the daily acute intakes at the 99.9% percentile for adults and children were 11.2 and 28.2 microg kg(-1) body weight day(-1), representing 5.6% and 14.1% of the ARfD for maneb, respectively. When comparing the point estimate approach with the probabilistic approach, the outcome of the point estimate calculations was generally higher or comparable with the outcome of the probabilistic approach at the 99.9 percentile (consumers only). The chronic exposures for adults and children were 0.35 and 0.76 microg kg(-1) body weight day(-1) at the 99.9 percentile, representing 0.7% and 1.5%, respectively, of the acceptable daily intake for mancozeb and maneb at 50 microg kg(-1) body weight.     

Effects of long-term supplementation of policosanol on blood cholesterol/glucose levels and 3-hydroxy-3-methylglutaryl coenzyme a reductase activity in a rat model fed high cholesterol diets

Food Science and Biotechnology - Policosanol is a well-defined nutraceutical for the management of blood cholesterol levels. The present study examined (i) the effect of policosanol supplementation...     

Dietary exposure assessment of Danish consumers to dithiocarbamate residues in food: a comparison of the deterministic and probabilistic approach.

Probabilistic and deterministic estimates of the acute and chronic exposure of the Danish populations to dithiocarbamate residues were performed. The Monte Carlo Risk Assessment programme (MCRA 4.0) was used for the probabilistic risk assessment. Food consumption data were obtained from the nationwide dietary survey conducted in 2000--02. Residue data for 5721 samples from the monitoring programme conducted in the period 1998--2003 were used for dithiocarbamates, which had been determined as carbon disulphide. Contributions from 26 commodities were included in the calculations. Using the probabilistic approach, the daily acute intakes at the 99.9% percentile for adults and children were 11.2 and 28.2 microg kg(-1) body weight day(-1), representing 5.6% and 14.1% of the ARfD for maneb, respectively. When comparing the point estimate approach with the probabilistic approach, the outcome of the point estimate calculations was generally higher or comparable with the outcome of the probabilistic approach at the 99.9 percentile (consumers only). The chronic exposures for adults and children were 0.35 and 0.76 microg kg(-1) body weight day(-1) at the 99.9 percentile, representing 0.7% and 1.5%, respectively, of the acceptable daily intake for mancozeb and maneb at 50 microg kg(-1) body weight.     

Dietary exposure to trace elements and health risk assessment in the region of Valencia,Spain: a total diet study

ABSTRACT

Dietary exposure of the Valencian region population to lead, cadmium, inorganic arsenic (iAs), chromium, copper, tin and methylmercury (meHg) was assessed in a total diet study carried out in the region of Valencia in 2010–11. A total of 8100 food samples were collected and analysed. Occurrence data were combined with consumption data to estimate dietary exposure in adults (> 15 years of age) and young children (6–15 years of age). The estimated intake was calculated by a probabilistic approach. Average intake levels (optimistic scenario) for lead, iAs, chromium and tin were 0.21, 0.08, 1.79 and 1.87 µg kg^?1 bw day^?1 respectively; for Cd and meHg average intake levels were 0.77 and 0.54 µg kg^–1 bw week^?1, respectively, and for Cu, 1.60 mg day^?1. In terms of risk characterisation, the results showed that 2.84% of the adult population may exceed the BMDL₁₀ (benchmark dose lower confidence limit) established for Pb, which is linked to renal effects; whereas 28.01% of the young children population may exceed the BMDL₀₁ related to neurodevelopment effects. In addition, 8.47% of the adult population and 12.32% of young children exceeded the meHg tolerable weekly intake (TWI).     

Inactivation kinetics of a piezotolerant Staphylococcus aureus isolated from high-pressure-treated sliced ham by high pressure in buffer and in a ham model system: Evaluation in selective and non-selective medium

The kinetics of inactivation by high pressure of a pressure-resistant strain of Staphylococcus aureus isolated from pressure-treated packaged sliced ham, in buffer and in a ham model system was studied. Selective (BP agar) and enrichment media (BHI agar) were used for enumeration in order to count healthy and sublethally injured cells of the pathogen. A first-order kinetic inactivation was observed in both suspension media, and a very significant increase in D values was apparent when the microorganism was suspended and pressurized in the model food system compared to buffer. In the case of phosphate buffer as suspension medium, the z_p values obtained were 107.5 and 113.6 MPa for the two recovery media, i.e. BP and BHI agars, respectively. In contrast, in the case of the food model system, a two-phase linear relation was apparent and the PDT (Pressure Death Time) curve can be divided into two linear sections, so that two z_p values could be defined, one for each section. Z_p values of 100 and 79.4 MPa correspond to pressures < 500 MPa for the BP and BHI counts, respectively, while z_p values of 416.7 and 333.3 MPa correspond to higher pressures > 500 MPa for the selective and non-selective medium, respectively. When S. aureus had been pressurized in phosphate buffer, the BHI agar was slightly better in cell recovery, while in the case of the ham model system, the BP agar proved superior and gave significantly higher colony counts.

Industrial relevance

The paper provides significant information for the food processing industry as it deals with the effect of high-pressure technology on a piezotolerant pathogen that may survive in sliced ham. This technology is already applied in ham products and this paper supports the need for the use of real food in pressure studies in order to avoid underestimation of the effect and hence the processing times. It is also shown that different recovery media, i.e. selective and non-selective, should be used to avoid underestimation of the surviving cells.     

Bioaccumulation and metabolism of polybrominated diphenyl ethers in carp (Cyprinus carpio) in a water/sediment microcosm: important role of particulate matter exposure

Microcosms were built up to simulate a pond system with polybrominated diphenyl ether (PBDE) contaminated sediment and bioorganisms. The microcosms were divided into groups A and B. In group A, both benthic invertebrates (tubificid worms) and carp (Cyprinu carpio) were added, while in group B, only fish were added. After exposure for 20 d, the fish were sampled (exposure I). A net was fixed in the microcosms, and new fish were added (exposure II). These fish were prohibited from contacting the sediment by the net, and the accumulation and depuration of PBDEs in the fish were investigated. Among 11 monitored PBDE congeners (BDE-28, BDE-47, BDE-99, BDE-100, BDE-153, BDE-154, BDE-183, BDE-206, BDE-207, BDE-208, and BDE-209), only 5 congeners (BDE-28, BDE-47, BDE-100, BDE-153, and BDE-154) were detected in the carp fillets and liver. BDE-99 and BDE-183 were not detected in the fish because of the efficient metabolic debromination in carp tissues. The uptake of PBDEs in exposure I was significantly higher/faster than that in exposure II, since the fish in exposure I had an opportunity to take in more of the highly contaminated particles. The uptake kinetics (k(s)) and elimination (k(e)) rate coefficients showed a general trend of decreasing with increasing log K(ow). No significant difference was observed in uptake/depuration kinetics between groups A and B, indicating that the tubificids' reworking does not affect the bioaccumulation of sediment-associated PBDEs in fish significantly. All the PBDE congeners, including nona- and deca-BDEs, were bioaccumulated in the tubificid worms. The PBDE concentrations in the worms were significantly higher than those in the fish, and the congener profile of the sevem major congeners (BDE-28, BDE-47, BDE-99, BDE-100, BDE-153, BDE-154, and BDE-183) was distinctly different from that of fish tissues. The biota-sediment accumulation factors in the worms ranged from 0.01 to 5.89 and declined with increasing bromination and log K(ow.).