Effects of chlorpyrifos and trichloropyridinol on HEK 293 human embryonic kidney cells.

Chlorpyrifos (CPF) [O, O-diethyl -O-3, 5, 6-trichloro-2-pyridyl phosphorothioate] is an organophosphate insecticide widely used for agricultural and urban pest control. Trichloropyridinol (TCP; 3,5,6-trichloro-2-pyridinol), the primary metabolite of CPF, is often used as a generic biomarker of exposure for CPF and related compounds. Human embryonic kidney 293 (HEK 293) cells were exposed to CPF and TCP with varying concentrations and exposure periods. Cell cultures enable the cost-effective study of specific biomarkers to help determine toxicity pathways to predict the effects of chemical exposures without relying on whole animals. Both CPF and TCP were found to induce cytotoxic effects with CPF being more toxic than TCP with EC50 values of 68.82 µg/mL and 146.87 µg·ml-1 respectively. Cell flow cytometric analyses revealed that exposure to either CPF or TCP leads to an initial burst of apoptotic induction followed by a slow recruitment of cells leading towards further apoptosis. CPF produced a strong induction of IL6, while TCP exposure resulted in a strong induction of IL1α. Importantly, the concentrations of CPF and TCP required for these cytokine inductions were higher than those required to induce apoptosis. These data suggest CPF and TCP are cytotoxic to HEK 293 cells but that the mechanism may not be related to an inflammatory response. CPF and TCP also varied in their effects on the HEK 293 proteome with 5 unique proteins detected after exposure to CPF and 31 unique proteins after TCP exposure.

The Omics Revolution in Agricultural Research.

The Agrochemicals Division cosponsored the 13th International Union of Pure and Applied Chemistry International Congress of Pesticide Chemistry held as part of the 248th National Meeting and Exposition of the American Chemical Society in San Francisco, CA, USA, August 10-14, 2014. The topic of the Congress was Crop, Environment, and Public Health Protection; Technologies for a Changing World. Over 1000 delegates participated in the Congress with interactive scientific programming in nine major topic areas including the challenges and opportunities of agricultural biotechnology. Plenary speakers addressed global issues related to the Congress theme prior to the daily technical sessions. The plenary lecture addressing the challenges and opportunities that omic technologies provide agricultural research is presented here. The plenary lecture provided the diverse audience with information on a complex subject to stimulate research ideas and provide a glimpse of the impact of omics on agricultural research.

Developing Global Leaders for Research, Regulation, and Stewardship of Crop Protection Chemistry in the 21st Century.

To provide sufficient food and fiber to the increasing global population, the technologies associated with crop protection are growing ever more sophisticated but, at the same time, societal expectations for the safe use of crop protection chemistry tools are also increasing. The goal of this perspective is to highlight the key issues that face future leaders in crop protection, based on presentations made during a symposium titled "Developing Global Leaders for Research, Regulation and Stewardship of Crop Protection Chemistry in the 21st Century", held in conjunction with the IUPAC 13th International Congress of Pesticide Chemistry in San Francisco, CA, USA, during August 2014. The presentations highlighted the fact that leaders in crop protection must have a good basic scientific training and understand new and evolving technologies, are aware of the needs of both developed and developing countries, and have good communication skills. Concern is expressed over the apparent lack of resources to meet these needs, and ideas are put forward to remedy these deficiencies.

Determination of polychlorinated biphenyls in soil and sediment by selective pressurized liquid extraction with immunochemical detection.

A selective pressurized liquid extraction (SPLE) method was developed for a streamlined sample preparation/cleanup to determine Aroclors and coplanar polychlorinated biphenyls (PCBs) in soil and sediment. The SPLE was coupled with an enzyme-linked immunosorbent assay (ELISA) for an effective analytical approach for environmental monitoring. Sediment or soil samples were extracted with alumina, 10% AgNO3 in silica, and sulfuric acid impregnated silica with dichloromethane at 100°C and 2000 psi. The SPLE offered simultaneous extraction and cleanup of the PCBs and Aroclors, eliminating the need for a post-extraction cleanup prior to ELISA. Two different ELISA methods: (1) an Aroclor ELISA and (2) a coplanar PCB ELISA were evaluated. The Aroclor ELISA utilized a polyclonal antibody (Ab) with Aroclor 1254 as the calibrant and the coplanar PCB ELISA kit used a rabbit coplanar PCB Ab with PCB-126 as the calibrant. Recoveries of Aroclor 1254 in two reference soil samples were 92±2% and 106±5% by off-line coupling of SPLE with ELISA. The average recovery of Aroclor 1254 in spiked soil and sediment samples was 92±17%. Quantitative recoveries of coplanar PCBs (107-117%) in spiked samples were obtained with the combined SPLE-ELISA. The estimated method detection limit was 10 ng g(-1) for Aroclor 1254 and 125 pg g(-1) for PCB-126. Estimated sample throughput for the SPLE-ELISA was about twice that of the stepwise extraction/cleanup needed for gas chromatography (GC) or GC/mass spectrometry (MS) detection. ELISA-derived uncorrected and corrected Aroclor 1254 levels correlated well (r=0.9973 and 0.9996) with the total Aroclor concentrations as measured by GC for samples from five different contaminated sites. ELISA-derived PCB-126 concentrations were higher than the sums of the 12 coplanar PCBs generated by GC/MS with a positive correlation (r=0.9441). Results indicate that the SPLE-ELISA approach can be used for quantitative or qualitative analysis of PCBs in soil and sediments. To our knowledge, this is the first report of an SPLE-ELISA approach not requiring a post-extraction cleanup step for detecting Aroclors and coplanar PCBs in soil and sediment.

Development and application of immunoaffinity chromatography for coplanar PCBs in soil and sediment.

An immunoaffinity chromatography (IAC) column was developed as a simple cleanup procedure for preparing environmental samples for analysis of polychlorinated biphenyls (PCBs). Soil and sediment samples were prepared using pressurized liquid extraction (PLE), followed by the IAC cleanup, with detection by an enzyme-linked immunosorbent assay (ELISA). Quantitative recoveries (84-130%) of PCB-126 were obtained in fortified sediment and soil samples using the PLE/IAC/ELISA method. These results demonstrated that the IAC procedure effectively removed interferences from the soil and sediment matrices. The IAC column could be reused more than 20 times with no change in performance with 99.9% methanol/0.1% Triton X-100 as the elution solvent. Results of 17 soil and sediment samples prepared by PLE/IAC/ELISA correlated well with those obtained from a conventional multi-step cleanup with gas chromatography/mass spectrometry detection.

Development of a simultaneous extraction and cleanup method for pyrethroid pesticides from indoor house dust samples.

An efficient and reliable analytical method was developed for the sensitive and selective quantification of pyrethroid pesticides (PYRs) in house dust samples. The method is based on selective pressurized liquid extraction (SPLE) of the dust-bound PYRs into dichloromethane (DCM) with analysis by gas chromatography/mass spectrometry. Various adsorbents and combinations of extraction solvents and temperatures were evaluated to achieve a high-throughput sample preparation that eliminates the post-extraction cleanup step. The final method used sulfuric acid-impregnated silica (acid silica) and neutral silica together in the extraction cell with the dust sample to provide both extraction and cleanup simultaneously. The optimal ratio of dust/acid silica/silica was 1:0.8:8. The extraction was performed at 2000 psi, at 100°C with DCM for 5 min in three cycles. Method precision and accuracy were evaluated by the analysis of triplicate aliquots of the dust samples and the samples fortified with the target PYRs. The accuracy measured as the recoveries of the PYRs in the fortified samples ranged from 85% to 120%. The precision measured as the relative standard deviation of replicate samples was within ±25%. The SPLE method was applied to 20 house dust samples collected from households that participated in two field studies regarding exposures to pesticides and other pollutants. Similar concentrations of target PYRs were obtained for the SPLE and a stepwise extraction/cleanup procedure. The SPLE procedure reduces organic solvent consumption and increases the sample throughput when compared with a traditional stepwise extraction and cleanup procedure. This study demonstrates that the SPLE procedure can be applied to complex dust matrices for analysis of PYRs for large scale exposure or environmental monitoring studies.

Development of a multianalyte enzyme-linked immunosorbent assay for permethrin and aroclors and its implementation for analysis of soil/sediment and house dust extracts.

Development of a multianalyte enzyme-linked immunosorbent assay (ELISA) for detection of permethrin and aroclors 1248 or 1254 and implementation of the assay for analysis of soil/sediment samples are described. The feasibility of using the multianalyte ELISA to monitor aroclors 1254 and permethrin simultaneously was tested with permethrin and aroclor standards and with aroclor- and permethrin-containing soil/sediment and house dust samples. Comparison of the I50 and I20 values of the multianalyte with those of a single-analyte assay revealed similar results, and multianalyte ELISA determination of analyte amounts in soil/sediment dust samples yielded similar results to those of a single-analyte assay. A single-analyte assay of permethrin content in permethrin-containing dust samples showed that the ELISA can determine the analyte accurately in samples with dust matrix contents ranging from 6.25 to 100 mg as indicated by the good correlation between the results of the immunoassay and those of the gas chromatography analysis.

Biological monitoring of 3-phenoxybenzoic acid in urine by an enzyme-linked immunosorbent assay.

An enzyme-linked immunosorbent assay (ELISA) method was employed for determination of the pyrethroid biomarker, 3-phenoxybenzoic acid (3-PBA) in human urine samples. The optimized coating antigen concentration was 0.5 ng/mL with a dilution of 1:4000 for the 3-PBA antibody and 1:6000 for the enzyme conjugate. Urine samples were hydrolyzed with concentrated hydrochloric acid; extracted with dichloromethane and solvent-exchanged into a methanol/buffer solution, prior to analysis in a 96-microwell plate immunoassay. Quantitative recoveries of 3-PBA were obtained for fortified urine samples by ELISA (92±18%) as well as by gas chromatography/mass spectrometry (GC/MS) (90±13%). The overall method precision of these samples was within ±20% for both the ELISA and GC/MS methods. Analytical results from over one hundred urine samples showed that the ELISA and GC/MS data were highly correlated, with a correlation coefficient of 0.95. At the 10 ng/mL comparative concentration level, the false positive rate was 0% and the false negative rate was 0.8% for ELISA when using GC/MS as the reference method. The ELISA method has a suitable low detection limit for 3-PBA to assess pyrethroid exposures in non-occupational settings.

Development of an immunoassay and a sol-gel-based immunoaffinity cleanup method for coplanar polychlorinated biphenyls from soil and sediment samples.

Two polychlorinated biphenyls (PCB) enzyme linked immunosorbent assays (ELISAs) were developed using goat PCB purified immunoglobulin (IgG) antibodies (Abs). The IgGs exhibited the highest affinity toward PCB-77 (24 ng mL(-1)) with sensitivities in the range of 6-11 ng mL(-1). The Abs cross-reacted with PCB-126 and the heptachlorodibenzofuran 1,2,3,4,6,7,8-HpCDF but not with PCB-169, PCB-118, Aroclor 1232, 1248, 1260 or the hexachlorodibenzofuran 2,3,4,6,7,8-HxCDF. The IgGs were also used to develop a sol-gel-based immunoaffinity purification (IAP) method for cleanup of PCB-126. Recovery efficiencies depended on the sol-gel formats; a 1:12 format resulted in the highest binding capacity. Net binding capacity ranged from 112 to 257 ng, and 90% of the analyte could be eluted with only 2 mL of ethanol. The method was also very efficient in purifying PCB-126 from spiked soil and sediment samples from contaminated sites; and eliminating matrix interferences to a degree that enabled analysis of the purified samples by ELISA. The approaches developed in the course of the study form a basis for the development of additional IAP methods for other PCBs, and their implementation in high-throughput screening programs for PCB in food, soil, and other environmental and biological samples.

Application of a permethrin immunosorbent assay method to residential soil and dust samples.

A low-cost, high throughput bioanalytical screening method was developed for monitoring cis/trans-permethrin in dust and soil samples. The method consisted of a simple sample preparation procedure [sonication with dichloromethane followed by a solvent exchange into methanol:water (1:1)] with bioanalytical detection using a magnetic particle enzyme-linked immunosorbent assay (ELISA). Quantitative recoveries (83-126%) of cis/trans-permethrin were obtained for spiked soil and dust samples. The percent difference of duplicate ELISA analyses was within +/- 20% for standards and +/- 35% for samples. Similar sample preparation procedures were used for the conventional gas chromatography/mass spectrometry (GC/MS) analysis except that additional cleanup steps were required. Recoveries of cis/trans-permethrin ranged from 81 to 108% for spiked soil and dust samples by GC/MS. The ELISA-derived permethrin concentrations were highly correlated with the GC/MS-derived sum of cis/trans-permethrin concentrations with a correlation coefficient (r) of 0.986. The ELISA method provided a rapid qualitative screen for cis/trans-permethrin in soil and dust while providing a higher sample throughput with a lower cost as compared to the GC/MS method. The ELISA can be applied as a complementary, low-cost screening tool to prioritize and rank samples prior to instrumental analysis for exposure studies.

Bioanalytical methods for food contaminant analysis.

Foods are complex mixtures of lipids, carbohydrates, proteins, vitamins, organic compounds, and other naturally occurring substances. Sometimes added to this mixture are residues of pesticides, veterinary and human drugs, microbial toxins, preservatives, contaminants from food processing and packaging, and other residues. This milieu of compounds can pose difficulties in the analysis of food contaminants. There is an expanding need for rapid and cost-effective residue methods for difficult food matrixes to safeguard our food supply. Bioanalytical methods are established for many food contaminants such as mycotoxins and are the method of choice for many food allergens. Bioanalytical methods are often more cost-effective and sensitive than instrumental procedures. Recent developments in bioanalytical methods may provide more applications for their use in food analysis.

An overview of measurement method tools available to communities for conducting exposure and cumulative risk assessments.

Community-based programs for assessing and mitigating environmental risks represent a challenge to participants because each brings a different level of understanding of the issues affecting the community. These programs often require the collaboration of several community sectors, such as community leaders, local governments and researchers. Once the primary concerns, community vulnerabilities and assets are identified, participants plan on how to address immediate actions, rank known risks, collect information to support decision making, set priorities and determine an evaluation process to assess the success of the actions taken. The evaluation process allows the community to develop new action plans based on the results obtained from earlier actions. Tracking the success of the community actions may be as simple as a visual/tangible result (e.g., cleaning a park) or as complex as the collection of specific measurements to track the reduction of toxic pollutants or to determine the presence of a specific contaminant. Recognizing that communities may need to perform measurements to meet their goals, this paper provides an overview of the available measurement methods for several chemicals and biologicals in relevant environmental samples to a community setting. The measurement methods are organized into several categories according to their level of complexity, estimated cost and sources. Community project technical advisors are encouraged to examine the objective(s) of the community to be addressed by a measurement collection effort and the level of confidence that needed for the data to make appropriate decisions. The tables provide a starting point for determining which measurement method may be appropriate for specific community needs.

High-throughput screening of dioxins in sediment and soil using selective pressurized liquid extraction with immunochemical detection.

A high-throughput screening method using selective pressurized liquid extraction (SPLE) and enzyme-linked immunosorbent assay (ELISA) for monitoring dioxins in sediment and soil is described. SPLE conditions were developed by extracting sediment or soil together with alumina, 10% AgNO3 in silica, and sulfuric acid impregnated silica (acid silica) using dichloromethane (DCM) as the solvent at 100 degrees C and 2000 psi. Post-extraction cleanups were not required for ELISA. Two reference sediments (National Institute of Standards and Technology SRM 1944 and Wellington Laboratories WMS01) were analyzed by the SPLE-ELISA method. The ELISA utilized a polyclonal antibody and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) as the calibrant. Recoveries of ELISA-derived TCDD equivalents (EQ) relative to the expected gas chromatography/high resolution mass spectrometry (GC/HRMS) derived dioxin toxic equivalent (TEQ) values were 116+/-11% for SRM 1944 and 102+/-13% for WMS01. ELISA TCDD EQs were consistent with the dioxin TEQs as measured by GC/HRMS for 25 soil/sediment samples from seven different contaminated sites. The ELISA had an approximate method detection limit of 10 pg g(-1) with a precision of 2.6-29% based on the relative percentage difference (%RPD) for duplicate samples. Estimated sample throughput for the SPLE-ELISA was three times or more than that of the GC/HRMS method employing PLE with a multi-column cleanup.

Development and application of immunoaffinity column chromatography for atrazine in complex sample media.

A rabbit antibody immunoaffinity (IA) column procedure was evaluated as a cleanup method for the determination of atrazine in soil, sediment, and food. Four IA columns were prepared by immobilizing a polyclonal rabbit anti-atrazine antibody solution to HiTrap Sepharose columns. Atrazine was bound to the IA columns when the loading solvents were either 100% water, 2% acetonitrile in water, or 10% methanol in phosphate buffered saline (PBS). Quantitative removal of atrazine from the IA columns was achieved with elution solvents of either 70% ethanol in water, 70% methanol in water, or 100% methanol. One control column was prepared using nonspecific rabbit IgG antibody. This control column did not retain any applied atrazine indicating atrazine did not bind indiscriminately to protein or the Sepharose support. The four IA columns showed reproducible coupling efficiency for the immobilization of the atrazine antibody and consistent binding and releasing of atrazine. The coupling efficiency (4.25 mg of antibody in 1 mL of resin bed) for the four IA columns ranged from 93 to 97% with an average of 96+/-2% (2.1%). Recoveries of the 500, 50, and 5 ng mL(-1) atrazine standard solutions from the four IA columns were 107+/-7% (6.5%), 122+/-14% (12%), and 114+/-9% (8.0%) respectively, based on enzyme-linked immunosorbent assay (ELISA) data. The maximum loading was approximately 700 ng of atrazine for each IA column (approximately 0.16 microg of atrazine per mg of antibody). The IA columns could withstand 100% methanol as the elution solvent and could be reused more than 50 times with no change in performance. The IA columns were challenged with soil, sediment, and duplicate-diet food samples and effectively removed interferences from these various matrices for subsequent gas chromatography/mass spectrometry (GC/MS) or ELISA analysis. The log-transformed ELISA and GC/MS data were significantly correlated for soil, sediment and food samples although the ELISA values were slightly higher than those obtained by GC/MS. The IA column cleanup procedure coupled with ELISA analysis could be used as an alternative effective analytical method for the determination of atrazine in complex sample media such as soil, sediment, and food samples.

Enzyme-linked immunosorbent assay (ELISA) and sol-gel-based immunoaffinity purification (IAP) of the pyrethroid bioallethrin in food and environmental samples.

Enzyme-linked immunosorbent assay (ELISA) and sol-gel-based immunoaffinity purification (IAP) methods for the pyrethroid bioallethrin were developed and applied for monitoring bioallethrin in spiked food, soil, and dust samples. Attempts to determine bioallethrin content in fruit and vegetable extracts revealed high variability between sample preparations and marked interferences with the assay. Sol-gel IAP followed by solid-phase sample concentration was effective in removing the interfering components and resulted in high recovery of bioallethrin from spiked crude acetonic extracts of fruits and vegetables, even in the presence of high extract concentrations (28%). Solid-phase treatment alone failed to remove the interfering components from the spiked sample. Gas chromatography-mass spectrometry analysis of the IAP samples revealed bioallethrin as a doublet unsolved peak because of the cis and trans isomer present in the standard with confirmation of its mass. Unlike fruit and vegetable extracts, soil and dust samples did not interfere with the ELISA, and the bioallethrin content in those samples could be determined with high precision without the need of any further purification.

ELISA measurement of stachylysin in serum to quantify human exposures to the indoor mold Stachybotrys chartarum.

The goal of this research was to develop a measurable indicator of human exposure to Stachyborys chartarum. Antibodies were produced against the hemolytic agent stachylysin obtained from the mold S. chartarum. These antibodies were used to develop two enzyme-linked immunosorbent assay methods for the analysis of stachylysin in human and rat sera and environmental samples. Stachylysin was measured in rat pups that received nasal instillations of S. chartarum conidia but not in control rat serum. Stachylysin in the serum of five human adults exposed to S. chartarum in water-damaged environments was 371 ng/mL but none was detected in the control serum. Stachylysin was also quantified in spore, wallboard, mycelial, and dust samples. The measurement of stachylysin may be a useful indicator in assessing human exposure to S. chartarum and in determining the presence of this indoor mold.