Pesticides monitoring in surface water of a subsistence agricultural catchment in Uganda using passive samplers.

Pesticides are intensely used in the agricultural sector worldwide including smallholder farming. Poor pesticide use practices in this agronomic setting are well documented and may impair the quality of water resources. However, empirical data on pesticide occurrence in water bodies of tropical smallholder agriculture is scarce. Many available data are focusing on apolar organochlorine compounds which are globally banned. We address this gap by studying the occurrence of a broad range of more modern pesticides in an agricultural watershed in Uganda. During 2.5 months of the rainy season in 2017, three passive sampler systems were deployed at five locations in River Mayanja to collect 14 days of composite samples. Grab samples were taken from drinking water resources. In these samples, 27 compounds out of 265 organic pesticides including 60 transformation products were detected. In the drinking water resources, we detected eight pesticides and two insecticide transformation products in low concentrations between 1 and 50 ng/L. Also, in the small streams and open fetch ponds, detected concentrations were generally low with a few exceptions for the herbicide 2,4-D and the fungicide carbendazim exceeding 1 ug/L. The widespread occurrence of chlorpyrifos posed the largest risk for macroinvertebrates. The extensive detection of this compound and its transformation product 3,4,5-trichloro-2-pyridinol was unexpected and called for a better understanding of the use and fate of this pesticide.

Exposure to Pesticides and Health Effects on Farm Owners and Workers From Conventional and Organic Agricultural Farms in Costa Rica: Protocol for a Cross-Sectional Study.

BACKGROUND: Pesticide use is increasing in low- and middle-income countries (LMICs) including Costa Rica. This increase poses health risks to farm owners, farm workers, and communities living near agricultural farms. OBJECTIVE: We aimed to examine the health effects associated with occupational pesticide exposure in farm owners and workers from conventional and organic smallholder farms in Costa Rica. METHODS: We conducted a cross-sectional study involving 300 owners and workers from organic and conventional horticultural smallholder farms in Zarcero County, Costa Rica. During the baseline study visit, we administered a structured, tablet-based questionnaire to collect data on sociodemographic characteristics, pesticide exposure, and health conditions (eg, respiratory and allergic outcomes and acute pesticide intoxication symptoms) and administered a neurobehavioral test battery (eg, Finger Tapping Test and Purdue Pegboard); we measured blood pressure, anthropometry (height, weight, and waist circumference), and erythrocytic acetylcholinesterase activity and also collected urine samples. In addition, a functional neuroimaging assessment using near-infrared spectroscopy was conducted with a subset of 50 study participants. During the follow-up study visit (~2-4 weeks after the baseline), we administered participants a short questionnaire on recent pesticide exposure and farming practices and collected hair, toenail, and urine samples. Urine samples will be analyzed for various pesticide metabolites, whereas toenails and hair will be analyzed for manganese (Mn), a biomarker of exposure to Mn-containing fungicides. Self-reported pesticide exposure data will be used to develop exposure intensity scores using an exposure algorithm. Furthermore, exposure-outcome associations will be examined using linear and logistic mixed-effects regression models. RESULTS: Fieldwork for our study was conducted between May 2016 and August 2016. In total, 113 farm owners and 187 workers from 9 organic and 83 conventional horticultural smallholder farms were enrolled. Data analyses are ongoing and expected to be published between 2019 and 2020. CONCLUSIONS: This study is one of the first to examine differences in health effects due to pesticide exposure between farm owners and workers from organic and conventional smallholder farms in an LMIC. We expect that this study will provide critical data on farming practices, exposure pathways, and how occupational exposure to pesticides may affect farm owners and workers' health. Finally, we hope that this study will allow us to identify strategies to reduce pesticide exposure in farm owners and workers and will potentially lay the groundwork for a future longitudinal study of health outcomes in farm owners and workers exposed to pesticides. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/10914.

Peptide-Based Sandwich Immunoassay for the Quantification of the Membrane Transporter Multidrug Resistance Protein 1.

Multitransmembrane proteins are notoriously difficult to analyze. To date, rapid, and cost-efficient detection methods are lacking and only mass spectrometry-based systems allow reliable quantification of these proteins. Here, we present a novel type of sandwich immunoassay that is capable of sensitively detecting multidrug resistance protein 1 (MDR1), a prototypic 12-transmembrane-domains transporter. In a first assay step, complex samples are enzymatically fragmented into peptides as routinely done for mass spectrometry. A proteotypic peptide derived from MDR1 was chosen and antibodies targeting this peptide were used to build a sandwich immunoassay. Validation of the optimized assay showed good sensitivity, reproducibility and it allowed reliable quantification of MDR1; cross-validation by mass spectrometry demonstrated the applicability for routine analyses in clinical and pharmaceutical research. MDR1 was quantified in primary human renal cell carcinoma and corresponding normal tissue and down-regulation or expression loss was found in tumor tissue corroborating its importance in drug resistance and efficacy.

Direct Quantification of Cytochromes P450 and Drug Transporters-A Rapid, Targeted Mass Spectrometry-Based Immunoassay Panel for Tissues and Cell Culture Lysates.

The quantification of drug metabolizing enzymes and transporters has recently been revolutionized on the basis of targeted proteomic approaches. Isotope-labeled peptides are used as standards for the quantification of the corresponding proteins in enzymatically fragmented samples. However, hurdles in these approaches are low throughput and tedious sample prefractionation steps prior to mass spectrometry (MS) readout. We have developed an assay platform using sensitive and selective immunoprecipitation coupled with mass spectrometric readout allowing the quantification of proteins directly from whole cell lysates using less than 20,000 cells per analysis. Peptide group-specific antibodies (triple X proteomics antibodies) enable the enrichment of proteotypic peptides sharing a common terminus. These antibodies were employed to establish a MS-based immunoassay panel for the quantification of 14 cytochrome P450 (P450) enzymes and nine transporters. We analyzed the P450 enzyme and transporter levels in genotyped liver tissue homogenates and microsomes, and in samples from a time course induction experiment in human hepatocytes addressing different induction pathways. For the analysis of P450 enzymes and transporters only a minute amount of sample is required and no prefractionation is necessary, thus the assay platform bears the potential to bridge cell culture model experiments and results from whole organ tissue studies.

Inhibition of ß-catenin signaling by phenobarbital in hepatoma cells in vitro.

The antiepileptic drug phenobarbital (PB) exerts hepatic effect based on indirect activation of the constitutive androstane receptor (CAR) via inhibition of the epidermal growth factor receptor (EGFR) and the kinase Src. It has furthermore been observed that in mice PB suppresses the growth of hepatocellular carcinoma with overactive signaling through the oncogenic Wnt/ß-catenin pathway, thus suggesting an interference of PB with ß-catenin signaling. The present work was aimed to characterize effects of PB on ß-catenin signaling at different cellular levels and to elucidate molecular details of the interaction of PB and ß-catenin in an in vitro system of mouse hepatoma cells. PB efficiently inhibited signaling through ß-catenin. This phenomenon was in-depth characterized at the levels of ß-catenin protein accumulation and transcriptional activity. Mechanistic analyses revealed that the effect of PB on ß-catenin signaling was independent of the activation of CAR and also independent of the cytosolic multi-protein complex responsible for physiological post-translation control of the ß-catenin pathway via initiation of ß-catenin degradation. Instead, evidence is provided that PB diminishes ß-catenin protein production by inhibition of protein synthesis via signal transduction through EGFR and Src. The proposed mechanism is well in agreement with previously published activities of PB at the EGFR and Src-mediated regulation of ß-catenin mRNA translation. Inhibition of ß-catenin signaling by PB through the proposed mechanism might explain the inhibitory effect of PB on the growth of specific sub-populations of mouse liver tumors. In conclusion, the present data comprehensively characterize the effect of PB on ß-catenin signaling in mouse hepatoma cells in vitro and provides mechanistic insight into the molecular processes underlying the observed effect.

Peptides in headlock--a novel high-affinity and versatile peptide-binding nanobody for proteomics and microscopy.

Nanobodies are highly valuable tools for numerous bioanalytical and biotechnical applications. Here, we report the characterization of a nanobody that binds a short peptide epitope with extraordinary affinity. Structural analysis reveals an unusual binding mode where the extended peptide becomes part of a ß-sheet structure in the nanobody. This interaction relies on sequence-independent backbone interactions augmented by a small number of specificity-determining side chain contacts. Once bound, the peptide is fastened by two nanobody side chains that clamp it in a headlock fashion. Exploiting this unusual binding mode, we generated a novel nanobody-derived capture and detection system. Matrix-coupled nanobody enables the fast and efficient isolation of epitope-tagged proteins from prokaryotic and eukaryotic expression systems. Additionally, the fluorescently labeled nanobody visualizes subcellular structures in different cellular compartments. The high-affinity-binding and modifiable peptide tag of this system renders it a versatile and robust tool to combine biochemical analysis with microscopic studies.

Indirect protein quantification of drug-transforming enzymes using peptide group-specific immunoaffinity enrichment and mass spectrometry.

Immunoaffinity enrichment of proteotypic peptides, coupled with selected reaction monitoring, enables indirect protein quantification. However the lack of suitable antibodies limits its widespread application. We developed a method in which multi-specific antibodies are used to enrich groups of peptides, thus facilitating multiplexed quantitative protein assays. We tested this strategy in a pharmacokinetic experiment by targeting a group of homologous drug transforming proteins in human hepatocytes. Our results indicate the generic applicability of this method to any biological system.