Combined effects of chlorpyrifos and cyfluthrin on neurobehavior and neurotransmitter levels in larval zebrafish.

Chlorpyrifos and cyfluthrin are insecticides commonly used in agriculture. The mixed residues of chlorpyrifos and cyfluthrin in the aquatic environment may have combined effects on nontarget species. Therefore, studying the combined toxic effects and mechanisms of pesticide mixtures is of great significance to environmental risk assessment. To evaluate the risk of combined exposure, we examined the effects of both compounds, separately and together, on motor activity, acetylcholinesterase (AChE) activity, and neurotransmitter levels in larval zebrafish. Chlorpyrifos exposure significantly reduced functional motor capacity (swim distance and velocity) and enhanced meandering, while cyfluthrin exposure alone had no significant effects on swim parameters. However, combined exposure significantly reduced total swimming distance and mean velocity and increased meandering. Both compounds alone and the combination significantly reduced AChE activity, and the combined effect was antagonistic. Combined exposure also significantly altered the concentrations of serotonin, serotonin precursors, and dopamine precursors, as well as concentrations of the amino acid neurotransmitters glycine, alanine, and aspartic acid. Combined exposure to chlorpyrifos and cyfluthrin exhibited distinct joint action modes in terms of neurobehavior, AChE activity, and neurotransmitter levels, thereby providing an experimental basis for assessing the combined exposure to chlorpyrifos and cyfluthrin's environmental risk.

Lateral flow immunoassay for 5-hydroxyflunixin based on near-infrared fluorescence molecule as an alternative label to gold nanoparticles.

A high-affinity monoclonal antibody (mAb) has been prepared and separately a gold nanoparticle (AuNP)-based and a near-infrared (NIR) fluorescence-based lateral flow immunoassay (LFA) developed for determination of 5-hydroxyflunixin residue in raw milk. The AuNP and IRDye® 800CW were used to label anti-5-hydroxyflunixin mAb to form the AuNP-mAb and NIR dye-mAb conjugates, respectively. Quantitative determination of 5-hydroxyflunixin was achieved by imaging the optical or fluorescence intensity of the AuNP-mAb and NIR dye-mAb captured on the test line. As a result, the detection limits of the AuNP-based LFA and NIR dye-based LFA were 0.82 and 0.073 ng/mL in raw milk, respectively. The considerable improvement on assay sensitivity of the NIR-based LFA can be attributed to the lower background and less antibody consumption per test than that of the AuNP-based LFA. The spiking experiment by the NIR-based LFA yielded 85.7-112.6% recovery with a relative standard deviation below 14%, indicating that it has satisfactory assay accuracy and precision. Furthermore, the analytical results of actual samples by the NIR dye-based LFA were consistent with that by instrumental analysis. Therefore, these results demonstrated that the NIR dye is an ideal alternative label to the conventional AuNP for the development of LFA for veterinary drugs in animal-origin food. Graphical abstract.

Immunoaffinity Cleanup and Isotope Dilution-Based Liquid Chromatography Tandem Mass Spectrometry for the Determination of Six Major Mycotoxins in Feed and Feedstuff.

In this study, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous determination of deoxynivalenol, aflatoxin B1, zearalenone, ochratoxin A, T-2 toxin and fumonisin B1 in feed and feedstuff was established. The sample was extracted with an acetonitrile-water mixture (60:40, v/v), purified by an immunoaffinity column, eluted with a methanol-acetic acid mixture (98:2, v/v), and reconstituted with a methanol-water mixture (50:50, v/v) after drying with nitrogen. Finally, the reconstituted solution was detected by LC-MS/MS and quantified by isotope internal standard method. The six mycotoxins had a good linear relationship in a certain concentration range, the correlation coefficients were all greater than 0.99, the limits of detection were between 0.075 and 1.5 µg·kg-1, and the limits of quantification were between 0.5 and 5 µg·kg-1. The average spike recoveries in the four feed matrices ranged from 84.2% to 117.1% with relative standard deviations less than 11.6%. Thirty-six actual feed samples were analyzed for mycotoxins, and at least one mycotoxin was detected in each sample. The proposed method is reliable and suitable for detecting common mycotoxins in feed samples.

Antagonistic effects and mechanisms of carbendazim and chlorpyrifos on the neurobehavior of larval zebrafish.

Residues from multiple pesticides are frequently detected on vegetables, which may produce combined toxicity not predicted by individual toxicity data. As these combined effects present additional dangers to food safety, we have compared individual to combined effects for a variety of pesticides. Carbendazim and chlorpyrifos are the two most commonly detected pesticides in vegetables, and previous studies reported that combined exposure results in synergistic developmental toxicity to zebrafish embryos. In this study, individual and combined effects on zebrafish motor activity were examined following individual and combined exposure to assess nervous system toxicity. Further, transcriptomics methods were used to identify potential molecular mechanisms for individual and combined toxicity. Carbendazim alone induced a disorganized swim pattern characterized by increased angular velocity, turn angle, meander, and acceleration during light-dark transition, while chlorpyrifos alone reduced average swim speed and light-dark acceleration. Combined treatment significantly reduced average swim velocity and total distance traveled. Combination indices indicated strong antagonism between compounds for average speed and light-dark acceleration. Transcriptomics (RNA-seq) showed that carbendazim significantly altered the expression of genes involved in antigen processing and presentation, apoptosis, autophagy, and metabolism, including ctslb, cyp7a1, hsp70l, and ugt1a1. Alternatively, chlorpyrifos significantly altered genes involved in various nervous system-related pathways, including glutamatergic, GABAergic, dopaminergic, and calcium signaling. Protein-protein interaction (PPI) network analysis suggested that chlorpyrifos significantly downregulated genes related to light transduction, resulting in decreased sensitivity to light-dark transitions, while antagonism mainly reflected divergent effects on phototransduction and retinol metabolism. Carbendazim had no significant effects on vision-related genes such as gnat1 and gngt1, while chlorpyrifos downregulated expression, an effect reversed by the combination. Comprehensive toxicity analyses must include joint effects of co-applied pesticides for enhanced food safety.

Antagonistic Effects of Enrofloxacin on Carbendazim-Induced Developmental Toxicity in Zebrafish Embryos.

Carbendazim (CAR) and enrofloxacin (ENF) are frequently detected in fruits and meat products, respectively. Since most people consume fruits, vegetables, and meat products, combined exposure is possible, necessitating further evaluation of toxic interactions. In this study, the developmental toxicity of separate and combined exposure was examined in zebrafish embryos. Carbendazim exposure at 0.79 mg/L and above significantly affected developmental parameters, while enrofloxacin alone had no substantial effects on these developmental parameters within the selected concentration range (0.10-0.40 mg/L). Surprisingly, ENF antagonized the CAR-evoked reduction in the 48 hpf (hours post-fertilization) hatching rate and the increases in the 96 hpf malformation and lethality rates. The results revealed that the antagonism might be associated with reciprocal effects of these compounds on metabolism-related genes, such as cyp7a1 and apoa1a. These results reveal a complex interaction between ENF and CAR on metabolic regulation during development and highlight the importance of combined assessment for agents with the potential for simultaneous exposure.

Dual near-infrared fluorescence-based lateral flow immunosensor for the detection of zearalenone and deoxynivalenol in maize.

A novel dual near-infrared fluorescence-based lateral flow immunosensor was developed to determine zearalenone and deoxynivalenol in maize. Two near-infrared dyes with distinct fluorescence characteristics were utilized to separately label the anti-zearalenone and anti-deoxynivalenol antibodies as detection reagents. The capture antigens zearalenone-BSA and deoxynivalenol-BSA were mixed and immobilized on the same test line of nitrocellulose membrane. This assay format facilitates simultaneous detection of the two mycotoxins on a single test line. After optimizing experimental parameters, the limits of detection for zearalenone and deoxynivalenol were as low as 0.55 µg/kg and 3.8 µg/kg in maize, respectively. The spiking experiment yielded recovery ratios ranging from 81.7% to 107.3% with coefficients of variation less than 14% demonstrating high assay accuracy and precision. Moreover, the actual sample analysis produced consistent results between this method and instrumental method. Therefore, the developed immunosensor can serve as an accurate and efficient approach for monitoring mycotoxins in agricultural products.

Surface enhanced Raman scattering-based lateral flow immunosensor for sensitive detection of aflatoxin M1 in urine.

Aflatoxin M1 (AFM1) is generally used as a biomarker in urine for the assessment of aflatoxin exposure in humans and animals. However, there is no approach for the rapid and on-site monitoring of AFM1 level in urine. Here, we report a surface enhanced Raman scattering (SERS)-based lateral flow immunosensor built for such a purpose. Raman molecule 5,5-dithiobis-2-nitrobenzoic acid and anti-AFM1 monoclonal antibody were conjugated with Au (core)@Ag (shell) nanoparticle to serve as SERS nanoprobe. AFM1-bovine serum albumin was conjugated with gold nanoparticle and then applied onto nitrocellulose membrane as a visible "GOLD" test line. Quantitation of AFM1 was performed by the readout of Raman signal from the SERS nanoprobes captured on the test line. After optimizing experimental parameters, the detection limit of this immunosensor can achieve as low as 1.7 pg/mL of AFM1 in urine, which is far below the recommended tolerable level (30 pg/mL) of AFM1 in urine. The spiking experiment yielded 93.8%-111.3% recovery with coefficients of variation below 17% demonstrating high assay accuracy and precision. Moreover, this immunosensing assay is fast with an assay time below 20 min. Therefore, the developed immunosensor is a promising tool for the rapid assessment of aflatoxin exposure in the field.

Multiplex SERS-based lateral flow immunosensor for the detection of major mycotoxins in maize utilizing dual Raman labels and triple test lines.

A multiplex surface-enhanced Raman scattering (SERS)-based lateral flow immunosensor was developed to determine six major mycotoxins in maize. Two characteristic Raman reporter molecules-5,5-dithiobis-2-nitrobenzoic acid (DTNB) and 4-mercaptobenzoic acid (MBA)-were used to label the synthesized Au@Ag core-shell nanoparticles for the preparation of SERS nanoprobes as detection reagents. Six corresponding hapten-protein conjugates were prepared and dispensed on three test lines of nitrocellulose membrane with two conjugates on each line as capture antigens. This design facilitates the simultaneous detection of the six mycotoxins in a single test. After optimizing the experimental parameters of immunosensor, the limits of detection were as low as 0.96 pg/mL for aflatoxin B1, 6.2 pg/mL for zearalenone, 0.26 ng/mL for fumonisin B1, 0.11 ng/mL for deoxynivalenol, 15.7 pg/mL for ochratoxin A, and 8.6 pg/mL for T-2 toxin, respectively. The spiking experiment showed high accuracy with recovery of 78.9-106.2 % and satisfactory assay precision with the coefficient of variations below 16 %. Moreover, this assay can be completed in less than 20 min, and its detection results were consistent with that of liquid chromatography-mass spectrometry. Therefore, the developed SERS-based lateral flow immunosensor is a promising approach for mycotoxin detection in the field.

Rapid one-step enzyme immunoassay and lateral flow immunochromatographic assay for colistin in animal feed and food.

BACKGROUND: Colistin (polymyxin E) is a kind of peptide antibiotic which has been approved in animal production for the purposes of disease prevention, treatment, and growth promotion. However, the wide use of colistin in animal feed may accelerate the spread of colistin-resistance gene MCR-1 from animal production to human beings, and its residue in animal-origin food may also pose serious health hazards to humans. Thus, it is necessary to develop corresponding analytical methods to monitor the addition of colistin in animal feed and the colistin residue in animal-origin food. RESULTS: A one-step enzyme-linked immunosorbent assay (ELISA) and a lateral flow immunochromatographic assay (LFIA) for colistin were developed based on a newly developed monoclonal antibody. The ELISA showed a 50% inhibition value (IC50) of 9.7 ng/mL with assay time less than 60 min, while the LFIA had a strip reader-based detection limit of 0.87 ng/mL in phosphate buffer with assay time less than 15 min. For reducing the non-specific adsorption of colistin onto sample vial, the components of sample extraction solution were optimized and proved to greatly improve the assay accuracy. The spiked recovery experiment showed that the recoveries of colistin from feed, milk and meat samples were in the range of 77.83% to 113.38% with coefficient of variations less than 13% by ELISA analysis and less than 18% by LFIA analysis, respectively. Furthermore, actual sample analysis indicated that the two immunoassays can produce results consistent with instrumental analysis. CONCLUSIONS: The developed assays can be used for rapid qualitative or quantitative detection of colistin in animal feed and food.