Pei Zhu

A novel and simple cell-based electrochemical impedance biosensor for evaluating the combined toxicity of DON and ZEN.

In this study, a novel and simple cell-based electrochemical biosensor was developed to assess the individual and combined toxicity of deoxynivalenol (DON) and zearalenone (ZEN) on BEL-7402 cells. The sensor was fabricated by modification with AuNPs, p-aminothiophenol, and folic acid in succession. The BEL-7402 cells which had a good activity were adhered on the electrode through the high affinity between the folate receptor and folic acid selectivity. We used the collagen to maintain the cell adhesion and viability. Electrochemical impedance spectroscopy (EIS) was developed to evaluate the individual and combined toxicity of DON and ZEN. Our results indicate that DON and ZEN caused a marked decrease in the cell viability in a dose-dependent manner. The value of electrochemical impedance spectroscopy decreased with the concentration of DON and ZEN in range of 0.1-20, 0.1-50 μg/ml with the detection limit as 0.03, 0.05 μg/ml, respectively, the IC50 for DON and ZEN as obtained by the proposed electrochemical method were 7.1 μg/ml and 24.6 μg/ml, respectively, and the combination of two mycotoxins appears to generate an additive response. The electrochemical cytotoxicity evaluation result was confirmed by biological assays. Compared to conventional methods, this electrochemical test is inexpensive, highly sensitive, and fast to respond, with long-term monitoring and real-time measurements. The proposed method provides a new avenue for evaluating the toxicity of mycotoxins.

Fluorescent magnetic bead-based mast cell biosensor for electrochemical detection of allergens in foodstuffs.

In this study, a novel electrochemical rat basophilic leukemia cell (RBL-2H3) cell sensor, based on fluorescent magnetic beads, has been developed for the detection and evaluation of different allergens in foodstuffs. Fluorescein isothiocyanate (FITC) was successfully fused inside the SiO2 layer of SiO2 shell-coated Fe3O4 nanoparticles, which was superior to the traditional Fe3O4@SiO2@FITC modification process. The as-synthesized fluorescent magnetic beads were then encapsulated with lipidosome to form cationic magnetic fluorescent nanoparticles (CMFNPs) for mast cell magnetofection. The CMFNPs were then characterized by SEM, TEM, VSM, FTIR, and XRD analyses, and transfected into RBL-2H3 cells through a highly efficient, lipid-mediated magnetofection procedure. Magnetic glassy carbon electrode (MGCE), which possesses excellent reproducibility and regeneration qualities, was then employed to adsorb the CMFNP-transfected RBL-2H3 cells activated by an allergen antigen for electrochemical assay. Results show that the exposure of model antigen-dinitrophenol-bovine serum albumin (DNP-BSA) to anti-DNP IgE-sensitized mast cells induced a robust and long-lasting electrochemical impedance signal in a dose-dependent manner. The detection limit was identified at 3.3×10(-4) ng/mL. To demonstrate the utility of this mast cell-based biosensor for detection of real allergens in foodstuffs, Anti-Pen a1 IgE and Anti-PV IgE-activated cells were employed to quantify both shrimp allergen tropomyosin (Pen a 1) and fish allergen parvalbumin (PV). Results show high detection accuracy for these targets, with a limit of 0.03 μg/mL (shrimp Pen a 1) and 0.16 ng/mL (fish PV), respectively. To this effect, we conclude the proposed method is a facile, highly sensitive, innovative electrochemical method for the evaluation of food allergens.

A Sensitive and simple macrophage-based electrochemical biosensor for evaluating lipopolysaccharide cytotoxicity of pathogenic bacteria.

In this study, a sensitive and simple electrochemical murine macrophage (Ana-1) cell sensor has been developed for early detection of lipopolysaccharides (LPS) to evaluate the toxicity of pathogenic bacteria. Magnetic glassy carbon electrode (MGCE), which possesses excellent reproducibility and regeneration qualities, was modified with a nanocomposite to improve electrochemical signals and enhance the sensitivity. The synthesized magnetic nanoparticles (MNPs) were internalized into murine macrophages, which completed the immobilization of macrophages onto the modified electrode for evaluating the cytotoxicity of LPS by electrochemical impedance spectroscopy (EIS). The MNPs facilitated reusability of the proposed sensor by allowing removal of the magnetic core from the electrode. Our results indicated that LPS caused a marked decrease in electrochemical impedance in a dose-dependent manner in range of 1-5μg/mL. By SEM, we found that microvilli on the plasma membrane became scarce and the membrane became smooth on cells incubated with LPS, which lessens the absorption of cells to reduce the impedance. And biological assay indicated that EIS patterns were correlated with the calcium concentration in cells, and suggested that [Ca(2+)]i production increased in cells incubated with LPS and its mobilization altered electrochemical signals. Compared with conventional methods, this electrochemical test is inexpensive, highly sensitive, and has a quick response, and thus provides a new avenue for evaluating the cytotoxicity of pathogens.

Development of a simple and convenient cell-based electrochemical biosensor for evaluating the individual and combined toxicity of DON, ZEN, and AFB1

A simple and convenient cell-based electrochemical biosensor was developed to assess the individual and combined toxicity of deoxynivalenol (DON), zearalenone (ZEN), and Aflatoxin B1 (AFB1) on Hep G2 cells. The sensor was modified in succession with AuNPs (gold nanoparticles), cysteamine, and laminin. The cells interacting with laminin formed tight cell-to-electrode contacts, and collagen was used to maintain cell adhesion and viability. Electrochemical impedance spectroscopy (EIS) was developed to evaluate mycotoxin toxicity. Experimental results show that DON, ZEN, and AFB1 caused a significant decrease in cell viability in a dose dependent manner. The EIS value decreased with concentrations of DON, ZEN, and AFB1 in the range of 0.01-20, 0.1-50, and 0.1-3.5μg/mL, and IC50 obtained using the developed method was 48.5, 59.0, and 3.10μg/mL, respectively. A synergistic effect was observed between DON and ZEN, an additive effect was observed between DON and AFB1, and an antagonism effect was found in the binary mixtures of ZEN and AFB1 and ternary mixtures. These results were confirmed via CCK-8 assay. Utilizing SEM, we found that cells treated with mycotoxins caused significant changes in cell morphology, thus lessening cell adsorption and impedance reduction. Biological assay indicated that EIS patterns correlated with [Ca2+]i concentrations and apoptosis and necrotic cells ratios, thus effecting electrochemical signals. This method is simpler, more convenient, sensitive, and has a quicker response rate than most conventional cytotoxicity evaluation methods.

The Antagonistic Effect of Mycotoxins Deoxynivalenol and Zearalenone on Metabolic Profiling in Serum and Liver of Mice

Metabolic profiling in liver and serum of mice was studied for the combined toxic effects of deoxynivalenol (DON) and zearalenone (ZEN), through gas chromatography mass spectrum. The spectrum of serum and liver sample of mice, treated with individual 2 mg/kg DON, 20 mg/kg ZEN, and the combined DON + ZEN with final concentration 2 mg/kg DON and 20 mg/kg ZEN for 21 days, were deconvoluted, aligned and identified with MS DIAL. The data matrix was processed with univariate analysis and multivariate analysis for selection of metabolites with variable importance for the projection (VIP) > 1, t-test p value < 0.05. The metabolic pathway analysis was performed with MetaMapp and drawn by CytoScape. Results show that the combined DON and ZEN treatment has an obvious “antagonistic effect” in serum and liver tissue metabolic profiling of mice. The blood biochemical indexes, like alkaline phosphatase, alanine transaminase, and albumin (ALB)/globulin (GLO), reveal a moderated trend in the combined DON + ZEN treatment group, which is consistent with histopathological examination. The metabolic pathway analysis demonstrated that the combined DON and ZEN treatment could down-regulate the valine, leucine and isoleucine biosynthesis, glycine, serine and threonine metabolism, and O-glycosyl compounds related glucose metabolism in liver tissue. The metabolic profiling in serum confirmed the finding that the combined DON and ZEN treatment has an “antagonistic effect” on liver metabolism of mice.

Explaining combinatorial effects of mycotoxins Deoxynivalenol and Zearalenone in mice with urinary metabolomic profiling

Urine metabolic profiling of mice was conducted utilizing gas chromatography-mass spectrometry (GC-MS) to investigate the combinatory effect of mycotoxins deoxynivalenol (DON) and zearalenone (ZEN) on the metabolism of the mice. Experiments were conducted by means of five-week-old mice which were individually exposed to 2 mg/kg DON, 20 mg/kg ZEN and the mixture of DON and ZEN (2 mg/kg and 20 mg/kg, respectively). The intragastric administration was applied for three weeks and urine samples were collected for metabolic analysis. Univariate and multivariate analysis were applied to data matrix processing along with respective pathway analysis by MetaMapp and CytoScape. The results showed that the combined DON and ZEN administration resulted in lower significant changes, compared to the individual mycotoxin treated groups verified by heatmap. Metabolic pathways network mapping indicated that the combined mycotoxins treated groups showed a little effect on the metabolites in most pathways, especially in glucose metabolism and its downstream amino acid metabolism. In glucose metabolism, the content of galactose, mannitol, galactonic acid, myo-inositol, tagatose was drastically down-regulated. Furthermore, the organic acids, pyruvate, and amino acids metabolism displayed the same phenomenon. In conclusion, the combined DON/ZEN administration might lead to an “antagonistic effect” in mice metabolism.

Cell Based-Green Fluorescent Biosensor using Cytotoxic Pathway for Bacterial Lipopolysaccharide Recognition

Lipopolysaccharide (LPS), a characteristic component of the outer membrane of Gram-negative bacteria, can be used as an effective biomarker to detect bacterial contamination. Here, we reported a 293/hTLR4A-MD2-CD14 cell-based fluorescent biosensor to detect and identify LPS, which is carried out in a 96-well microplate which is nondestructive, user-friendly, and highly efficient. The promoter sequence of the critical signaling pathway gene ZC3H12A (encoding MCPIP1 protein) and enhanced green fluorescence protein (EGFP) were combined to construct a recombinant plasmid, which was transferred into 293/hTLR4A-MD2-CD14 cells through lipid-mediated, DNA-transfection way. LPS was able to bind to TLR4 and coreceptors-induced signaling pathway could result in green fluorescent protein expression. Results show that stable transfected 293/hTLR4A-MD2-CD14 cells with LPS treatment could be directly and continually observed under a high content screening imaging system. The novel cell-based biosensor detects LPS at low concentration, along with the detection limit of 0.075 μg/mL. The cell-based biosensor was evaluated by differentiating Gram-negative and Gram-positive bacteria and detecting LPS in fruit juices as well. This proposed fluorescent biosensor has potential in sensing LPS optically in foodstuff and biological products, as well as bacteria identification, contributing to the control of foodborne diseases and ensurance of public food safety with its high throughput detection way.

Gas chromatography-mass spectrometry metabolomic study of lipopolysaccharides toxicity on rat basophilic leukemia cells.

Lipopolysaccharide (LPS) can lead to uncontrollable cytokine production, fatal sepsis syndrome and depression/multiple organ failure, as pathophysiologic demonstration. Various toxic effects of LPS have been extensively reported, mainly on the toxicity of LPS in cellular level, macrophages or tumor cells, etc. This work aimed on the impact of LPS on mast cell metabolism, which focused on LPS-induced cellular metabolic profiles. Gas chromatography-mass spectrometry (GC-MS) based metabolomics strategy was implemented for the endo-metabolites detection in rat basophilic leukemia (RBL-2H3) cells, treated with 10 μg/mL LPS for 24 h, along with multiple time-dose tests of cells viability/apoptosis. Significantly changes metabolites were mainly involved the metabolism of glycine, serine, threonine and the biosynthesis of phenylalanine, tyrosine, tryptophan and pentose phosphate pathway. The endo-metabolism results illustrated that LPS treatment led to downregulation of glycine, serine and threonine metabolism besides pentose phosphate pathway in RBL-2H3 cells. This novel insight into LPS cellular metabolism, provides some heuristic guidance for elucidating the underlying mechanism of LPS-mediated disease.

Pathway of 3-MCPD-induced apoptosis in human embryonic kidney cells

3-Chloropropane-1,2-diol (3-MCPD) is a heat-produced contaminant formed during the preparation of soy sauce worldwide. The present investigation was conducted to determine the molecular aspects of 3-MCPD toxicity on human embryonic kidney cells (HEK293). Cell viability and apoptosis were assessed in response to exposure to 3-MCPD using the MTT assay and high-content screening (HCS). DNA damage, intracellular reactive oxygen species (ROS) and apoptosis-related proteins were evaluated. Genes related with apoptosis were detected by qPCR-array for further understanding the 3-MCPD induced cell apoptosis signaling pathway. Our results clearly showed that 3-MCPD treatment inhibits cell proliferation and reactive oxygen species generation. qPCR-array indicated that nine apoptotic genes were up-regulated more than 2-fold and six down-regulated more than 2-fold. Genes associated with the mitochondrial apoptotic pathway, especially BCL2 family genes, changed significantly, indicating that the mitochondrial apoptotic pathway is activated. Death receptor pathway-related genes, TNFRSF11B and TNFRSF1A, changed significantly, indicating that the death receptor pathway is also activated, resulting in the inhibition of cell growth and proliferation as well as induction of apoptosis. To sum up, the experiment results indicated that 3-MCPD induced HEK293 cell toxicity through the death receptor pathway and mitochondrial pathway.