Olaquindox Research Articles

Europium Nanoparticle-Based Lateral Flow Strip Biosensors for the Detection of Quinoxaline Antibiotics and Their Main Metabolites in Fish Feeds and Tissues.

Olaquindox (OLA) and quinocetone (QCT) have been prohibited in aquatic products due to their significant toxicity and side effects. In this study, rapid and visual europium nanoparticle (EuNP)-based lateral flow strip biosensors (LFSBs) were developed for the simultaneous quantitative detection of OLA, QCT, and 3-methyl-quinoxaline-2-carboxylic acid (MQCA) in fish feed and tissue. The EuNP-LFSBs enabled sensitive detection for OLA, QCT, and MQCA with a limit of detection of 0.067, 0.017, and 0.099 ng/mL (R2 ≥ 0.9776) within 10 min. The average recovery of the EuNP-LFSBs was 95.13%, and relative standard deviations were below 9.38%. The method was verified by high-performance liquid chromatography (HPLC), and the test results were consistent. Therefore, the proposed LFSBs serve as a powerful tool to monitor quinoxalines in fish feeds and their residues in fish tissues.

Advances in Metabolism and Metabolic Toxicology of Quinoxaline 1,4-Di-N-oxides.

Quinoxaline 1,4-di-N-Oxides (QdNOs) have been used as synthetic antimicrobial agents in animal husbandry and aquaculture. The metabolism and potential toxicity have been also concerns in recently years. The metabolism investigations showed that there were 8 metabolites of Carbadox (CBX), 34 metabolites of Cyadox (CYA), 33 metabolites of Mequindox (MEQ), 35 metabolites of Olaquindox (OLA), and 56 metabolites of Quinocetone (QCT) in different animals. Among them, Cb3 and Cb8, M6, and O9 are metabolic residual markers of CBX, MEQ and OLA, which are associated with N → O reduction. Toxicity studies revealed that QdNOs exhibited severe tumorigenicity, cytotoxicity, and adrenal toxicity. Metabolic toxicology showed that toxicity of QdNOs metabolites might be related to the N → O group reduction, and some metabolites exhibited higher toxic effects than the precursor, which could provide guidance for further research on the metabolic toxicology of QdNOs and provide a wealth of information for food safety evaluation.

Four birds with one stone: Aggregation-induced emission-type zeolitic imidazolate framework-8 based bionic nanoreactor for portable detection of olaquindox in environmental water and swine urine by smartphone

The abuse of olaquindox (OLA) as both an antimicrobial agent and a growth promoter poses significant threats to the environment and human health. While nanoreactors have proven effective in hazard detection, their widespread adoption has been hindered by tedious chemical processes and limited functionality. In this study, we introduce a novel green self-assembly strategy utilizing invertase, horseradish peroxidase, antibodies, and gold nanoclusters to form an aggregation-induced emission-type zeolitic imidazolate framework-8 nanoreactor. The results demonstrate that the lateral flow immunoassay not only allows for qualitative naked eye detection but also enables optical analysis through the fluorescence generated by aggregated gold nanoclusters and enzyme-catalyzed enhancement of visible colorimetric signals. To accommodate more detection scenarios, the photothermal effects and redox reactions of the nanoreactor can fulfill the requirements of thermal sensing and electrochemical analysis for smartphone applications. Remarkably, the proposed approach achieves a detection limit 17 times lower than conventional methods. Besides, the maximum linear range spans from 0.25 to 5 μg/L with high specificity, and the recovery is 85.2–112.9% in environmental water and swine urine. The application of this high-performance nanoreactor opens up avenues for the construction of multifunctional biosensors with great potential in monitoring hazardous materials.

A fluorescent lateral flow immunoassay based on CdSe/CdS/ZnS quantum dots for sensitive detection of olaquindox in feedstuff.

A portable fluorescence immunosensor based on the CdSe/CdS/ZnS quantum dots (QDs) with multiple-shell structure was fabricated for the precise quantification of olaquindox (OLA). The QDs labeled anti-OLA antibody used as bioprobe played an important role in the design and preparation of a lateral flow test strip. Due to the strong fluorescent intensity of QDs, the sensitivity is greatly improved. The quantitative results were obtained using a fluorescent strip scan reader within 8min, and the calculated limit of detection for OLA at 0.12µg/kg, which was 2.7 times more sensitive than that of the conventional colloidal gold-based strips method. Acceptable recovery of 85.0%-95.5% was obtained by the spiked samples. This newly established QDs-based strip immunoassay method is suitable for the on-site detection and rapid initial screening of OLA in swine feedstuff, and is potentially applied for the detection of other veterinary drugs to ensure food safety.

Molecularly imprinted electrochemical sensor based on synergistic interaction of honeycomb-like Ni-MOF decorated with AgNPs and N-GQDs for ultra-sensitive detection of olaquindox in animal-origin food

Olaquindox (OLA) in food from its illegal use possesses great harmful effects on humans, making it important to develop sensitive, inexpensive, and convenient methods for OLA detection. This study innovatively presented a molecularly imprinted electrochemical sensor based on the synergistic effects of nitrogen-doped graphene quantum dots (N-GQDs) and a nickel-based metal–organic framework functionalized with silver nanoparticles (Ag/Ni-MOF) for OLA detection. N-GQDs and Ag/Ni-MOF with unique honeycomb structures were sequentially modified on the glassy carbon electrode (GCE) surface to accelerate the electron transfer rate and increase the available region of the electrode. Molecularly imprinted polymers were further grown on the Ag/Ni-MOF/N-GQDs/GCE by electropolymerization to significantly enhance the selective recognition of OLA. The constructed sensor showed excellent performance for selective OLA determination, with a wide linear range (5–600 nmol·L-1) and exceedingly low detection limit (2.2 nmol·L-1). The sensor was successfully applied to detect OLA in animal-origin food with satisfactory recoveries (96.22–101.02%).

Off-line magnetic Fe3O4@SiO2@MIPs-based solid phase dispersion extraction coupling with HPLC for the simultaneous determination of olaquindox and its metabolite in fish muscle and milk samples

An innovative core–shell magnetic molecularly imprinted polymers, Fe3O4@SiO2@MIPs, was elaborately tailored for specific separation and enrichment of olaquindox (OLA) and its metabolic marker methyl-3-quinoxaline-2carboxylic acid (MQCA). Herein, benefiting from the combination of functionalized magnetic nanoparticles (Fe3O4@SiO2) and MIPs, Fe3O4@SiO2@MIPs not only possessed favorable magnetic properties and stability, avoiding tedious sample pretreatment process, but also demonstrated exceptional selective recognition ability and adsorption capacity, suppressing influence of coexisting interfering substances. Encouraged by prominent merits, Fe3O4@SiO2@MIPs-based magnetic solid phase extraction with HPLC method was developed, realizing simultaneous measurement of OLA and MQCA. Under optimal conditions, excellent linear ranges of 0–100 μg/L with detection limit of 0.175–0.271 μg/L were obtained. The proposed method was finally utilized for determination of OLA and MQCA in fish muscle and milk samples with satisfactory recoveries (80.56–95.26%) and relative standard deviation below 8.1%, furnishing a reliable and sensitive strategy for enrichment and detection residual veterinary drugs in food samples.

Administration of olaquindox impairs spermatogenesis and sperm quality by increasing oxidative stress and early apoptosis in mice

Olaquindox (OLA), a potent antibacterial agent, has been widely used as a feed additive and growth promoter in animal husbandry. Our previous study has shown that OLA administration in female mice could markedly cause sub-fertility. Here we established the model in male mice to investigate the toxic effects of OLA on mammalian spermatozoa quality and fetal development. After continuous 45 days of OLA gavage, the dosage of 60 mg/kg/day (high dose) significantly affected body weight, organ weights and coefficients, and the morphology of the testis seminiferous tubule in male mice. Dosage of 60 mg/kg/day also reduced sperm count, motility, and viability. OLA at both low-dose (5 mg/kg/day) and high-dose induced peroxidation, early apoptosis, and abnormal mitochondrial membrane potential in sperm. Significantly, high-dose OLA impaired in vitro fertilized embryo development, indicated by the decreased percentages of 2-cell and blastocyst formation. Surprisingly, the natural fertility of males was unaffected after OLA gavage, which was indicated by the comparable litter size after mating. However, paternal gavage of OLA significantly decreased the survival rate of the offspring from the age of 4 weeks. In sum, our study showed that OLA gavage in male mice damages sperm quality and offspring survival, illustrating the use of OLA as a feed additive should be strictly restricted.

A label-free electrochemical immunosensor based on AuNPs/GO-PEI-Ag-Nf for olaquindox detection in feedstuffs

For the first time, a label-free electrochemical immunosensor was developed to detect olaquindox (OLA) in feedstuffs using graphene oxide (GO) nanocomposites (GO-PEI-Ag) decorated by silver nanoparticles (AgNPs) and functionalized by polyethyleneimine (PEI) as electrode modification materials. In this work, GO with huge specific surface area was used as a carrier to load abundant AgNPs, which exhibited good redox activity and perfect conductivity. The stability of GO-Ag in solution was ameliorated by PEI, which was utilized as a solubilizer. Nafion (Nf) was applied to attach tightly and uniformly GO-PEI-Ag nanomaterials on the electrode surface. The gold nanoparticles (AuNPs) were electrodeposited on the GO-PEI-Ag-Nf modified electrode surface for signal amplification and antibody immobilization. Above all, the electrochemical signal significantly improved. Under the optimal conditions, the signal intensity showed linear correlation with the logarithm of OLA concentration ranging from 0.01 ng mL−1 to 150 ng mL−1, along with the low detection limit of 4.8 pg mL−1. Moreover, the analytical performance in feedstuff samples was evaluated, showing favorable recovery of 90.0%-100.2% with RSD of 2.2%-6.9%. The fabricated biosensor can provide a suitable approach to measure OLA and is promising to detect other additives in feedstuffs.

Rapid fabrication of partially exfoliated graphite foil with 3D hierarchical structure and its application in electrochemical detection of olaquindox

An ultra-fast and controllable electrochemical method for preparation of partially exfoliated graphite foil (EG) was developed. The exfoliation process was realized in NaH 2 PO 4 aqueous by a multi potential step (STEP) technology. The EG electrode possesses a 3D hierarchical structure with the graphene-like nanosheets seamlessly linked with the graphite substrate. The EG electrode can be directly used as working electrode of electrochemical sensor. The performed sensor displayed high performance for electrochemical detection of olaquindox (OLA), with limit of detection (LOD) of 15 nM and a linear range of 0.05–18.0 μM. In this work, an ultra-fast (150 s) and controllable electrochemical method for preparation of partially exfoliated graphite foil (EG) was developed. The exfoliation process was realized in NaH 2 PO 4 aqueous by a multi potential step (STEP) technology. The graphite foil (G) was switched periodically between the mild oxidation potential and the reduction potential, avoiding completely exfoliation of the graphene sheets. The exfoliation degree of G electrode can be strictly controlled by the electrochemical parameters. The EG electrode possesses a hierarchical structure with the graphene-like nanosheets seamlessly linked with the graphite substrate, which can ensure its direct use as working electrodes for electrochemical sensors. Owing to its large surface area (6.1 times higher than G electrode), extremely low resistance ( R ct = 0.247 Ω), and promotion of adsorption of molecules, the EG electrode displayed surpassed catalytic effect toward the electrochemical redox of olaquindox (OLA). Based on the EG electrode, a high-performance electrochemical sensor for detection of OLA was constructed. The limit of detection (LOD) of the proposed sensor was achieved to be 15 nM (S/N=3), with a linear range of 0.05 – 18.0 μM and ultra-high sensitivity of 2.94 × 10 −3 A cm −2 μM −1 . Further, the low cost, flexible, small volume and easy integration make the EG electrode based sensor promise perspectives in the field of electroanalysis.

Olaquindox-Induced Liver Damage Involved the Crosstalk of Oxidative Stress and p53 In Vivo and In Vitro.

Olaquindox (OLA), a member of the quinoxaline-N,N-dioxide family, has been widely used as a growth-promoting feed additive and treatment for bacterial infections. The toxicity has been a major concern, and the precise molecular mechanism remains poorly understood. The present study was aimed at investigating the roles of oxidative stress and p53 in OLA-caused liver damage. In a mouse model, OLA administration could markedly cause liver injury as well as the induction of oxidative stress and activation of p53. Antioxidant N-acetylcysteine (NAC) inhibited OLA-induced oxidative stress and p53 activation in vivo. Furthermore, knockout of the p53 gene could significantly inhibit OLA-induced liver damage by inhibiting oxidative stress and the mitochondria apoptotic pathway, compared to the p53 wild-type liver tissue. The cell model in vitro further demonstrated that p53 knockout or knockdown in the HCT116 cell and L02 cell significantly inhibited cell apoptosis and increased cell viability, presented by suppressing ROS production, oxidative stress, and the Nrf2/HO-1 pathway. Moreover, loss of p53 decreased OLA-induced mitochondrial dysfunction and caspase activations, with the evidence of inhibited activation of phosphorylation- (p-) p38 and p-JNK and upregulated cell autophagy via activation of the LC3 and Beclin1 pathway in HCT116 and L02 cells. Taken together, our findings provided a support that p53 primarily played a proapoptotic role in OLA-induced liver damage against oxidative stress and mitochondrial dysfunction, which were largely dependent on suppression of the JNK/p38 pathway and upregulation of the autophagy pathway via activation of LC3 and Beclin1.

Development of Resistance in Escherichia coli ATCC25922 under Exposure of Sub-Inhibitory Concentration of Olaquindox.

Quinoxaline1,4-di-N-oxides (QdNOs) are a class of important antibacterial drugs of veterinary use, of which the drug resistance mechanism has not yet been clearly explained. This study investigated the molecular mechanism of development of resistance in Escherichia coli (E. coli) under the pressure of sub-inhibitory concentration (sub-MIC) of olaquindox (OLA), a representative QdNOs drug. In vitro challenge of E. coli with 1/100× MIC to 1/2× MIC of OLA showed that the bacteria needed a longer time to develop resistance and could only achieve low to moderate levels of resistance as well as form weak biofilms. The transcriptomic and genomic profiles of the resistant E. coli induced by sub-MIC of OLA demonstrated that genes involved in tricarboxylic acid cycle, oxidation-reduction process, biofilm formation, and efflux pumps were up-regulated, while genes involved in DNA repair and outer membrane porin were down-regulated. Mutation rates were significantly increased in the sub-MIC OLA-treated bacteria and the mutated genes were mainly involved in the oxidation-reduction process, DNA repair, and replication. The SNPs were found in degQ, ks71A, vgrG, bigA, cusA, and DR76-4702 genes, which were covered in both transcriptomic and genomic profiles. This study provides new insights into the resistance mechanism of QdNOs and increases the current data pertaining to the development of bacterial resistance under the stress of antibacterials at sub-MIC concentrations.

Molecular mechanism of olaquindox-induced hepatotoxicity and the hepatic protective role of curcumin

Olaquindox (OLA) is a chemosynthetic growth promoter, which could promote the treatment of bacterial infections and improve feed energy efficiency. Hepatotoxicity is still a poor feature associated with the adverse effects of OLA. The present study aimed to investigate the molecular mechanism of OLA-induced hepatotoxicity and the protective role of curcumin in mice and HepG2 cells. The result showed that representative biomarkers involved in mitochondrial pathway, p53 pathway, mitogen-activated protein kinase (MAPK) pathway, autophagy and antioxidant pathway were activated. Furthermore, curcumin attenuated OLA-induced serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and liver damage in mice. In addition, cell viability of HepG2 was enhanced by curcumin pretreatment at 5, 10 and 20 μM. Meanwhile, curcumin markedly ameliorated OLA-induced oxidative stress, apoptosis and mitochondrial dysfunction. Moreover, curcumin pretreatment significantly up-regulated the expressions of nuclear factor erythroid-2-related factor 2 (Nrf2) and heme oxygenase-1(HO-1) and down-regulated the expressions of nuclear factor-kappaB (NF-kB) and p53 through reduced the nuclear translocation of NF-kB induced by OLA. In summary, our findings indicated that OLA-induced hepatotoxicity involved in mitochondrial apoptosis, autophagy, p53 pathway, Nrf2/HO-1 pathways, and curcumin regulated OLA-induced liver damage, oxidative stress and apoptosis via activation of Nrf2/HO-1 pathway and suppression of p53 and NF-kB pathway.

Oral administration of olaquindox negatively affects oocytes quality and reproductive ability in female mice.

As an effective feed additive in the livestock industry, olaquindox (OLA) has been widely used in domestic animal production. However, it is unclear whether OLA has negative effects on mammalian oocyte quality and fetal development. In this study, toxic effects of OLA were tested by intragastric gavage ICR mice with water, low-dose OLA (5mg/kg/day), or high-dose OLA (60mg/kg/day) for continuous 45 days. Results showed that high-dose OLA gavage severely affected the offspring birth and growth. Significantly, high-dose OLA impaired oocyte maturation and early embryo development, indicated by the decreased percentage of germinal vesicle breakdown, first polar body extrusion and blastocyst formation. Meanwhile, oxidative stress levels were increased in oocytes or ovaries, indexed by the increased levels of ROS, MDA, H2O2, NO, and decreased levels of GSH, SOD, CAT, GSH-Px and GSH-Rd. Furthermore, aberrant mitochondria distribution, defective spindle assembly, abnormal H3K4me2/H3K9me3 levels, increased DNA double-strand breaks and early apoptosis rate, were observed after high-dose OLA gavage. Taken together, our results for the first time illustrated that high-dose OLA gavage led to sub-fertility of females, which means that restricted utilization of OLA as feed additive should be considered.

Molecularly imprinted electrochemical sensor based on polypyrrole/dopamine@graphene incorporated with surface molecularly imprinted polymers thin film for recognition of olaquindox

In this paper, an advanced molecularly imprinted electrochemical sensor (MIECS) based on electropolymerized olaquindox (OLA) surface molecularly imprinted polymer thin film on a modified glassy carbon electrode (GCE) was developed for the detection of OLA. It was fabricated by coating dopamine@graphene (DGr) on GCE, then electropolymerizing pyrrole (Py) and molecularly imprinted polymers (MIPs). Graphene (Gr) was introduced for improving conductivity and sensitivity. Dopamine (DA) was used for dispersion and adhesion of Gr. Polypyrrole (PPy) could fix DGr and enhance the current response evidently. The established sensor could selectively recognize OLA but not the analogs of OLA. Some essential parameters controlling the performance of the developed sensor were investigated and optimized. Under optimal conditions, the linear relationship between the current intensity and OLA concentration was obtained from 50 nmol L−1 to 500 nmol L−1 with a limit of detection (LOD) of 7.5 nmol L−1. Analytical results of OLA based on the developed MIECS for fish and feedstuffs showed a good agreement with the results based on high performance liquid chromatography (HPLC).

Novel Electrochemical Sensor Fabricated for Individual and Simultaneous Ultrasensitive Determination of Olaquindox and Carbadox Based on MWCNT-OH/CMK-8 Hybrid Nanocomposite Film.

A hybrid nanocomposite consisting of hydroxylated multi-walled carbon nanotubes (MWCNTs−OH) and cube mesoporous carbon (CMK−8) was applied in this study to construct an MWCNT−OH/CMK−8/gold electrode (GE) electrochemical sensor and simultaneously perform the electro-reduction of olaquindox (OLA) and carbadox (CBX). The respective peak currents of CBX and OLA on the modified electrode increased by 720- and 595-fold relative to the peak current of GE. The performances of the modified electrode were investigated with electrochemical impedance spectroscopy, cyclic voltammetry, and differential pulse voltammetry. Then, the modified electrodes were used for the individual and simultaneous determination of OLA and CBX. The fabricated sensor demonstrated a linear response at 0.2–500 nmol/L in optimum experimental conditions, and the detection limits were 104.1 and 62.9 pmol/L for the simultaneous determination of OLA and CBX, respectively. As for individual determination, wide linear relationships were obtained for the detected OLA with levels of 0.05–500 nmol/L with LOD of 20.7 pmol/L and the detected CBX with levels of 0.10–500 nmol/L with LOD of 50.2 pmol/L. The fabricated sensor was successfully used in the independent and simultaneous determination of OLA and CBX in spiked pork samples.

A “Two-in-One” Tandem Immunoaffinity Column for the Sensitive and Selective Purification and Determination of Trace/Ultra-Trace Olaquindox and Its Major Metabolite in Fish Tissues by LC–MS/MS

A “two-in-one” bilayer immunoaffinity column (IAC) coupled in tandem was prepared for the highly selective purification of olaquindox (OLA) and its major metabolite, 3-methyl-quinoxaline-2-carboxylic acid (MQCA), and their determinations were carried out with the developed high-performance liquid chromatography–tandem mass spectrometry (LC–MS/MS) method. The prepared highly selective two-in-one IAC was used for fish tissue sample clean-up after acidolysis. Then, the purified samples were analyzed by LC–MS/MS in ESI+ and multiple reaction monitoring mode. The calibration curves showed excellent linearity, with a correlation coefficient (R2) greater than 0.999. Under the optimal conditions, the average recoveries at the spiked levels of 1.0, 5.0, and 10.0 μg/kg were 81.2–94.6% with intra- and inter-day relative standard deviations (RSD; intra-day, n = 5; inter-day, n = 3) that were less than 8.1% and 11.7%, respectively. The limits of detection (LODs) were 0.01 and 0.3 μg kg−1, and the limits of quantifications (LOQs) were 0.04 and 0.5 μg kg−1 for OLA and MQCA, respectively. The validated method was successfully applied to the qualitative and quantitative detection of real samples collected from several local markets.

Discovery of the Marker Residue of Olaquindox in Pigs, Broilers, and Carp.

The excretion, metabolism, distribution, and residue depletion of olaquindox (OLA), an antibacterial and growth-promoting agent used in food-producing animals for decades without a clear understanding of metabolic fate, was completely studied in pigs, broilers, carp, and rats using a radio-tracing approach combined with liquid chromatography-ion trap/time-of-flight mass spectroscopy to define the scientific marker residue (MR). After a single gavage of [3H]OLA, over 92% of the dose was excreted via urine. OLA was transformed into eight metabolites (O1-O8) in pigs and broilers, four metabolites (O1, O2, O4, and O7) in carp, and nine metabolites (O1-O9) in rats. O2 was the major residue in edible tissues of four species and persisted for the longest time in the kidneys with the longest half-life of 3.52-4.6 d. Bisdesoxyolaquindox (O2) is designated to be the MR, and the kidneys are considered to be the target tissue for OLA in food producing animals. Monitoring for this metabolite would improve the food safety evaluation and residue control of this drug.

One Step Synthesis of Covalent Connected Three-dimensional Graphene/Carbon Nanotube for Olaquindox Electrochemical Sensor

An electrochemical sensor based on a three-dimensional (3D) graphene/carbon nanotube (G/CNT) hybrid was developed for the sensitive detection of olaquindox (OLA). 3D G/CNT hybrid through covalent C-C bonding was fabricated by one-step synthesis using catalytic chemical vapor deposition. To construct the base of the sensor, a novel composite was fabricated by a functionalized 3D G/CNT with poly-(dimethy diallyl ammonium chloride) and Nafion via simple grind and ultrasonication dispersion methods. The electrochemical determination of OLA exhibited an oxidation peak at −0.685 V with a higher current response. Due to the effective surface area and active sites, a good linear relationship was observed between the OLA concentration and peak current over the range of 1.5 × 10−6–2.7 × 10−3 mg mL−1 with a detection limit of 6.12 × 10−8 mg mL−1 (3σ). The proposed electrochemical sensor was used to detect OLA in swine samples at a recovery ranging from 97.78% to 104.25%, which exhibits a broader prospect to supervise animal products and a high potential for food security applications.

Development of an icELISA and Immunochromatographic Assay for Methyl-3-Quinoxaline-2-Carboxylic Acid Residues in Fish

To evaluate residues of olaquindox (OLA) and its marker compound methyl-3-quinoxaline-2-carboxylic acid (MQCA) in aquatic products in the field, five types of hapten and corresponding antigens with exposure of different antigenic determinants were designed and synthesized. The optimal immunogen-coating combinations were obtained by antiserum detection and heterologous coating screening, successfully achieving a highly specific monoclonal antibody (mAb) against MQCA. Based on the highly specific mAb 1A11 showing no cross-reactivity with other analogs, an indirect competitive ELISA and an immunochromatographic strip for MQCA detection in fish were developed using the MQCA derivative MQCA-(NH2)4 coupled to KLH as the immunogen and MQCA-NH2 coupled to OVA as the heterologous coating antigen. The IC50 value of the indirect competitive enzyme-linked immunosorbent assay (icELISA) was 3.17 ng/mL, and the linear range was 0.58–17.29 ng/mL for MQCA under optimized concentrations of coating antigen and antibody of 0.03 and 0.3 μg/mL, respectively. The recoveries of MQCA by the developed icELISA in fish samples ranged from 81.6 to 90.7%. The cutoff value of the strip was 5 ng/mL, and the limit of detection was 0.25 ng/mL under optimal working conditions of 4 μL 0.1 M K2CO3 and 50 μL 0.2 mg/mL per 1 mL colloidal gold solution. The developed icELISA and immunochromatographic strip will be very useful tools for the primary screening of MQCA in fish.

Mechanisms of Antibacterial Action of Quinoxaline 1,4-di-N-oxides against Clostridium perfringens and Brachyspira hyodysenteriae.

Quinoxaline 1,4-di-N-oxides (QdNOs) are a class of bioreductive compounds, however, their antibacterial mechanisms are still unclarified. The aim of this study was to assess the ability of two representative QdNO drugs, cyadox (CYA) and olaquindox (OLA), to produce reactive oxide species (ROS) in Gram-positive anaerobe Clostridium perfringens CVCC1125 and Gram-negative anaerobe Brachyspira hyodysenteriae B204. In addition, the effects of QdNOs on the integrity of bacterial cell walls and membranes as well as the morphological alterations and DNA oxidative damage in C. perfringens and B. hyodysenteriae were analyzed. It was demonstrated that under anaerobic conditions, QdNOs were metabolized into the reduced products which did not show any antibacterial activity. A significant dose-related increase of intracellular ROS level and intracellular hydroxyl radicals were evident in bacteria exposed to QdNOs. The result of biochemical assay showed that the cell walls and membranes of the bacteria treated with QdNOs were damaged. After exposure to 1/2MIC to 4MIC of CYA and OLA, C. perfringens and B. hyodysenteriae became elongated and filamentous. Morphological observation with scanning and transmission electron microscopes revealed rupture, loss of cytoplasmic material and cell lysis in QdNO-treated bacteria, indicating serious damage of cells. There was an increase of 8-OHdG in the two strains treated by QdNOs, but it was lower in C. perfringens CVCC1125 than in B. hyodysenteriae B204. Agarose gel electrophoresis showed the degradation of chromosomal DNA in both of the two anaerobes treated by QdNOs. The results suggest that QdNOs may kill C. perfringens and B. hyodysenteriae via the generation of ROS and hydroxyl radicals from the bacterial metabolism of QdNOs, which cause oxidative damage in bacteria under anaerobic conditions.