Novel enzyme-linked aptamer-antibody sandwich assay and hybrid lateral flow strip for SARS-CoV-2 detection.

We have successfully generated oligonucleotide aptamers (Apts) and monoclonal antibodies (mAbs) targeting the recombinant nucleocapsid (N) protein of SARS-CoV-2. Apts were obtained through seven rounds of systematic evolution of ligands by exponential enrichment (SELEX), while mAbs were derived from the 6F6E11 hybridoma cell line. Leveraging these Apts and mAbs, we have successfully devised two innovative and remarkably sensitive detection techniques for the rapid identification of SARS-CoV-2 N protein in nasopharyngeal samples: the enzyme-linked aptamer-antibody sandwich assay (ELAAA) and the hybrid lateral flow strip (hybrid-LFS). ELAAA exhibited an impressive detection limit of 0.1 ng/mL, while hybrid-LFS offered a detection range of 0.1 - 0.5 ng/mL. In the evaluation using ten nasopharyngeal samples spiked with known N protein concentrations, ELAAA demonstrated an average recovery rate of 92%. Additionally, during the assessment of five nasopharyngeal samples from infected individuals and ten samples from healthy volunteers, hybrid-LFS displayed excellent sensitivity and specificity. Our study introduces a novel and efficient on-site approach for SARS-CoV-2 detection in nasopharyngeal samples. The reliable hybrid Apt-mAb strategy not only advances virus diagnostic methods but also holds promise in combating the spread of related diseases.

Disruption of liver development and coagulation pathway by ochratoxin A in embryonic zebrafish.

Ochratoxin A (OTA) is a mycotoxin that is found in various food and feed products. The molecular mechanisms that are associated with OTA hepatotoxicity and teratogenicity have not been extensively elucidated in a developing organism. In this study, the transcriptomic profile of zebrafish embryos indicates that hemostasis and blood coagulation are the top two pathways affected by OTA. The treatment of embryos with OTA was able to decrease the expression of genes that encode coagulation factors and liver markers, including f7, f9b, cp and vtna. OTA also weakened the signal of liver-specific microRNA-122. OTA administration not only reduced the size of a developing embryonic liver, but also decreased the number of phosphorylated histone H3-positive cells by immunohistochemical staining. OTA suppressed the expression of hhex and prox1, two critical transcriptional factors during hepatoblast specification, in the developing liver, but did not alter the insulin signal in the pancreas. In vitro analysis with zebrafish liver (ZFL) cells indicated that OTA blocked the expression of f7, fgb and liver markers. In summary, OTA exposure resulted in the generation of small livers which led to deficiency of coagulation factors in embryonic zebrafish. Impairment of hhex and prox1 gene expression and hepatocyte proliferation contributed to the disruption of liver development mediated by OTA.

Citrinin disrupts microtubule assembly in cardiac cells: Impact on mitochondrial organization and function.

Citrinin (CTN) is a mycotoxin commonly present in various foods and feeds worldwide, as well as dietary supplements in Asian countries, but the risks and cellular mechanisms associated with its cardiotoxicity remains unclear. In this study, RNA-seq analysis of CTN-treated H9c2 cardiac cells demonstrated significant perturbations in pathways related to microtubule cytoskeleton and mitochondrial network organization. CTN disrupted microtubule polymerization and downregulated mRNA levels of microtubule-assembling genes, Map2 and Tpx2, in H9c2 cardiac cells. Additionally, CTN interfered with the distribution of mitochondrial network along the microtubules, leading to the accumulation of dysfunctional mitochondria characterized by elevated superoxide levels and reduced membrane potential. This disruption also caused the buildup of lysosomes and ubiquitinated proteins, which hindered waste clearance in microtubule-disassembled H9c2 cells. Molecular docking analysis indicated that CTN could bind to the colchicine binding site on ß-tubulin, thereby mimicking the microtubule-disrupting effect of colchicine. This study provides morphological, transcriptomic, and mechanistic evidence to elucidate the cardiotoxic mechanisms of CTN, which involve the dysregulated microtubule network, subsequent mitochondrial mislocalization, and impaired proteolysis of damaged proteins/organelles in cardiac cells. Our findings may enhance the fundamental understanding and facilitate future risk assessment of CTN.

Novel antibody- and aptamer-based approaches for sensitive detection of mycotoxin fusaric acid in cereal.

This study presents the first successful generation of polyclonal antibodies (pAbs) and oligonucleotide aptamers specifically targeting fusaric acid (FA). Utilizing these pAbs and aptamers, three highly sensitive and specific assays were developed for the detection of FA in cereals with limits of detection (LOD) ranging from 5 to 50 ng/g: an antibody-based enzyme-linked immunosorbent assay (ELISA), an aptamer-based enzyme-linked aptamer-sorbent assay (ELASA), and a hybrid enzyme-linked aptamer-antibody sandwich assay (ELAAA). The recovery rates of FA in spiked cereal samples ranged from 87 % to 112 % across all assays. Analysis of 15 cereal feed samples revealed FA contamination levels of 459 to 1743 ng/g (ELISA), 427 to 1960 ng/g (ELASA), and 381 to 1987 ng/g (ELAAA). These results were further validated by HPLC analysis, confirming high consistency within developed assays. Overall, the ELISA, ELASA, and ELAAA are promising tools for the rapid detection of FA, significantly contributing to food safety monitoring.

Neurotoxicity of mycotoxin citrinin: Novel evidence in developing zebrafish and underlying mechanisms in human neuron cells.

Citrinin (CTN) is a mycotoxin that is found as a contaminant in various types of food/feed grains and fermented food supplements. Previous studies have already established the nephrotoxicity and hepatotoxicity of CTN, but the neurotoxicity of CTN has not been clearly examined. In this study, CTN at 2-20 µM was first found to interfere with the neural ganglia formation and locomotive behavior of embryonic zebrafish, a vertebrate animal model, at 24 hpf and 6 dpf, respectively. Further exposure of human neuroblastoma SH-SY5Y cells to 10 and 20 µM CTN for 72 h indicated that pathways responsible for neuron differentiation and projection guidance were down-regulated while oxidative stress and electron transport chain pathways were up-regulated based on the enrichment results of GSEA in the transcriptomic profiling. PCR analysis verified that CTN significantly down-regulated the expression of marker genes involved in neuron differentiation and synaptic signaling. CTN at the doses impairing cellular neurite outgrowth did not trigger mitochondrial oxidative stress and dysfunction. The neurotoxic mechanisms of CTN provide new information that is valuable in the assessment of CTN-related health risk for the general public.

Exposure to Mycotoxin Citrinin Promotes Carcinogenic Potential of Human Renal Cells.

Mycotoxin citrinin (CTN), commonly found in food and health supplements, may induce chromosomal instability. In this study, human renal proximal tubule epithelial cells (hRPTECs) that were exposed to CTN (10 and 20 µM) over 3 days exhibited numerical chromosomal aberrations. Short-term (3 days) and long-term (30 days) exposures to CTN significantly promoted mitotic spindle abnormalities, wound healing, cell migration, and anchorage-independent growth in human embryonic kidney 293 (HEK293) cells. Short-term exposure to 10 and 20 µM CTN increased the number of migrated cells on day 10 by 1.7 and 1.9 times, respectively. The number of anchorage-independent colonies increased from 2.2 ± 1.3 to 7.8 ± 0.6 after short-term exposure to 20 µM CTN and from 2.0 ± 1.0 to 12.0 ± 1.2 after long-term exposure. The transcriptomic profiles of CTN-treated HEK293 were subjected to over-representative analysis (ORA), gene set enrichment analysis (GSEA), and Ingenuity pathway analysis (IPA). Short-term exposure to CTN promoted the RTK/KRAS/RAF/MAPK cascade, while long-term exposure altered the extracellular matrix organization. Both short- and long-term CTN exposure activated cancer and cell cycle-related signaling pathways. These results demonstrate the carcinogenic potential of CTN in human cells and provide valuable insights into the cancer risk associated with CTN.

Pilot production of a sensitive ELISA kit and an immunochromatographic strip for rapid detecting citrinin in fermented rice.

Citrinin (CTN) is a mycotoxin primarily produced by Monascus species. Excess consumption of CTN may lead to nephrotoxicity and hepatotoxicity. A pilot study for commercial production of competitive direct enzyme-linked immunosorbent assay (cdELISA) kit and an immunochromatographic strip (immunostrip) for screening CTN in red yeast rice is established in this study. The coating antibody and the CTN-horse radish peroxidase (HRP) concentrations were optimized to increase the sensitivity and specificity of cdELISA kit. The conjugation methods/ratios of CTN to HRP as well as the long-term stability of kit components were also evaluated. The IC50 and detection limit of the ELISA kit were determined to be 4.1 and 0.2 ng mL-1, respectively. Analysis of 20 red yeast rice samples using ELISA kits revealed the contamination levels of CTN from 64 to 29 404 ng g-1. The on-site rapid detection of CTN with the immunostrip showed that CTN levels in seven samples exceeded the regulatory limit of 5 ppm. Additionally, the coefficient correlation between the results of HPLC and ELISA kits of 20 samples was 0.96. Sensitive and convenient tools at commercial levels for detection of CTN contamination in food are established herein to protect the health of the public.

Aflatoxin B1 interferes with embryonic liver development: Involvement of p53 signaling and apoptosis in zebrafish.

Aflatoxin B1 (AFB1), a naturally occurring mycotoxin, is present in human placenta and cord blood. AFB1 at concentrations found in contaminated food commodities (0.25 and 0.5 µM) did not alter the spontaneous movement, heart rate, hatchability, or morphology of embryonic zebrafish. However, around 86 % of 0.25 µM AFB1-treated embryos had livers of reduced size, and AFB1 disrupted the hepatocyte structures, according to histological analysis. Additionally, AFB1 treatment that begins at any stage before 72 h post-fertilization (hpf) effectively reduced the size of embryonic livers. In hepatic areas, AFB1 suppressed the expression of Hhex and Prox1, which are two critical transcriptional factors for initiating hepatoblast specification. KEGG analysis based on transcriptome profiling indicated that p53 signaling and apoptosis are the only observed pathways in AFB1-treated embryos. AFB1 at 0.5 µM significantly activated the expression of tp53, mdm2, puma, noxa, pidd1, and gadd45aa genes that are related to the p53 pathway and also that of baxa, casp 8 and casp 3a in the apoptotic process. TUNEL staining demonstrated that AFB1 triggered the apoptosis of embryonic hepatocytes in a dose-dependent manner. These results indicate that the deficiency of both hhex and prox1 as well as hepatocyte apoptosis via the p53-Puma/Noxa-Bax axis may contribute to the embryonic liver shrinkage that is caused by AFB1.

Systematic Characterization of the Group 2 House Dust Mite Allergen in Dermatophagoides microceras.

BACKGROUND: Allergic asthma, a chronic airway inflammatory disease, is a critical public health problem. Indoor house dust mites (HDMs) could cause allergic asthma. The prevalence of sensitization to Dermatophagoides microceras (Der m) was approximately 80% and is related to the immunoglobulin E crossing-reactivity of mites belonging to the same genus, Dermatophagoides pteronyssinus (Der p) and Dermatophagoides farina (Der f). However, studies on Der m are scant. METHODS: We used integrated OMICs approaches to identify and characterize the group 2 mite allergen-like protein in Der m (Der m 2). We established a Der m 2-induced allergic asthma mouse model and treated the mice with a fungal immunomodulatory protein (FIP-fve) isolated from Flammulina veluptipes to evaluate the allergenicity of Der m 2 and the immunomodulatory effects of FIP-fve. RESULTS: By performing de novo draft genome assembly and comparative genome analysis, we identified the putative 144-amino acid Der m 2 in silico and further confirmed its existence through liquid chromatography-tandem mass spectrometry. Der m 2 is a lipopolysaccharides (LPS)-binding protein. Thus, we examined the LPS-binding activity of recombinant Der m 2 by performing molecular docking analysis, co-immunoprecipitation (Co-IP), and a pull-down assay. Der m 2 elicited the production of pro-inflammatory cytokines, interleukin (IL)-6, and IL-8 in BEAS-2B cells, a human bronchial epithelial cell line, and induced airway hyperresponsiveness in mice. Furthermore, in mice sensitized with Der m 2, the administration of FIP-fve in either the earlier stage or the late stage, FIP-fve alleviated allergic asthma by moderating airway inflammation and remodeling. CONCLUSIONS: Der m 2 induced inflammatory responses in cell and mouse models. FIP-fve alleviated inflammation in Der m 2-induced asthma in mice by exerting an immunomodulatory effect.

Sensitive enzyme-linked immunosorbent assay and rapid one-step immunochromatographic strip for fumonisin B1 in grain-based food and feed samples.

BACKGROUND: Maize contaminated with mycotoxin fumonisin B1 poses a global threat to agricultural production. In this study, polyclonal antibodies (pAb) specific to fumonisin B1 were generated from rabbits immunised with fumonisin B1-keyhole limpet haemocyanin (KLH). These antibodies were used to establish a sensitive competitive direct enzyme-linked immunosorbent assay (cdELISA) and gold nanoparticle immunochromatographic strip for detecting fumonisin B1 levels in maize-based foods and feeds. RESULTS: In cdELISA, fumonisins B1, B2 and B3 at concentrations of 0.42, 0.58 and 81.5 ng mL(-1) respectively caused 50% inhibition (IC(50)) of binding of fumonisin B1-horseradish peroxidase (HRP) to the antibodies. Effective on-site detection of fumonisin B1 was achieved by developing a rapid and sensitive pAb-based gold nanoparticle immunochromatographic strip. This strip had a detection limit of 5 ng mL(-1) for fumonisin B1 in maize-based samples. Additionally, the whole analytical process could be completed within 10 min. Close examination of 15 maize-based samples by cdELISA revealed that 11 were fumonisin-positive, with a mean concentration of 435 +/- 20.1 ng g(-1). These results correlated well with those obtained by immunochromatographic strip. CONCLUSION: Both cdELISA and immunochromatographic strip methods established in this study are sensitive for rapid detection of fumonisins in agricultural commodities.

A Sensitive Two-Analyte Immunochromatographic Strip for Simultaneously Detecting Aflatoxin M1 and Chloramphenicol in Milk.

A two-analyte immunochromatographic strip (immunostrip) was developed for the simultaneous detection of aflatoxin M1 (AFM1) and chloramphenicol (CAP) in milk. Protein conjugates (AFM1-ovalbumin (OVA) and CAP-OVA) and goat anti-rabbit IgG were respectively drawn on nitrocellulose membrane as two test lines (T1 and T2) and a control line (C). The immunostrip was dipped into a well that contained a 200 µL milk sample, 5 µL AFM1 antibody-gold conjugates, and 8 µL CAP antibody-gold conjugates; the whole assay was completed in 15 min and the results could be interpreted visually or using a reader. This immunostrip has cut-off levels of 0.1 ng/mL and 0.5 ng/mL for AFM1 and CAP, respectively. Analysis of CAP and AFM1 in milk samples revealed that data from the immunostrip test agreed closely with those obtained from ELISA. The two-analyte immunostrip is a rapid way for on-site simultaneous detection of AFM1 and CAP in milk.

Ochratoxin A triggered intracerebral hemorrhage in embryonic zebrafish: Involvement of microRNA-731 and prolactin receptor.

Ochratoxin A (OTA), a mycotoxin widely found in foodstuffs, reportedly damages multiple brain regions in developing rodents, but the corresponding mechanisms have not been elucidated. In this study, zebrafish embryos at 6 h post fertilization (hpf) were exposed to various concentrations of OTA and the phenomenon associated with intracerebral hemorrhage was observed at 72 hpf. Exposure of embryos to OTA significantly increased their hemorrhagic rate in a dose-dependent manner. Large numbers of extravagated erythrocytes were observed in the midbrain/hindbrain areas of Tg(fli-1a:EGFP; gata1:DsRed) embryos following exposure to OTA. OTA also disrupted the vascular patterning, especially the arch-shaped central arteries (CtAs), in treated embryos. Histological analysis revealed a cavity-like pattern in their hindbrain ventricles, implying the possibility of cerebral edema. OTA-induced intracerebral hemorrhage and CtA vessel defects were partially reversed by the presence of miR-731 antagomir or the overexpression of prolactin receptor a (prlra); prlra is a downstream target of miR-731. These results suggest that exposure to OTA has a negative effect on cerebral vasculature development by interfering with the miR-731/PRLR axis in zebrafish.

Activation of ERK and JNK signaling pathways by mycotoxin citrinin in human cells.

Mycotoxin citrinin (CTN) is commonly found in foods and feeds that are contaminated/inoculated with Penicillium, Aspergillus and Monascus species. The exposure of human embryonic kidney (HEK293) and HeLa cells to CTN resulted in a dose-dependent increase in the phosphorylation of two major mitogen-activated protein kinases (MAPKs), ERK1/2 and JNK. In HEK293 cultures, the administering of CTN increased both the mRNA and protein levels of egr-1, c-fos and c-jun genes; additionally, the ERK1/2 pathway contributed to the upregulation of Egr-1 and c-Fos protein expression. CTN treatment also induced the transcription activity of Egr-1 and AP-1 proteins, as evidenced by luciferase reporter assays. Bioinformatic analyses indicated two genes Gadd45 beta and MMP3 have Egr-1 and AP-1 response elements in their promoters, respectively. Furthermore, co-exposure of HEK293 cells to CTN and MAPK pathway inhibitors demonstrated that CTN increased the levels of Gadd45 beta mRNA through ERK1/2 signaling pathway and up-regulated the MMP3 transcripts majorly via JNK pathway. Finally, CTN-triggered caspase 3 activity was significantly reduced in the presence of MAPK inhibitors. Our results suggest that CTN positively regulates ERK1/2 and JNK pathways as well as their downstream effectors in human cells; activated MAPK pathways are also involved in CTN-induced apoptosis.

Exposure to aflatoxin B1 interferes with locomotion and neural development in zebrafish embryos and larvae.

Aflatoxin B1 (AFB1) is the major mycotoxin that contaminates aquafeeds and regarded as a causative agent in illnesses and the mortality of aquacultural species. However, the effects of AFB1 on developing fish and associated toxic mechanism are still unknown. This study examines the behavioral changes, neuronal morphology and gene expression in zebrafish embryos and larvae upon exposure to aflatoxin solutions. Treatment of 6 h post fertilization (hpf) embryos with AFB1 at 15-75 ng/mL significantly changed the swimming patterns of seven days post-fertilization (dpf) zebrafish larvae. Larvae in the 15 ng/mL group demonstrated a hypolocomotor activity in free swimming, but hyperlocomotion was observed in the larvae exposed to 30-75 ng/mL AFB1. AFB1 at 75 ng/mL also significantly reduced the startle response of 7 dpf larvae after tapping stimulus. Exposure to AFB1 resulted in an aberrant morphology of trigeminal ganglion and hindbrain neurons in transgenic embryos (HuC:eGFP); this finding was supported by acetylated alpha-tubulin staining in wild-type fish. Additionally, AFB1 altered the levels of neurotoxic markers, including gfap and huC. The transcriptomic profile of AFB1-treated embryos revealed several differentially expressed genes that are related to neuroactivity and neurogenesis. PCR analysis verified that AFB1 significantly down-regulated the expression of ngfa and atp1b1b genes and increased that of prtga gene. The results herein indicate the toxicological impacts of AFB1 on the behaviors and neurodevelopment of fish in the early embryonic stage. Disruption of neural formation and synapse dysfunction may be responsible for the behavioral alteration.

Development of a monoclonal antibody against ochratoxin A and its application in enzyme-linked immunosorbent assay and gold nanoparticle immunochromatographic strip.

A monoclonal antibody (mAb) specific to ochratoxin A (OTA) was produced from a stable hybridoma cell line, 9C9H9, generated by the fusion of P3/NS1/1-AG4-1 myeloma cells with spleen cells isolated from a BALB/c mouse immunized with OTA-keyhole limpet hemocyanin. The 9C9H9 mAb belongs to the immunoglobulin G1 (kappa chain) isotype. A competitive direct enzyme-linked immunosorbent assay (cdELISA) and a competitive indirect ELISA were established for antibody characterization. The concentrations causing 50% inhibition of binding of OTA-horseradish peroxidase to the antibody by OTA, OTB, and OTC were found to be 0.32, 0.17, and 0.28 ng/mL, respectively, in the cdELISA. A sensitive and rapid mAb-based gold nanoparticle immunochromatographic strip was also developed using this mAb. This strip has a detection limit of 5 ng/mL for OTA and can be completed in 10 min. Analysis of OTA in coffee samples revealed that data obtained from immunochromatographic strip were in a good agreement with those obtained from cdELISA. The mAb-based cdELISA and immunochromatographic strip assay established in this study were sensitive and accurate for rapid screening of OTA in coffee samples.

Mechanism of patulin-induced apoptosis in human leukemia cells (HL-60).

Patulin (PAT) is a fungal secondary metabolite that exhibits potential cellular and animal toxicities. In this study, human promyelocytic leukemia (HL-60) cells were used to elucidate the mechanism and death mode associated with PAT. Morphological evidence of apoptosis, including membrane blebbing, nuclei fragmentation and DNA laddering formation was clearly observed 6h after exposure to PAT. The results of Western blotting indicated that PAT activated various processed caspases, and cleaved DFF45 and poly (ADP-ribose) polymerase (PARP) in a dose-dependent manner; it also induced a time-dependent increase in caspase 3 and 9 catalytic activities. The apoptosis mediated by PAT in HL-60 was accompanied with cytochrome c release from mitochondria and Bcl-2 expression decrease. The presence of thiol-containing compounds with PAT dramatically reduced the caspase 3 activity that was triggered by PAT; the addition of antioxidants, including mannitol and Tiron, had a similar effect. However, the suppression of p53 protein expression by RNA interference (RNAi) in human embryonic kidney (HEK293) cells did not significantly modify PAT-elicited caspase 3 activity. These findings suggest that PAT-induced apoptosis is mediated through the mitochondrial pathway without the involvement of p53; the interaction with sulfhydryl groups of macromolecules by PAT and the subsequent generation of reactive oxygen species (ROS) plays a primary role in the apoptotic process.

Development of a Sensitive Enzyme-Linked Immunosorbent Assay and Rapid Gold Nanoparticle Immunochromatographic Strip for Detecting Citrinin in Monascus Fermented Food.

Antibodies against citrinin (CTN) were generated from rabbits, which were injected with CTN-keyhole limpet hemocyanin (KLH). This work involved the development of a sensitive competitive direct enzyme-linked immunosorbent assay (cdELISA) and a rapid gold nanoparticle immunochromatographic strip (immunostrip) method for analyzing CTN in Monascus-fermented food. CTN at a concentration of 5.0 ng/mL caused 50% inhibition (IC50) of CTN-horseradish peroxidase (CTN-HRP) binding to the antibodies in the cdELISA. The capable on-site detection of CTN was accomplished by a rapid antibody-gold nanoparticle immunostrip with a detection limit of 20 ng/mL and that was completed within 15 min. A close inspection of 19 Monascus-fermented foods by cdELISA confirmed that 14 were contaminated with citrinin at levels from 28.6⁻9454 ng/g. Further analysis with the immunostrip is consistent with those results obtained using cdELISA. Both means are sensitive enough for the rapid examination of CTN in Monascus-fermented food products.

Aristolochic acid I interferes with the expression of BLCAP tumor suppressor gene in human cells.

Aristolochic acid I (AAI) is a phytocompound that is linked to the progressive renal disease and development of human urothelial carcinoma. The bladder cancer-associated protein (BLCAP) gene exhibits a tumor suppressor function in various tumors, including bladder carcinoma. This study evaluated the effect of AAI on BLCAP expression and its associated mechanism in human cells. Administering AAI to human embryonic kidney cells (HEK293), human proximal tubule epithelial cells (HK-2) and urinary bladder cancer cells (HT-1376) significantly reduced the expression of BLCAP mRNA and protein. AAI also effectively suppressed the luciferase activities driven by BLCAP promoters of various lengths in HEK293 cells. AAI significantly reduced both activator protein 1 (AP-1) and nuclear factor-κB (NF-κB) activities in reporter assays, but further point mutations revealed that Ap-1 and NF-κB binding sites on the BLCAP promoter were not AAI-responsive elements. Application of the DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine (5-aza-dC), reversed the decline of BLCAP expression that had been induced by AAI. However, AAI exposure did not alter hypermethylation of the BLCAP promoter, determined by methyl-specific polymerase chain reaction (PCR) and bisulfate sequencing. Knocking down BLCAP in HEK293 cell line enhanced the potential for cellular migration, invasion, and proliferation, along with the induction of a capacity for anchorage-independent growth. In conclusion, AAI down-regulated the expression of BLCAP gene and the deficiency in BLCAP expression contributed to the malignant transformation of human cells, implying that BLCAP may have a role in mediating AAI-associated carcinogenesis.

Induction of oxidative stress response by the mycotoxin patulin in mammalian cells.

Patulin (PAT), a mycotoxin mainly produced by Penicillium and Aspergillus, is found in various foods and feeds. In the present study, its effects on oxidative stress in various mammalian cell lines were investigated. When cell-permeating fluorescent dyes were used as indicators of the generation of reactive oxygen species (ROS), we found that PAT treatment directly increased intracellular oxidative stress in human embryonic kidney (HEK293) and human promyelocytic leukemia (HL-60) cells. Lipid peroxidation levels were also significantly increased in HL-60 cells and mouse kidney homogenates treated with PAT. Suppression of CuZn-superoxide dismutase (SOD) expression in mammalian cells by small interfering RNA resulted in an increase in PAT-mediated membrane damage, while overexpression of human CuZn-SOD or catalase led to a reduction in damage, indicating the involvement of ROS in PAT toxicity. Pretreatment of HEK293 cells with Tiron, a free radical scavenger, reduced the phosphorylation levels of extracellular signal-regulated kinase (ERK) 1/2 elicited by PAT. The ERK1/2 signaling pathway inhibitor, U0126, also significantly decreased the levels of ROS associated with PAT treatment. These findings indicate that PAT treatment results in the ROS production in mammalian cells, and ROS partially contributes to PAT-induced cytotoxicity. Activation of ERK1/2 signaling pathway is correlated with PAT-mediated ROS.

Development of a monoclonal antibody against domoic acid and its application in enzyme-linked immunosorbent assay and colloidal gold immunostrip.

A monoclonal antibody (mAb) specific to domoic acid was produced from a stable hybridoma cell line, 9F1F11, generated by the fusion of P3/NS1/1-AG4-1 myeloma cells with spleen cells isolated from a Balb/c mouse immunized with domoic acid--keyhole limpet hemocyanin. The 9F1F11 mAb belongs to the immunoglobulin G1 (kappa-chain) isotype. A competitive direct enzyme-linked immunosorbent assay (cdELISA) and a competitive indirect ELISA were established for antibody characterization. In the cdELISA, the concentration causing 50% inhibition (IC50) of binding of domoic acid-horseradish peroxidase to the antibody by domoic acid was found to be 0.58 ng/mL. A sensitive and rapid mAb-based colloidal gold immunostrip was also developed. The immunostrip assay, which has a detection limit of 5 ng/mL for domoic acid, can be completed in 10 min. Analysis of domoic acid in blue mussel samples revealed that data obtained from immunostrip were in a good agreement with those obtained from cdELISA. The mAb-based cdELISA and immunostrip assay established in this study were sensitive and accurate for rapid screening of domoic acid in shellfish samples.