Biing-Hui Liu

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.

Evaluation of genotoxic risk and oxidative DNA damage in mammalian cells exposed to mycotoxins, patulin and citrinin

Mycotoxins are fungal secondary metabolites with very diversified toxic effects in humans and animals. In the present study, patulin (PAT) and citrinin (CTN), two prevalent mycotoxins, were evaluated for their genotoxic effects and oxidative damage to mammalian cells, including Chinese hamster ovary cells (CHO-K1), human peripheral blood lymphocytes, and human embryonic kidney cells (HEK293). PAT, but not CTN, caused a significant dose-dependent increase in sister chromatid exchange (SCE) frequency in both CHO-K1 and human lymphocytes. PAT also elevated the levels of DNA gap and break in treated CHO-K1. In the single cell gel electrophoresis (SCGE) assay, exposure of HEK293 to concentrations above 15 microM of PAT induced DNA strand breaks; the tail moment values also greatly increased after posttreatment with formamidopyrimidine-DNA glycosylase (Fpg). This suggests that in human cells PAT is a potent clastogen with the ability to cause oxidative damage to DNA. However, no significant change in the tail moment values in CTN-treated cultures was found, suggesting that CTN is not genotoxic to HEK293. Incubation of HEK293 with CTN increased the mRNA level of heat shock protein 70 (HSP70), but not that of human 8-hydroxyguanine DNA glycosylase 1 (hOGG1). PAT treatment did not modulate the expression of either HSP70 or hOGG1 mRNA.

Characterization of Aspergillus spores by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

The intact fungal spores of several strains of four Aspergillus species, Aspergillus flavus, A. oryzae, A. parasiticus, and A. sojae, were directly analyzed by matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry. Very simple MALDI mass spectra are obtained by directly mixing spores with a matrix such as alpha-cyano-4-hydroxycinnamic acid or sinapinic acid. The mass spectra are obtained from the ablation of cell walls of spores owing to the acidity of the matrix solution. The MALDI results show that aflatoxigenic strains and non-aflatoxigenic strains have different mass peak profiles. Furthermore, the MALDI results of non-aflatoxigenic A. flavus and A. parasiticus spores resemble those of the closely related A. oryzae and A. sojae spores, respectively.

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.

Mycotoxin Citrinin Induced Cell Cycle G2/M Arrest and Numerical Chromosomal Aberration Associated with Disruption of Microtubule Formation in Human Cells

As a nephrotoxic mycotoxin, citrinin (CTN) contaminates various foodstuffs and animal feed commodities. In the present study, the effects of CTN on cell cycle arrest and microtubule formation were investigated by applying human embryonic kidney (HEK293) cells as a model. Exposure of HEK293 cells to CTN resulted in an arrest of cell cycle G2/M in a concentration-dependent increase. Administrating CTN elevated the expression levels of p53 and p21 proteins, yet attenuated the signals of phosphorylated cell division cycle 2 (cdc2). Furthermore, treating HEK293 with CTN increased both the value of mitotic index and the population of cells recognized by antibody mitotic protein monoclonal 2, suggesting that arrest of CTN-induced cell cycle occurred mainly during the mitotic phase. With the assistance of immunocytostaining of α-tubulin, CTN was found to disrupt the stable microtubule skeleton during the interphase of cell cycle and also interfere with the mitotic spindle integrity during mitosis. Additionally, for either in vivo or in vitro assays, CTN effectively inhibited tubulin polymerization in a concentration-dependent manner. When human peripheral blood mononuclear cells were exposed to CTN, the percentage of cells with numerical chromosome changes was increased by 4.3-fold over that of vehicle-treated group. Results of this study suggest that CTN-activated G2/M arrest primarily arises from the inhibition of tubulin polymerization and associated mitotic spindle formation. Additionally, disruption of microtubule organization by CTN also contributes to the induction of numerical chromosome aberration in human cells.

Aristolochic acid I induced oxidative DNA damage associated with glutathione depletion and ERK1/2 activation in human cells

Aristolochic acid I (AAI) has been widely found in herbal remedies and linked to the development of nephropathy and urothelial carcinoma in humans. This study elucidated the mechanism of oxidative stress and DNA damage mediated by AAI in human cells. Treatment of human promyelocytic leukemia cells (HL-60) and human renal proximal tubular cells (HK-2) with AAI led to a dose-dependent increase of reactive oxygen species (ROS). AAI also elevated the levels of DNA strand breaks and 8-hydroxy guanosine in HL-60 and HK-2 cells. Antioxidants, including Tiron, N-acetyl-l-cysteine (NAC) and glutathione (GSH), effectively suppressed the AAI-induced ROS and AAI-elicited genotoxicity, indicating that AAI induced the DNA damage through oxidative stress. GSH depletion was also found in AAI-treated cultures and proceeded prior to ROS formation. Exposure of HL-60 cells with AAI activated both ERK1/2 and p38 kinase phosphorylation, while only MEK1/2 inhibitor, U0126, significantly decreased AAI-mediated ROS. Preincubation of cells with thiol-containing compounds (NAC and GSH) inhibited the caspase 3 activity triggered by AAI, but non-thiol Tiron did not show a similar effect. This study demonstrated that AAI treatment results in oxidative stress-related DNA damage through GSH depletion and ERK1/2 activation; AAI-induced apoptosis is associated with GSH loss, but is independent of ROS generation.

Evaluation of nephrotoxic effects of mycotoxins, citrinin and patulin, on zebrafish (Danio rerio) embryos

Citrinin (CTN) and patulin (PAT) are fungal secondary metabolites which are found in food and feed and showed organotoxicity in mature animals. In this study zebrafish embryos were applied to investigate the developmental toxicity of CTN and PAT on embryonic kidney. In the presence of CTN and PAT, the gross morphology of kidneys from embryos with green fluorescent kidney (wt1b:GFP) was not apparently altered. Histological analysis of CTN-treated embryos indicated cystic glomerular and tubular lesions; a disorganized arrangement of renal cells was also found in the PAT-treated group. From the view point of renal function, dextran clearance abilities of embryos exposed to CTN and PAT were significantly reduced. The damaged renal function caused by CTN could be partially rescued by the administration of pentoxifylline, suggesting the reduction of glomerular blood flow contributes to CTN-induced renal dysfunction. Additionally, CTN induced the expression of proinflammation genes, including COX2a, TNF-α and IL-1β, but failed to modify the levels and distribution of wt1a transcript and Na(+)/K(+)-ATPase protein. In summary, CTN and PAT caused profound nephrotoxicity in histological structure and biological function of zebrafish embryos; the inflammatory pathway and blood rheology may involve in CTN-induced renal impairment.

Monitoring of microcystin-protein phosphatase adduct formation with immunochemical methods.

Using anti-microcystin-LR monoclonal antibodies, an immunoblotting procedure was developed to monitor the formation of microcystin-protein phosphatase adducts in vitro and in vivo. The detection limits for the covalent binding of MCYST-LR with the recombinant protein phosphatase 1 (PP1) and rabbit liver cytosol proteins were found to be 0.1 ng and 0.3 ng per assay, respectively. MCYST-PP1 adducts were detected 30 s after the addition of MCYST-LR into the reaction mixture. Reduction of the methyldehydroalanine (Mdha) residue of MCYST-LR with ethanethiol totally abolished the covalent binding of the toxin to PP1, but retained its inhibitory toxicity on PP1. Immunoblotting analyses and enzyme-linked immunosorbant assay showed that between 5 min to 16 h after i.p. injection of single dose (35 microg/kg) of MCYST-LR into mice, approximately 0-27% of the injected toxin was found covalently bound while 0.2-9.2% existed as free form in liver cytosol.

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.

Production of monoclonal antibody for okadaic acid and its utilization in an ultrasensitive enzyme-linked immunosorbent assay and one-step immunochromatographic strip.

Okadaic acid (OA) is a common marine biotoxin that accumulates in bivalves and causes diarrhetic shellfish poisoning (DSP). This study generated a monoclonal antibody (mAb) specific to OA from a hybridoma cell line, 6B1A3, which was obtained by fusion of myeloma cells (P3/NS1/1-AG4-1) with spleen cells isolated from a BALB/c mouse immunized with OA-γ-globulin. The 6B1A3 mAb belongs to the immunoglobulin G1 (κ chain) isotype. Both competitive direct and indirect enzyme-linked immunosorbent assays (ELISAs) were established for characterization of the antibody. The concentrations causing 50% inhibition of binding of OA-horseradish peroxidase to the antibody by OA were calculated to be 0.077 ng/mL in the cdELISA. A rapid and sensitive mAb-based gold nanoparticle immunochromatographic strip was also established. This proposed strip has a detection limit of 5 ng/mL for OA and can be finished in 10 min. Extensive analyses of 20 seafood samples with ELISA revealed that 10 were slightly contaminated with OA, with a mean concentration of 0.892 ng/g. Analysis of OA in shellfish samples showed that data acquired by the immunochromatographic strip agreed well with those acquired by the ELISA. The mAb-based ELISA and immunochromatographic strip assay developed in this study have adequate sensitivity and accuracy for rapid screening of OA in shellfish samples.