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ABSTRACT: 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.
Food and chemical toxicology: an international journal published for the British Industrial Biological Research Association 07/2012; 50(12):4398-4404. · 2.99 Impact Factor
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ABSTRACT: 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.
Toxicology in Vitro 02/2011; 25(4):810-6. · 2.78 Impact Factor
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ABSTRACT: Aristolochic acid I (AAI) is a phytotoxin that has been found in various herbal remedies and linked to the development of human carcinogenesis. To investigate the playing role of AAI in the function of macrophages, lipopolysaccharide (LPS)-stimulated macrophage cells RAW264.7 were employed as a model to examine the effect of AAI on the expression of the inducible nitric oxide synthase (iNOS) gene. AAI reduced the expression of iNOS mRNA and protein, as well as the production of NO in LPS-stimulated macrophages. Treatment of transfected macrophages with AAI effectively suppressed the luciferase activities of the iNOS promoter which is activated by LPS. The results of promoter deletion and electrophoretic gel mobility shift assay (EMSA) indicated that the NF-κB binding site at nucleotides -86 to -76 was the major site that was most responsible for the inhibitory effect of AAI. Moreover, the presence of AAI substantially reduced the phosphorylation of the inhibitory κBα (IκBα) protein in LPS-stimulated cultures. AAI also down-regulated the LPS-induction of TNF-α, a NF-κB regulated gene. On the other hand, AAI did not modulate the luciferase activities of reporter construct that contained iNOS mRNA 3'-UTR. Taken together, the data herein suggest that in activated macrophages, AAI effectively down-regulated the expression of iNOS gene by interfering with the activation of NF-κB at the transcription level. The stability of iNOS mRNA was not the target of AAI inhibition.
Toxicology Letters 02/2011; 202(2):93-9. · 3.23 Impact Factor
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ABSTRACT: 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.
Toxicology Letters 12/2008; 183(1-3):105-11. · 3.23 Impact Factor
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ABSTRACT: 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.
Toxicological Sciences 03/2007; 95(2):340-7. · 4.65 Impact Factor
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ABSTRACT: Patulin (PAT), a mycotoxin mainly produced by Penicillium and Aspergillus, is frequently detected in moldy fruits and fruit products. Exposure of human embryonic kidney (HEK293) cells to PAT led to a dose- and time-dependent increase in the phosphorylation of two major mitogen-activated protein kinases (MAPKs), p38 kinase and c-Jun N-terminal kinase (JNK). The phosphorylated forms of MAPK kinase 4 (MKK4), c-Jun, and ATF-2 were also seen in PAT-treated cultures. The cell death caused by PAT was significantly reduced by the p38 kinase inhibitor, SB203580, but not by the JNK inhibitor, SP600125. Neither p38 kinase nor JNK played a role in the PAT-induced DNA damage. In PAT-treated cells, inactivation of double-stranded RNA-activated protein kinase R (PKR) by the inhibitor, adenine, markedly suppressed JNK and ERK phosphorylation. Treatment of HEK293 cells with PAT-cysteine adduct, a chemical derivative of PAT, showed no effect on MAPK signaling pathways, cell viability, or DNA integrity. These results indicate that PAT causes rapid activation of p38 kinase and JNK in HEK293 cells, but only the p38 kinase signaling pathway contributes to the PAT-induced cell death. PKR also plays a role in PAT-mediated MAPK activation.
Toxicological Sciences 03/2006; 89(2):423-30. · 4.65 Impact Factor
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ABSTRACT: Patulin (PAT), a mycotoxin produced by certain species of Penicillium and Aspergillus, is often detectable in moldy fruits and their derivative products. PAT led to a concentration-dependent and time-dependent increase in phosphorylation of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) in human embryonic kidney (HEK293) cells, human peripheral blood mononuclear cells (PBMCs), and Madin-Darby canine kidney (MDCK) cells. Exposure of HEK293 cells to concentrations above 5 microM PAT for 30 min induced ERK1/2 phosphorylation; activation of ERK1/2 was also observed after 24 h incubation with 0.05 microM of PAT. Treatment of human PBMCs for 30 min with 30 microM PAT dramatically increased the phosphorylated ERK1/2 levels. Both MEK1/2 inhibitors, U0126 and PD98059, suppressed ERK1/2 activation in either HEK293 or MDCK cells. In HEK293 cells, U0126-mediated inhibition of PAT-induced ERK1/2 phosphorylation resulted in a significant decrease in levels of DNA damage, expressed as tail moment values, in the single cell gel electrophoresis assay. Conversely, U0126 did not affect cell viability, lactate dehydrogenase release, and the DNA synthesis rate in PAT-treated cultures. Exposure of HEK293 cells for 90 min to 15 microM PAT elevated the levels of early growth response gene-1 (egr-1) mRNA, but not of c-fos, fosB, and junB mRNAs. These results indicate that in human cells, PAT causes a rapid and persistent activation of ERK1/2 and this signaling pathway plays an important role in mediating PAT-induced DNA damage and egr-1 gene expression.
Toxicology and Applied Pharmacology 10/2005; 207(2):103-11. · 4.45 Impact Factor
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ABSTRACT: Monascus purpureus and its fermentation products have been used in food coloring and meat preservation in Asia for centuries and have also been recently used as dietary supplements because of their cholesterol-lowering ability. However, the presence of the mycotoxin citrinin (CTN), a secondary metabolite of Monascus species, in fermentation products is a potential threat to public health. In the present study, HPLC was used to analyze CTN levels in lipid and aqueous extracts of commercialized Monascus products. CTN was detected in lipid extracts of all examined samples at concentrations varying between 0.28 and 6.29 microg/g, but was not found in aqueous extracts. When human embryonic kidney cells (HEK293) were incubated for 72 h with Monascus extracts, the concentrations causing 50% cell death by all lipid extracts were in the range of 1.8-4.7 mg/mL, whereas aqueous extracts showed a lower cytotoxicity. Incubation of HEK293 cells with 60 microM pure CTN for 72 h caused cell viability to fall to 50% of control levels. In addition, coadministration of pure CTN and lipid extracts from Monascus samples significantly enhanced CTN cytotoxicity for HEK293 cells using the MTT assay. These results provide the first information about the cytotoxic effects of various Monascus samples and CTN-Monascus mixtures on a human cell line.
Journal of Agricultural and Food Chemistry 02/2005; 53(1):170-5. · 2.82 Impact Factor
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ABSTRACT: Polyclonal antibodies for domoic acid were generated from rabbits after the animals had been immunized with either domoic acid-keyhole limpet hemocyanin (KLH) or domoic acid-bovine serum albumin (BSA). A competitive direct enzyme-linked immunosorbent assay (cdELISA) and a competitive indirect ELISA (ciELISA) were used for the characterization of the antibodies and for analysis of domoic acid in blue mussels and clams. The antibody titers in the serum of rabbits immunized with domoic acid-KLH were considerably higher than those in rabbits immunized with domoic acid-BSA. The antibodies from the rabbits immunized with domoic acid-KLH were further characterized. In the cdELISA, the concentrations causing 50% inhibition (IC(50)) of binding of domoic acid-horseradish peroxidase to the antibodies by domoic acid and a domoic acid analogue, kainic acid, were found to be 0.75 and 200 ng/mL, respectively. In the presence of blue mussel matrix, the detection limit of domoic acid was <25 ng/g. The overall analytical recovery of domoic acid (25-500 ng/g) added to the blue mussels and then extracted with 50% aqueous methanol in the cdELISA was found to be 81.1%. The efficacy of cdELISA was also confirmed by the high-performance liquid chromatography method. Analysis of domoic acid in shellfish samples showed that 10 of the 15 shellfish examined were contaminated with domoic acid at levels of <50 ng/g.
Journal of Agricultural and Food Chemistry 08/2004; 52(17):5334-9. · 2.82 Impact Factor
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ABSTRACT: 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.
Toxicology and Applied Pharmacology 10/2003; 191(3):255-63. · 4.45 Impact Factor
