Ting-Shuan Wu

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.

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.

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.

Cardiotoxicity of Mycotoxin Citrinin and Involvement of MicroRNA-138 in Zebrafish Embryos

Citrinin (CTN) is a fungal secondary metabolite that contaminates various foodstuffs and animal feeds; it also exhibits organotoxicity in several animal models. In this study, the zebrafish was used to elucidate the mechanism of CTN cardiotoxicity in developing embryos. Following CTN administration, the gross morphology of the embryonic heart was apparently altered, including heart malformation, pericardial edema, and red blood accumulation. Whole-mount immunostaining and histological analysis of ventricle and atrium indicated incorrect heart looping and reduced size of heart chambers. From the perspective of cardiac function, the heartbeat and blood flow rate of embryos were significantly decreased in the presence of CTN. CTN also modulated the expression of tbx2a and jun B genes, but not that of bmp4 and nkx2.5. Furthermore, the heart areas of CTN-exposed embryos demonstrated an elevated levels of aldh1a2 and cspg2 messenger RNA; these 2 cardiac-related genes are known to be involved in retinoic acid (RA) pathway as well as downstream targets of microRNA-138 (miR-138) in zebrafish. CTN treatment also downregulated the expression of miR-138. Moreover, overexpression of miR-138 was able to rescue the heart defects generated by CTN. These results support the notion that CTN exposure has a severe impact on heart development, affecting heart morphogenesis through the dysregulation of miR-138, RA signaling, and tbx2a.