The effect of Citrinin and Patulin on development of heart and kidney on zebrafish embryos

Autor: Ting-Shuan Wu, 吳亭萱
Rok vydání: 2013
Druh dokumentu: 學位論文 ; thesis
Popis: 101
Citrinin (CTN) and Patulin (PAT) are secondary metabolites secreted by fungi that frequently contaminate food and feed. The organotoxicity of CTN and PAT has been reported in several animal models, and kidney is the major target organ of these two toxins. However, their early developmental toxicities to embryos remain unclear. Since, zebrafish shows the characteristics of embryo transparency, rapid development, and similarity to human, the zebrafish embryo was used as a vertebrate model to study the developmental toxicity of CTN and PAT in the present study. In the CTN study, the LC 50 values of embryos at 6-72 hpf and 24-72 hpf were found to be 57.9 and 60.4 μM, respectively, after exposed to CTN. When the embryos of Tg (wt1b:GFP), a transgenic line with fluorescent nephrons, were exposed to 50 μM CTN, the gross morphology of glomeruli and pronephric tubules was not changed. Further histological examination of 50 μM CTN-treated embryos revealed the disorganization of cell arrangement and the presence of cysts in the glomeruli and tubule. The glomerulus filtration rate, an indicator of renal function, was significantly decreased in 7.5 μM CTN-treated group. The co-administration of pentoxifylline, a renal medicine improving blood flow, was able to restore the renal dysfunction induced by CTN. From the viewpoint of molecular level, 50 μM CTN induced the mRNA expression of proinflammatory genes, including COX2, TNFα, and IL-1β; however, the expression and distribution of glomerular marker gene wt1a and the pronephric tubule functional protein Na+/K+ATPase were not altered. On the other hand, CTN at 50 μM resulted in various cardiotoxic phenomena, including abnormal heart tube looping, decreased chamber volume, pericardium edema and erythrocyte accumulation. Additionally, the heartbeat and blood circulation of embryos were significantly decreased with the increasing CTN concentrations, but the gross number of erythrocytes was unaffected. CTN also down-regulated the mRNA levels of tbx2a and up-regulated that of junB in 72 hpf embryos. The co-presence of heart medicine metoprolol with CTN restored the mRNA levels of tbx2a and junB, recovered the heartbeat rate, and rescued the CTN-induced lethality. Further evidence demonstrated that CTN exposure increased aldh1a2 and cspg2 mRNA levels in the heart areas of 72 hpf embryos. Both aldh1a2 and cspg2 genes are known to play important roles in the retinoic acid (RA) signaling pathway and served as the target genes of microRNA-138 (miR-138). The expression of miR-138 was also significantly decreased in the CTN-treated heart. Microinjection of the miR-138 mimic into embryos at 1-2 cell stages was able to reduce the proportion of 72 hpf embryos with cardiac defects which were caused by CTN treatment. When 6 hpf zebrafish embryos were treated with nephrotoxin PAT till 120 hpf, the LC50 value was found to be 176.8 μM. The PAT concentration as high as 200 μM did not affect the kidney morphology of 24-72 hpf Tg (wt1b:GFP), but the disorganization of cell arrangement was found in the glomeruli of embryos by histological examination. In addition, the glomerular filtration rate of 72 hpf embryos was significantly decreased after 50-100 μM PAT treatments. Co-administration of mannitol, a clinical medicine with osmosis modulation ability, was able to repair the renal function damaged by PAT. However, PAT exposure did not cause any evident heart defect, no matter from the perspectives of morphology, histology and cardiac function. PAT treatment also showed no effect on the development of jaw cartilage in 120 hpf embryos. In conclusion, treatment of zebrafish embryos with CTN led to developmental cardiotoxicity and nephrotoxicity. CTN exerts part of its adverse effects on developing heart through suppressing miR-138 expression, subsequently promotes RA-related aldh1a2 and cspg2 activities and attenuates tbx2a levels. Moreover, heart defects and decreased blood circulation may be the cause of pro-inflammatory genes (COX2, TNFα and IL-1β) activation which results in decreasing renal function. On the other hand, PAT was able to cause developmental nephrotoxicity, but not cardiotoxicity, in zebrafish embryos. This study is the first time to demonstrate the developmental toxicity of CTN and PAT by applying a zebrafish model. Results collected herein could provide a solid base to evaluate the adverse effect of mycotoxin on the general public, including pregnant women and developing individuals.
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