Effects Of Carbofuran&Chlorothalonil On Expression Pattern Of Tyrosine Aminotransferase And Mechanism Of Transcription Regulation In Zebrafish

There’re toxic effects to metabolism of carbohydrate, protein and lipid inorganisms produced by pesticides. The stress linked with pesticides is characterizedby alteration of hypothalamic–pituitary–adrenal (HPA) axis hormones, the autonomicnervous system, cytokines, and activation of immune-neuroendocrine axis. Thetyrosine aminotransferase （EC2.6.1.5， TAT） that is the first rate-limiting enzymein the catabolic pathway of tyrosine is involved in gluconeogenesis and has beendescribed in several organisms. The TAT transcription is regulated byglycometabolism-related hormones. The glucocorticoid and glucagon induce TATexpression, and the insulin depresses TAT transcription. As a target gene ofglucocorticoid and key enzyme of gluconeogenesis, the expression pattern of TATcould be affected by pesticides exposure, which would influence the carbohydratemetabolism.The full-length cDNA sequence of TAT gene in zebrafish (Danio rerio) wasobtained in the present study. Based on an investigation on pesticide residues invegetables sampled from markets for one year, pesticides including Carbofuran andChlorothalonil were employed to expose zebrafish due to the highest detection rates.Effects of pesticides on the TAT mRNA, protein and enzyme activity were evaluated,and the mechanism of TAT expression regulated by pesticides as well as the influenceon carbohydrate metabolism was also discussed in the present study.(1) The full-length cDNA sequence of TAT gene was cloned by RT-PCR andRACE techniques from zebrafish. The TAT cDNA contained an open reading frame（ORF） of1407bp encoding a deduced polypeptide of468amino acids with amolecular weight of51.7kDa. Sequence comparison revealed that the protein ofzebrafish TAT shared the highest identity of76.6-86%with other teleosts. The phylogenetic tree constructed by a NJ method showed that zebrafish TAT wasclustered together with fish TATs. The temporal and spatial pattern of TATexpression was analysed by Real-time PCR technique. It was found that TATtranscripts were most abundant in liver of adult zebrafish, and a significant increase ofTAT transcription occurred in hatched zebrafish fry during embryonic and larvaldevelopment. The similar expression pattern of zebrafish TAT to mammalianindicated conserved functions of TAT between species.(2) Total2983vegetable samples were examined to detect pesticide residuesfrom markets in one year. Carbofuran and chlorothalonil were detected in somesamples. At present, effects of carbofuran and chlorothalonil on expression andregulation of TAT genes are seldom studied. Therefore, they were employed forpesticide exposure in zebrafish.After exposed to carbofuran at concentrations of10,30,50,100,200and500μg·L-1for4h, the expression of TAT mRNA was significantly decreased atconcentration over30μg·L-1, revealing a dose-response relationship. After exposed tocarbofuran for1,2,4,8,12and24h at concentrations of50μg·L-1, the expression ofTAT mRNA was significantly decreased after treatment of4h, showing atime-response relationship. After exposed to chlorothalonil at concentrations of5、7.5、10、15、20and30μg·L-1for8h, the expression of TAT mRNAwas significantlydecreased at concentration over10μg·L-1without a dose-response relationship. Afterexposed to chlorothalonil for2,4,6,8,12and24h at concentrations of15μg·L-1, theexpression of TAT mRNA was significantly decreased after treatment of8h without atime-response relationship. After exposed to carbofuran at concentration of50μg·L-1for4h or to chlorothalonil at concentration of15μg·L-1for8h, the protein contentand enzyme activity of zebrafish TAT were significantly reduced with a degree ofabout50%compared with the control group. Results showed that the transcriptionactivity of TAT in zebrafish liver could be significantly inhibited by carbofuran andchlorothalonil, leading to a significant reduction in protein content and enzymeactivity of TAT.(3) Effects of growth hormone (GH) and thyroid hormone (T4) on theexpression of zebrafish TAT were evaluated. By real time PCR technique, it wasproved that growth hormone (GH) and thyroid hormone T4might stimulate theexpression of zebrafish TAT. A promoter sequence (1575bp) of zebrafish TAT gene was obtained. Three potential binding sites for STAT5were found in the sequence,suggesting that GH might regulate the expression of zebrafish TAT by STAT5.Effects of carbofuran and chlorothalonil on levels of glycometabolism-relatedhormones, GH, T4, blood glucose, hepatic glycogen and AChE were evaluated. Afterexposed to carbofuran at concentration of50μg·L-1for4h or to chlorothalonil atconcentration of15μg·L-1for8h, cortisol levels were significantly decreased, whilelevels of glucagon and insulin were increased. The carbofuran resulted in increases ofGH and T4, and no effect was observed by the chlorothalonil. The content of hepaticglycogen was significantly decreased, while no significant change in blood glucosewas detected. The activity of zebrafish AChE was not significantly inhibited bypesticides. Results showed that pesticide exposure reduced the level of glucocorticoidhormone and increased the insulin level in zebrafish, which was involved thedown-regulated expression of TAT mRNA. The reduction in protein content andenzyme activity of TAT might inhibit the gluconeogenesis in zebrafish liver, but showno effect on blood glucose.