Endocrine Disrupting Effects Of Three Triazole Fungicides On Zebrafish (Danio Rerio)

Triazole fungicides represent the most important category of fungicides. They are applied on a number of crops such as rice, fruits and vegetables because of their broad-spectrum antifungal activity. Their antifungal activity is due to their ability to inhibit the P450enzyme (CYP51), which blocks the conversion of lanosterol to ergosterol causing disruption of fungal wall. But the inhibition potency of these triazole fungicides is not limited to fungi, they may also inhibit other P450-mediated activities resulting in various adverse effects. Several triazoles have been reported to alter the concentrations or genes transcription involved in steroid homoeostasis and show endocrine disrupting effects. This thesis took advantage of zebrafish model to investigate the endocrine disrupt effects of three triazole fungicides (tebuconazole, hexaconazole and difenoconazole). The largest used triazole fungicide in China is tebuconazole, hexaconazole is persistent in environment and the difenoconazole was registed in China in recent.The results of adult fish acute toxicity test showed that LC50(96h) values of tebuconazole, hexaconazole and difenoconazole were18.4,4.48and2.3mg/L respectively. These results indicated that tebuconazole was lowly toxic to adult fish which hexaconazole and difenoconazole were moderately toxic. The results of embryo acute toxicity test showed that LC50(96h) values of tebuconazole, hexaconazole and difenoconazole were10.03,7.07and1.7mg/L respectively, which indicated that embryos are more sensitivity than adult. Zebrafish embryos exhibited malformations of uninflated swim bladder, notochord deformation, pericardial edema and yolk sac edema after being exposed to various triazoles from6to120h post-fertilization (hpf). But only difenoconazole have significant teratogenic effect.In the the present study, thyroid hormone levels and the expression of related genes in the hypothalamic-pituitary-thyroid (HPT) axis were investigated in zebrafish embryos exposure to various triazoles until120hpf. After exposure, T3levels and most of the HPT related genes were increased, while hexaconazole and difenoconazole exposure decreased T4levels. That indicate all three triazoles are thyroid hormone disrupting chemicals. It seemed that those triazoles could induce the transcription of transthyretin (TTR), uridine diphosphate glucuronosyltransferase (UGTlab), thyronine deiodinase (Diol and Dio2) genes. This might increase T4elimination and the ratio of T4conver to T3, led to the reduction in the levels of circulating T4and increased T3levels. The upregulation of corticotrophin-releasing hormone (CRH), thyroid-stimulating hormone (TSHP) and sodium/iodide symporter (NIS) gene transcription could possibly be explained as a compensatory mechanism to the T4reduction. The upregulation of thyroid hormone receptors (TRa and TRβ) might due to the increased T3levels and would influence the transcription of other genes involved in thyroid function.The potential effects of various triazoles on hypothalamus-pituitary-gonad (HPG) axis of zebrafish were assessed in the present study. Plasma sex steroid levels were examined by ELISA in male adult zebrafish following a21-day exposure to various triazoles. To further understand the estrogenic response of triazoles, gonadosomatic index (GSI), hepatosomatic somatic index (HSI), the expression levels of estrogen receptor genes (ERa, ERβ1, and ERP2) and the vitellogenin genes (Vtg1and Vtg2) from the livers, the P450aromatase gene (cyp19alb) from the brains were examined. Tebuconazole, hexaconazloe and difenoconazole exposure reduced plasma concentrations of T and increased plasma levels of E2, the GSI were decreased while exposed to tebuconazole, all these indicated tebuconazole, hexaconazole and difenoconazole had estrogenic activities. Real-time reverse transcriptase polymerase chain reaction assay showed that three triazoles significantly increased hepatic mRNA expression of Vtgl and Vtg2in male zebrafish in a dose-dependent manner, Vtgl was more senstivity than Vtg2. Consistent with Vtg, hexaconazole and difenoconazole exposure could induce hepatic mRNA expression of estrogen receptor genes:ERa and ERβ1, and ERa was more senstivity than ERβ1. In addition, CYP19b mRNA expression in the hypothalamus were upregulated after various triazoles exposure. Our study indicated that hypothalamus CYP19b is the target of those three triazoles. Thus, we hypothesize that various triazoles treatment elevates the conversion of T into E2and thus causes the significant elevation of E2levels.