## 3 Results and Discussion
### Effects of BPA and Its Alternatives on HPT Axis
#### 3.3.2 Effects on Thyroid Hormones and Genes Related to HPT Axis  
Similar to the HPG axis, the thyrotropin-releasing hormone (TRH) with the corticotrophic-releasing hormone (CRH) of the HPT axis secrete thyroid-stimulating hormone (TSH) from the hypothalamus [89]. TSH secreted by the pituitary then regulates the synthesis of thyroid hormone (TH), that is thyroxine (T4) and triiodothyronine (T3). Measurement of thyroid hormones is commonly used in zebrafish, as it is the most integrated endpoint in assessing thyroid endocrine disruption [90]. Zebrafish exposed to BPA [80; 83], BPAF [84], BPF [81; 83], and BPS [83; 86; 87] were affected by either activating or suppressing hormone production. Interestingly, significant decreases in T4 concentrations along with increases in TSH concentration were commonly observed in fish exposed to BPF and BPS [81; 87]. Moreover, changes in thyroid hormones were more sensitive to the toxicity of BPS than BPA (Figure 5). These results suggest that the thyroid endocrine disruption of BPA alternatives is no less than that of BPA and may significantly affect on thyroid hormone homeostasis. Decreases in T4 could reduce the TH availability in target tissues and subsequently influence metabolic pathways [78; 84].  
Figure 5: _Cont._TSH has been measured to assess the potential mechanism of thyroid dysfunction. TSH levels increased significantly after BPA analog exposure [81, 87], which might be involved in the negative feedback mechanism. Significant up-regulation of tsh\(\beta\) genes is also well supported by changes in TSH levels. TRH and CRH stimulate TSH secretion, and transcription of trh and crh genes were significantly upregulated after exposure to BPAF, BPF, and BPS [81, 82, 83, 86, 87]. The results of these studies suggest that the increase in TSH concentrations and upregulation of trh and crh caused by bisphenol exposure may be attributed to the promotion of thyroid hormone synthesis and its subsequent release to compensate for the decreased T4 levels in zebrafish.  
The transcription of various genes in the HPT axis has been measured in zebrafish exposed to BPA, BPAF, BPF, BPS, and BPZ. Iodothyonine deiodinases (Dio) are the key regulators of T4 and T3 bioavailability [91], and three types of dio enzymes (Dio1, Dio2, and Dio3) and genes (dio1, dio2, and dio3) have been found in zebrafish [92]. Dio2 enzyme plays a vital role in catalyzing the conversion of T4 into biologically active T3 [91]. Significant increases in dio2 mRNA expression in zebrafish exposed to BPAF [82, 84], BPF [81], and BPS [86] have been reported, and an increase in T3 and a decrease in T4 were observed together in some studies [81, 86]. These results suggest that BPA analogs potentially affect dio2 gene transcription and thyroid hormone production.  
Overall, the results demonstrated that BPA analogs significantly changed thyroid hormone concentrations and modified the mRNA expression of key genes involved in the HPT axis, suggesting thyroid endocrine disruption in zebrafish. BPA analogs and thyroid hormones' chemical structures have similarities; therefore, these chemicals exhibit the property of binding to thyroid receptors and competing with the thyroid hormone [93]. Since endocrine systems interact with each other, further studies are needed to assess BPA alternatives' primary target among the endocrine axes.