## 3 Results and Discussion
### Effects of BPA and Its Alternatives on HPC Axis
#### 3.2.4 Effects on Sex Hormones and Genes Related to HPG Axis  
The concentration and ratio of 17\(\beta\)-estradiol (E2) and testosterone (T) hormones have been widely used as integrated biomarkers for reproduction. In the studies summarized here, the adverse effects of BPA and its alternatives on hormone levels were sex-dependent, and a significant increase in E2 and decrease in T concentrations were observed in male zebrafish exposed to BPA, BPAF, BPF, BPS, and BPSIP (Figure 4). Interestingly, changes in sex hormones in male zebrafish were more sensitive to the toxicity of its alternatives than BPA. For example, male zebrafish exposed to BPS significantly increased E2 compared to the control group even at 0.5-1 ug/L [62; 64]. The decrease in T hormone was up to 200 times more sensitive than BPA for most alternatives (e.g., BPAF, BPF, BPS, and BPSIP) [62; 63; 64; 65; 66]. These results suggest that the endocrine disruption of BPA alternatives is no less than that of BPA and may significantly affect sex hormones in males. Figure 4: _Cont._Significant increases in aromatase (_cyp19_) genes are well supported by changes in the two sex hormones. CYP19 enzyme is involved in the final step in converting of T to E2 [72], and enzyme activities are generally well correlated with their mRNA levels [73]. _Cyp19a_ mRNA expression increased significantly in male zebrafish exposed to BPA [37; 56], BPAF [57], BPF [61], and BPS [62] (Figure 4). Interestingly, the adverse effects of BPA alternatives on gene transcription were sex-dependent, with males being more sensitive than females. In the HPG axis, gonadotropin-releasing hormone (GnRH) in the hypothalamus stimulates luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in teleosts [74]. Gonadotropin hormones in the pituitary regulate the synthesis of sex hormones, E2 and T. In terms of homeostasis, it is meaningful to examine the effects of BPA and its alternatives on the GnRH, LH, and FSH of zebrafish. Zebrafish possess the hormones GnRH2 and GnRH3, as well as four different GnRH receptors [75]. While there are few studies are measuring the GnRH hormone [46], it was frequently confirmed that the expression of _gnrh2_ or _gnrh3_ genes in zebrafish exposed to BPA, BPAF, BPF, BPS, and BPSIP was significantly increased (Table 3) [46; 48; 57; 61; 62; 65]. These results suggest that BPA analogs can directly or indirectly (e.g., a negative feedback action in the hypothalamus compensates for the reduced E2 production) affect GnRH. LH and FSH, which bind to specific receptors and induce gametogenesis, were also confirmed by measuring hormone or gene expression in zebrafish exposed to BPA and its alternatives. The \(\beta\)-subunit mRNA encoding LH and FSH were generally upregulated in male zebrafish exposed to BPAF [57], BPF [61], and BPS [62]. Two possibilities can be responsible for the decrease in the T hormone, despite a significant increase in gonadotropin hormone-related mRNA expression in male zebrafish exposed to BPA alternatives. The first option suggests that the rate of E2 production with the aromatase enzyme is higher than that observed with the T hormone. In contrast, the second option proposes the possibility of decreased expression of several genes involved in steroidogenesis (e.g., _star_, 17_\(\beta\)_lsqd_, and _cyp17_). The commonalities and differences in toxicity of BPA analogs have been explained based on their chemical structure [65; 76]. Bisphenols have a phenolic group in common. Figure 4: Lowest observed adverse effect level (LOAEL) of 178-estradiol (E2) hormone (**a**), testosterone (T) hormone (**b**), and _cyp19a_ mRNA expression (**c**) in male zebrafish exposed to BPA, BPAF, BPF, BPS, and BPSIP. The hydrophobic group of the propane moiety and the 4-hydroxyl group on the A-phenyl ring are suggested regulatory factors that can cause differences in the BPA analog toxicity [76; 77]. If the estrogenicity and anti-androgenicity of BPA are due to the phenolic ring, the endocrine disruption would be possessed even if BPA analogs are used as substitutes.