## 4 Discussion  
Several types of chemicals were widely used as HDs in Korea until the end of 2011 [28]. A number of studies have been conducted on pulmonary inflammation and fibrosis as a toxic mechanism of lung disease [4; 10]. However, information developmental toxicity by thyroid endocrine disruption is limited. The results of the present study demonstrated that Vegetable HD and PHMG-H affected the survival, development, production of THs, and the transcription of key genes involved in the HPT axis. The toxicity of Vegetable HD was lower than that of Oxy(r) and Wiselect HDs, which were mainly composed of PHMG-P [12]. This result was based on the endpoints of coagulation of the embryo, hatchability, larval survival, body length, concentrations of THs, and gene transcription. The two counter ions of PHMG may be responsible for the observed results. A previous study also reported that people exposed to HD containing PHMG-H did not suffer from HD-associated lung disease, even though the concentration used was much higher than that of PHMG-P [29]. Differences in gene transcription related to the HPT axis between PHMG-H and PHMG-P can explain the differences in toxicity. Further research is required to determine whether the ionic interactions of PHMG are related to toxicity. As numerous chemicals constitute a product, the toxicity of the entire product is generally greater than that of a single chemical [30]. However, in the present study, the toxicity of PHMG-H was greater than that of Vegetable HD in all observed endpoints. These results suggest that the chemical reactions of the components present in the HD product reduced the toxicity of PHMG-H. One explanation is that due to the reaction  
Figure 3: Transcription of genes related to the hypothalamus-pituitary-thyroid axis in zebrafish larvae after exposure to Vegetable HD (**A**) and PHMG-H (**B**) for 7 d. Fold change values of gene transcription are expressed as mean \(\pm\) standard deviation of four replicates. Asterisk indicates significant difference from the control group (\(p<0.05\)). among different chemicals, the guanidine group does not bind to the cell membrane, which is composed of neutral phospholipids [31]. Further studies are required to fully understand the important factors that determine the product toxicity. In teleost fish, TSH is the primary stimulatory hormone for the thyroid gland. Circulating levels of T3 and T4 are regulated by negative feedback through the HPT axis by altering the release of TSH from the pituitary [32]. TRH and corticotropin-releasing hormone (CRH) are the main stimulators of TSH [18]. While there has been extensive debate on the sensitivity of molecular biomarkers, the measurement of gene transcription has been generally accepted as a sensitive biomarker for endocrine disruption [33]. The level of _tsh\(\beta\)_ mRNA is well correlated with the production of TSH [34]. In the present study, Vegetable HD and PHMG-H significantly downregulated the _tsh\(\beta\)_ gene and increased the concentration of T3. These results suggest that Vegetable HD and PHMG-H can modulate the concentrations of TSH, which can subsequently affect the production of THs. The ratio of T3/T4 has been used as an indicator of TH homeostasis in the zebrafish larvae after exposure to endocrine disrupting chemicals [35]. A significantly greater T3/T4 ratio was observed in zebrafish larvae after exposure to PHMG-H, indicating that balance of THs was disrupted by this compound. Altered thyroid hormone balance could further result in adverse effects on development, growth, regulation of stress, and energy metabolism in fish [36]. T4 is converted into T3, bioactive form of TH, by deiodination [37]. Therefore, increased T3 levels are mostly due to either increase of T4 synthesis or increase of deiodinase (deio)-mediated conversion of T4 to T3 [38]. The finding of significant increase of T3/T4 ratio, based on the decrease of T4, suggests that deiodination could play a major role in the regulation of circulating T3 levels.