## 4 Discussion  
To understand the effects of two NBFRs on sex hormones, male zebrafish for in vivo and H295R and MVLN cells for in vitro were employed in this study. Both in vivo and in vitro results suggest a line of evidence supporting sex endocrine-disruption by DBDPE and BTBPE. Results of male zebrafish and H295R cells showed that exposures to two NBFRs increased E2 levels partly through the transcriptional changes of steroidogenic genes. These observations are in similar agreement with some PBDEs such as deca-BDE and octa-BDE reported elsewhere [5; 6; 7]. Sex hormones play critical roles in sexual development, such as gonad gametogenesis and reproductive systems [42]. In the present observation, in male zebrafish, DBDPE and BTBPE exposure resulted in enhanced E2 levels and E2/11-KT ratios. The observations from H295R cells also showed a similar trend despite the fact that statistical significance was not evident. These results indicate that levels of sex hormone exposed to DBDPE and BTBPE might be mediated by mechanisms other than the alteration of the steroidogenesis pathway, e.g., metabolic activation from other systems [43] or hepatic metabolism and excretion [44]. Similarly, a previous study showed significant alteration of sex hormones after exposure to DE-71 in zebrafish [45], but another study reported no effects in H295R cell after DE-71 exposure [46]. In H295R cells, key genes that are translated to the enzymes related to sex hormone synthesis (_STAR_, _3_BHSD_, _CYP17_, and _CYP19_) were analyzed to explain the weak E2 increase (Figure 4). The _STAR_ gene regulates the transfer of cholesterol into the inner mitochondrial membrane [46]. _3_BHSD_ and _CYP17_ genes are translated to enzymes for steroidogenesis and androgen synthesis. The _CYP19_ gene, which is translated to aromatase, is responsible for a conversion of T to E2. In our study, transcription levels of all target genes in H295R cells after exposure to DBDPE did not change despite the increasing trend in the E2/T ratio. In a previous study, a few out of 19 different PBDEs showed the alteration in aromatase activity in H295R cells [47]. Following the exposure to BTBPE, up-regulations of _3_BHSD_ and _CYP19_ genes were observed, although levels of E2 were only slightly increased. These results were in correspondence with a previous paper that reported no significant change in sex hormone after the exposure to octa-BDE mixture despite an increase in aromatase activity [46]. However, our result of transcriptional levels in H925R cells are not comparable to the upregulation of E2 levels in male zebrafish, but the reasons for this discrepancy are not readily available. Figure 4: Transcriptional changes of (**A**) _STAR_, (**B**) _3_BHSD_, (**C**) _CYP17_, and (**D**) _CYP19_ genes in H295R cells exposed to DBDPE and BTBPE. The results are shown as mean \(\pm\) SD of triplicates (\(n=3\)). Asterisk indicates a significant difference in transcriptional level of treatment groups relative to the solvent control (0.1% DMSO, \(p<0.05\)). The value of \(p\) for trend was determined based on the linear regression analysis. If \(p\) for trend was \(<0.05\), a linear trend is indicated with a red dotted line. In MVLN cells exposed to DBDPE and BTBPE, ER-binding affinity was not observed while significant increases of \(er\alpha\) and \(er\beta\) gene expressions were observed in zebrafish liver. Our results on the MVLN cell line assay demonstrated that DBDPE and BTBPE may not bind directly with the ER. Likewise, a previous study reported that PBDE congeners such as BDE-209 did not alter E2-induced ER in MVLN cells [48]. Regardless of the increase in E2 levels or alteration of \(er\alpha\) and \(er\beta\) gene expression, transcription of \(vtg\) gene after exposure to DBDPE and BTBPE was not altered in the fish. Vitellogenin has been a well-known indicator of endocrine disruption in male fish [49; 50]. It is developed in the liver of female fish by E2 signal and is an essential protein for oocyte maturation and reproduction in female fish.