Effects of alternative flame retardants, TBB and TBPH on thyroid hormone system in zebrafish (Danio rerio) larvae and GH3 and FRTL-5 cell lines

Abstract

TBB (2-ethylhexyl-2,3,4,5-tetrabromobenzoate) and TBPH (bis(2-ethylhexyl)-2,3,4,5-tetrabromophthalate) are major components widely used as alternative flame retardants in several commercial products such as Firemaster® 550. Both have been detected in various environments such as indoor dust, atmosphere, municipal sewage, sediment, and even in wildlife. However, available information on their endocrine disrupting potential as well as toxicological mechanisms is limited. Therefore, we investigated the adverse effects of each compound on endocrine system, especially thyroid system. Zebrafish (Danio rerio) embryos/larvae were exposed to TBB (0, 0.05, 0.5, 5, or 50 mg/L) and TBPH (0, 0.05, 0.5, 5, or 50 mg/L), respectively until 144 hours post-fertilization (hpf) in order to observe the effects on both developmental and molecular endpoints. Significant differences in developmental toxicity including hatchability (%), time-to-hatch (day), survival (%), wet weight (mg), and malformation rate (%) compared to control group were not observed. However, thyroid hormones (THs

T3 and T4) and gene transcriptions involved in TH system were altered in zebrafish larvae. THs play crucial roles in regulating many physiological processes such as development and metabolism. Trends in T3 following TBB exposure increased while both T4 and T3 following TBPH exposure decreased. Change in T3 to T4 ratio (T3/T4) was only observed in TBB exposure with increasing trend. In gene analysis, most TH related genes were up-regulated following exposure to TBB while any significant changes were not observed following TBPH exposure. To elucidate the mechanism of each compound, GH3 (rat pituitary tumor cells) and FRTL-5 (rat thyroid follicular cells) were employed for assessment of thyroid hormone disruption. In GH3 cells, exposure to TBB (0, 0.05, 0.5, 5, or 50 mg/L) and TBPH (0, 0.1, 1, 10, or 100 mg/L) down-regulated the mRNA expression of tshβ, especially significant at 0.1, 1, 10, and 100 mg/L of TBPH exposure. Also, significant down-regulation of dio2 gene expression at the highest concentration of TBB (50 mg/L) exposure was observed. In general, transcriptional level of trα was significantly decreased following both TBB and TBPH exposure, while that of trβ and dio1 was not significantly altered. In FRTL-5 cells, exposure to TBB (0, 0.5, 5, or 50 μg/L) and TBPH (0, 0.01, 0.1, or 1 mg/L) altered several gene transcription involved in THs synthesis and thyroid development. Transcription of nis and nkx2.1 was significantly decreased at the highest concentration of TBB (50 μg/L). Also, significant decrease in tg was observed following exposure to TBB (0.5, 5, or 50 μg/L). In case of TBPH exposure, significant down-regulation of nkx2.1 was observed at 0.01, 0.1, or 1 mg/L of TBPH. As a result, TBB and TBPH changed hormonal and transcriptional levels related to TH in zebrafish, GH3, and FRTL-5 cells. Even if there were different responses between the organism and cell lines, changes in THs level and related gene expression demonstrated TBB and TBPH as potential endocrine disruptors. In zebrafish larvae, TBB and TBPH exposure did not significantly change developmental endpoints while causing alterations in THs levels and gene transcription. In GH3 and FRTL-5 cell assay, significant changes in gene transcription involved in THs synthesis, transportation, or metabolism were observed. Collectively, changes in molecular levels such as hormones and genes may help predict potential health effects on organismal levels. Therefore, this study will contribute to the general understanding of thyroid hormone disruption and related mechanism by TBB and TBPH.