Hexabromocyclododecane (HBCD) is a representative of alicyclic brominated flame retardan ts (CBFRs), which have been frequently found in environments and living organisms. HBCD p ossesses all the char a cterics of persistant organic pollutants and was listed in Annex A (POP for elimination) under the Stockholm Convention on Persistent Organic Po llutants in May 2013. T he c ommercially available HBCD (technical mainly consists of α --, β --, and γ HBCD It has been evidenced that d istribution in the environmental media , bioaccumulation, metabolism, food chain
amplification, and toxic effect s of HBCD are diastereoisomer selective. Therefore, it is necessary to investi gate the toxicities of HBCD at the level of individual isomers in order to evaluate its environmental safety and health risks. I t has been reported that HBCD can cause potential neurotoxicity however, there has been no study to address their diastereoisom er specific neurotoxicity so far. 2 dibromo 4 --(1,2 dibromoethyl) cyclohexane (TBECH) and 1,2,5,6 tetrabromocyclooctane (TBCO), with the same structures and properties as HBCD, were regarded as the potential alternatives of HBCD, which could have major imp lications for their significant increases in production, application and environmental accumulation in the near future. It is essential to assess the environmental and health effects of these potential alternatives .However, the understanding of neurotoxic ities of TBECH and TBCO remains lacking. Therefore, this dissertation includes the following parts of studies.
The neurotoxicit ies of α --, β --, and γ HBCD in SH SY5Y human neuroblastoma cells were investigated . The r esults showed that the HBCD diastereoisomers decreased cell viability, increased lactate dehydrogenase (LDH) release, and impaired cytoskeleton development. Typical morpholo gical features and apoptosis rates showed that all the HBCD diastereoisomers induced SH SY5Y cell apoptosis. The expression levels of several cell apoptosis related genes and proteins, including Bax, caspase 3,caspase 9, cytochrome c (Cyt c), Bcl 2, and X linked inhibitor of apoptosis (XIAP),as well as the cell cycle arrest, DNA damage, and adenosine triphosphate (ATP)consumption, were examined. The results showed that the HBCD diastereoisomer icneurotoxicity was ranked β HBCD > γ HBCD > α HBCD. The cell apoptosis and caspase expression levels of the three HBCD diastereoisomers followed the same order, suggesting that caspase dependent apoptosis may be one of the mechanisms responsible for the st ructure selective HBCD diastereoisomer neurotoxicity. To further explore the mechanisms of cell cytotoxicity, HBCD diastereoisomer induced reactive oxygen species (ROS) and the intracellular calcium Ca 2+2+) levels were examined. The levels of intracellular Ca 2+ and ROS increased significantly. The ROS levels followed the order β HBCD > γ HBCD > α HBCD, which is the same as their toxicities, whereas those of intracellular Ca 2+ were γ HBCD > β HBCD > α HBCD. Thus, ROS may be a key factor regulating the neurotoxici ty of HBCD diastereoisomers. To the best of our knowledge, this is the first study to report on the diastereoisomer specific toxicity of HBCD in human neural cells and on the possible mechanisms responsible for the selective neurotoxicity of HBCD diastereo isomers.
The neurotoxicity effects of HBCD, TBECH, and TBCO on human SH SY5Y cells were compared. The r esults showed that HBCD, TBECH, and TBCO induced cytotoxicit ies , including dose dependent cell viability decreases, cell membrane permeability increases , cell cytoskeleton development damage, and cell apoptosis induction. The order of toxicity was HBCD > TBCO > TBECH. The cell apoptosis and caspase (Bax, caspase 3, caspase 9, C yt c, Bcl 2, and XIAP) expression levels of HBCD, TBECH, and TBCO followed the same order, suggesting that caspase dependent apoptosis may be one of the mechanisms responsible for the neurotoxicities of HBCD, TBECH, and TBCO. To further explore the mechanisms of cell cytotoxicity, the levels of intracellular ROS and Ca 2+ induced by th e exposures to HBCD, TBECH, and TBCO were examined. The ROS levels w ere significantly elevated for all the exposures, and t he presence of antioxidant N acetyl L cysteine (NAC) significantly reduced the cytotoxicity The intracellular Ca 2+ concentration was significantly increased for the exposure to HBCD or TBCO, but not for TBECH treatment. The presence of Ca 2+ inhibitor BAPTA AM significantly reduced the cytotoxicit ies induced by HBCD and TBCO. The se observations suggested that oxidative stress contribute d to the cytotoxicit ies of HBCD, TBECH, and TBCO, and HBCD and TBCO m ight also induce Ca 2+ mediated apoptosis.
In summary diastereoisomer specific neurotoxicity of HBCD in human SH SY5Y neuroblastoma cells was investigated Moreover, the neur otoxicity of HBCD and its potential alternatives, TBCO and TBECH, were compared. This study gained some insight s into the structure dependent cytotoxicity for different CBFRs,provid ing valuable basis for the risk assessment of CBFRs.