Project title: Neurotoxicity of Polybrominated Dipheyl Ether (PBDE) Congeners in Mice Cerebellar Granule Neurons
Completed in: 2009 | Faculty advisor: Lucio G. Costa
Polybrominated diphenyl ethers (PBDEs) are a class of brominated flame retardants extensively used in a variety of consumer products. In the last 30 years, uses of PBDEs increased dramatically worldwide, as a result, levels of PBDEs found in the environment, animal and human tissues have also increased dramatically. PBDEs are persistent in the environment, bioaccumulate and biomagnify through the trophic levels. Infants and toddlers have the highest body burden, due to exposure via breast milk and house dust, during critical developmental stages. Animal studies have shown that PBDEs can cause developmental neurotoxicity, although the mechanisms are unknown. This study examined and compared the cytotoxicity of five environmentally relevant PBDE congeners: 47, 99, 100, 153, and 209. These congeners are the most commonly found in the environment and in humans at higher levels than any other congener. Previous studies have shown PBDEs to cause oxidative stress and apoptotic cell death in neuronal in vitro; however, no report exists on direct comparison of congener-specific cytotoxicity. Primary cereballar granule neurons (CGNs) cell cultures were used to establish dose-response curves of BDE-47, -99, -100, -153, and -209 for inhibition of cell viability, induction of apoptosis, reactive oxygen species (ROS) formation and lipid peroxidation. All congeners tested concentration dependent reduction in cell viability, assessed by measuring MTT reduction. Up to 96% of cytotoxicity was caused by apoptotic mechanisms, assessed by Hoechst 33258 staining. Comparing the ability of each congener to reduce cell viability and induce apoptosis, the order of cytotoxicity is BDE-100 > BDE-47 > BDE-99 > BDE-153 and > BDE-209. Additionally, all five congeners were observed to cause oxidative stress with increased ROS formation and lipid peroxidation in a concentration dependent manner. Corresponding to the order of cytotoxicity, the ability of each congener to induce oxidative stress is also BDE-100 > BDE-47 > BDE-99 > BDE-153 and > BDE-209. This suggests oxidative stress and apoptosis as possible mechanisms of cytotoxicity of PBDEs. Furthermore, addition of antioxidants, melatonin and glutathione monoethyl ester (GSHEE), reduced PBDEs toxicity observed by reduced induction of apoptosis, ROS formation, and lipid peroxidation. Lastly, intracellular accumulation of the five PBDE congeners was studied using a magnetic bead ELISA kit after 24 hr exposure. Exposure to 1 and 10 uM of BDE-47, -99, -100, -153, and -209 caused a concentration-dependent PBDE accumulation in cells. At 10 uM, intracellular concentration of PBDEs associated with cells was 200-times that of the applied dose for BDE-100 at the highest and 20-times for BDE-209 at the lowest. The results of this study showed that of the five environmentally relevant PBDE congeners, BDE-100 was consistently the most toxic. It may be speculated that the elevated toxicity of BDE-100 is the result of having three bromine atoms at ortho-positions. Correlation between number of ortho-substitution and cytotoxicity have been observed for PCBs in neuronal cultures, however, further research is required to confirm the same effect with poly-ortho-substituted PBDEs.