Neurotoxicity of past, present and future flame retardants - neurotoxic hazard characterization and risk assessment of (alternative) flame retardants

Brominated flame retardants (BFRs), and to a lesser extent polychlorinated biphenyls (PCBs), are widely used chemicals that prevent or slow the onset and spreading of fire in order to reduce injuries and death. Unfortunately, many of these compounds pose serious threats for human health and the environment. Previous research identified the nervous system as a sensitive target organ. This thesis therefore describes studies in which the neurotoxic mechanism of action of some selected PCBs and BFRs are (further) unraveled. The obtained data underline the need for safer and less persistent alternative halogen-free flame retardants (HFFRs). Unfortunately, the (neuro)toxic potential of these HFFRs is largely unknown. Therefore, available toxicity data of 13 selected HFFRs was reviewed and subsequently in vitro experiments were performed to identify the in vitro neurotoxic potential of these selected HFFRs. First, cytotoxic effects on the neuronal cell lines PC12 and B35 were investigated. The majority of the tested HFFRs induced negligible cytotoxicity. Next, effects of the compounds on reactive oxygen species (ROS) production was determined which showed that a considerable fraction of the HFFRs affects ROS production. Oxidative stress occurs when ROS levels in the cell dramatically increase, potentially resulting in significant damage to cell structures. Since an increase in intracellular calcium concentrations ([Ca2+]i) is an important trigger for neuronal (dys)function and cell damage, including apoptosis, effects on Ca2+-homeostasis were investigated by using single cell fluorescent microscopy. Some HFFRs were able to disturb basal Ca2+-homeostasis. Since [Ca2+]i is also essential for proper intra- and intercellular signaling, effects on depolarization-evoked increases in [Ca2+]i in PC12 cells were also determined. This proved to be a sensitive endpoint as 7 of 13 tested compounds were able to inhibit depolarization-evoked calcium influx. In the first part of this thesis it is also shown that postsynaptic inhibitory GABAA and excitatory α4β2 nicotinic acetylcholine (nACh) receptors are a direct target for PCBs and BFRs. Therefore, effects on the function of nACh-receptors, expressed in Xenopus oocytes, were measured using the two-electrode voltage-clamp technique. The results demonstrate that a few HFFRs can act as nACh-receptor antagonist. Using the obtained in vitro (neuro)toxicity data, the suitability of these HFFRs for replacement of BFRs was assessed using an initial rank ordering. Two HFFRs were classified as having a negligible, six as having a low, one as having a moderate, and four as having high in vitro neurotoxic potency. Since the in vivo (neuro)toxicity of these compounds is unknown, synaptic plasticity in hippocampal slices of mice neonatally exposed to the BFR tetrabromobisphenol-A (TBBPA) or the HFFRs aluminium diethylphosphinate (Alpi) or zinc stannate (ZS) was measured. Despite the clear in vitro neurotoxic effects of ZS, no (developmental) neurotoxicity was observed ex vivo following single oral exposure. The negligible neurotoxic potency of Alpi in vitro was confirmed in this study. Although additional data are required for complete (human) risk assessment, the findings described in this thesis demonstrate that several HFFRs could be suitable substitutes for BFRs