The Role of Ca2+ Imbalance in the Induction of Acute Oxidative Stress and Cytotoxicity in Cultured Rat Cerebellar Granule Cells Challenged with Tetrabromobisphenol A

Using primary cultures of rat cerebellar granule cells (CGC) we examined the role of calcium transients induced by tetrabromobisphenol A (TBBPA) in triggering oxidative stress and cytotoxicity. CGC were exposed for 30 min to 10 or 25 µM TBBPA. Changes in intracellular calcium concentration ([Ca2+]i), in the production of reactive oxygen species (ROS), and in the potential of mitochondria (∆Ψm) were measured fluorometrically during the exposure. The intracellular glutathione (GSH) and catalase activity were determined after the incubation; cell viability was evaluated 24 h later. TBBPA concentration-dependently increased [Ca2+]i and ROS production, and reduced GSH content, catalase activity, ∆Ψm and neuronal viability. The combination of NMDA and ryanodine receptor antagonists, MK-801 and bastadin 12 with ryanodine, respectively, prevented Ca2+ transients and partially reduced cytotoxicity induced by TBBPA at both concentrations. The antagonists also completely inhibited oxidative stress and depolarization of mitochondria evoked by 10 µM TBBPA, whereas these effects were only partially reduced in the 25 µM TBBPA treatment. Free radical scavengers prevented TBBPA-induced development of oxidative stress and improved CGC viability without having any effect on the rises in Ca2+ and drop in ∆Ψm. The co-administration of scavengers with NMDA and ryanodine receptor antagonists provided almost complete neuroprotection. These results indicate that Ca2+ imbalance and oxidative stress both mediate acute toxicity of TBBPA in CGC. At 10 µM TBBPA Ca2+ imbalance is a primary event, inducing oxidative stress, depolarization of mitochondria and cytotoxicity, whilst at a concentration of 25 µM TBBPA an additional Ca2+-independent portion of oxidative stress and cytotoxicity emerges. Electronic supplementary material The online version of this article (doi:10.1007/s11064-016-2075-x) contains supplementary material, which is available to authorized users.

]. In addition, the presence of bromine atoms in the structure of TBBPA means that it acts like a free radical molecule and is susceptible to reductive debromination [9]. Therefore, the interaction of TBBPA with other fluorescent probes used in this study cannot be excluded.
Moreover bastadin 12, one of the pharmacological tools used in this study that modulates the activity of RyR, is a brominated tyrosine derivative which could also react with the fluorescent probes.
The primary aim of these control experiments was to assess the direct interaction of In all the experiments TBBPA was applied at the same concentration that was used in the main article, 10 µM and 25 µM. In the initial experiment a 100 µM solution of DCFH-DA and DCF was used, which is the loading concentration of the DCF assay in CGC (see the article). In other experiments (Tables 2-4)  The results shown in Table 1 demonstrate that 0.5 % DMSO had no effect on the fluorescence of DCFH-DA solution, whereas the application of TBBPA resulted in an increase in the fluorescence to 110%. Under these conditions TBBPA had no effect on the fluorescence of 100 µM DCF (results not shown). There was little effect on the fluorescence using the combination of NMDAR and RyR antagonists, 0.5 µM MK-801, 200 µM ryanodine and 2.5 µM bastadin 12.
In subsequent experiments DCFH-DA and other fluorescent probes were used at a concentration of 1 µM. The results presented in Table 2 demonstrate that TBBPA at both concentrations, as well as the other brominated substance, bastadin 12 increased the fluorescence of the 1 µM cell-free solution of DCFH-DA by 32-40% and of DCF by 8-14 %.
The vehicle, DMSO, and the other test substances did not interfere with DCFH-DA and DCF fluorescence. Table 2. Effects of TBBPA and NMDAR/RyR antagonists on the fluorescence of 1 µM DCFH-DA and DCF solutions in the cell-free system   98  97  TBBPA 10 µM  107  106  TBBPA 25 µM  106  103  MK-801  100  98  Ryanodine  98  95  Bastadin 12 105 In the next experiment ( Table 3) the effects of the test substances on the fluorescence of cell-free solutions containing 1 µM fluo-3 AM and fluo-3 were measured. Table 3 shows that TBBPA very slightly increased the fluorescence by 3 -7%, whereas bastadin 12 enhanced fluo-3 fluorescence by 11%. Table 4   The question is whether the data obtained in CGC cultures using the DCF test are representative of a biological response, or whether they are artifacts of the purely chemical interaction between the test substances and the fluorescent probes. Other authors have warned that the increased DCF fluorescence observed with TBBPA in the presence of cells cannot be attributed to cellular ROS [7], and that the DCF test is not suitable for evaluating TBBPAinduced oxidative stress in cells [9]. We argue that the ROS using DCF test is still useful, provided the results are treated with caution. These results of biological experiments should be additionally confirmed using pharmacological tools that do not interfere directly with oxidative stress. Moreover assessing the level of oxidative stress with alternative methods should provide supporting data. In our study, these conditions have been met. One should also consider, that the combination of these antagonists also including bastadin 12, does not interfere with the fluorescence of DCF in the control, TBBPA-untreated CGC (Fig. 2). In turn, in the cell-free system, two of these antagonists, MK-801 and ryanodine, also had no effect on The presentation of increased production of ROS in the DCF test solely, without support of other indices, would be incomplete proof for TBBPA-induced oxidative stress in CGC. Our results using the DCF test are consistent with results where alternative methods have been used to evaluate oxidative stress. The results described in the main article (Figs 3 and 4) show that in addition to an increase in DCF fluorescence, TBBPA induces a concentration-dependent decrease in GSH level and catalase activity in CGC, and that these effects of 10 µM TBBPA are eliminated by the NMDAR and RyR antagonists. Based on literature data and our own results we consider a drop in GSH level and decreased catalase activity to be secondary to increased production of ROS. Our results strongly suggest that an increase in DCF fluorescence in CGC treated with 10 µM TBBPA is not an artefact, but reflects the increased production of ROS, which is an element of TBBPA-induced, Ca 2+mediated oxidative stress in neurons.