The Effect of a Brominated Flame Retardant, Tetrabromobisphenol-A, on Free Radical Formation in Human Neutrophil Granulocytes: The Involvement of the MAP Kinase Pathway and Protein Kinase C

Abstract

This study investigates the effects of one of the most frequently used brominated flame-retardants (BFR), tetrabromobisphenol-A (TBBPA), on formation of reactive oxygen species (ROS) and calcium levels in human neutrophil granulocytes. TBBPA enhanced ROS production in a concentration-depended manner (1-12 microM), measured as 2,7-dichlorofluorescein diacetate amplified (DCF) fluorescence. The results on ROS production by TBBPA was confirmed by lucigenin-amplified chemiluminescence. The TBBPA induced formation of ROS was due to activation of respiratory burst, as shown by the NADPH oxidase inhibitor DPI (10 microM). TBBPA induced activation of respiratory burst was also inhibited by the MEK 1/2 inhibitor U0126 (10 microM), the PKC inhibitor BIM (0.25 microM), and the tyrosine kinase inhibitor erbstatin-A (25 microM). We also found a small reduction in ROS formation in the absence of extracellular calcium and when verapamil was added. The phosphorylation of ERK 1/2 was confirmed by Western blotting. TBBPA also induced a concentration dependent increase in intracellular free calcium measured with Fura-2/AM. We suggest that exposure of human neutrophil granulocytes to the brominated flame retardant TBBPA leads to an activation of the NADPH oxidase primarily by an ERK 1/2 stimulated pathway. The data also show that PKC, calcium, and tyrosine kinases may be involved in the activation.

Synergistic neurotoxicity of oxygen-glucose deprivation and tetrabromobisphenol A in vitro: role of oxidative stress

“…# Differences statistically significant vs. 5 µM TBBPA (one-way ANOVA followed by Dunnett's test, p < 0.05) When examining the putative mechanism of TBBPA and OGD synergy we considered calcium imbalance, mitochondrial disfunction and oxidative stress. According to literature data, these processes might be common to both of toxic factors studied here [9,14,22,23,25,27,28,32]. Therefore, using standard methods we measured…”

“…The ability of TBBPA to activate the formation of ROS has been observed in vivo in animal models [35,39]. Previous studies by Reistad et al [27,28] demonstrated that TBBPA administered in vitro at low micromolar concentrations significantly increased the fluorescence of DCF in human neutrophil granulocytes and in CGC, which reflects the enhanced production of ROS and the generation of oxidative stress. This finding is corroborated by our present data.…”

“…The results of numerous studies have demonstrated the toxic properties of TBBPA, including its immunotoxicity and endocrine-disrupting abilities [18,27]. Experiments performed in vivo have shown that the acute exposure of adult mice to TBBPA results in its selective accumulation in the striatum, which is accompanied by significant behavioral effects [24].…”

“…There are also indications that TBBPA may act as a developmental neurotoxin [20], although there has been a lack of consistency in studies describing the neurodevelopmental effects of TBBPA [38]. Studies performed in vitro using various cell cultures demonstrated the cytotoxicity of TBBPA and have identified several putative mechanisms, including disturbances in the intracellular signaling pathways, calcium imbalance, oxidative stress and excitotoxicity in neurons [5,22,25,27,28]. In light of the above evidence, there is growing concern about the possible toxic effects of TBBPA.…”

“…Ca uptake and changes in fluorescence of the probes fluo-3, R123 and DCF, which are selective for intracellular calcium concentration, mitochondrial membrane potential and ROS production, respectively [15,27,28,34,40].…”