Determination of Apoptotic Mechanism of Action of Tetrabromobisphenol A and Tetrabromobisphenol S in Human Peripheral Blood Mononuclear Cells: A Comparative Study

Abstract

Background: Tetrabromobisphenol A (TBBPA) is the most commonly used brominated flame retardant (BFR) in the industry. TBBPA has been determined in environmental samples, food, tap water, dust as well as outdoor and indoor air and in the human body. Studies have also shown the toxic potential of this substance. In search of a better and less toxic BFR, tetrabromobisphenol S (TBBPS) has been developed in order to replace TBBPA in the industry. There is a lack of data on the toxic effects of TBBPS, while no study has explored apoptotic mechanism of action of TBBPA and TBBPS in human leukocytes.

Methods: The cells were separated from leucocyte-platelet buffy coat and were incubated with studied compounds in concentrations ranging from 0.01 to 50 µg/mL for 24 h. In order to explore the apoptotic mechanism of action of tested BFRs, phosphatidylserine externalization at cellular membrane (the number of apoptotic cells), cytosolic calcium ion and transmembrane mitochondrial potential levels, caspase-8, -9 and -3 activation, as well as PARP-1 cleavage, DNA fragmentation and chromatin condensation in PBMCs were determined.

Results: TBBPA and TBBPS triggered apoptosis in human PBMCs as they changed all tested parameters in the incubated cells. It was also observed that the mitochondrial pathway was mainly involved in the apoptotic action of studied compounds.

Conclusions: It was found that TBBPS, and more strongly TBBPA, triggered apoptosis in human PBMCs. Generally, the mitochondrial pathway was involved in the apoptotic action of tested compounds; nevertheless, TBBPS more strongly than TBBPA caused intrinsic pathway activation.

Keywords: PARP-1 cleavage; apoptosis; caspase activation; chromatin condensation; cytosolic calcium ion level; peripheral blood mononuclear cells; tetrabromobisphenol A; tetrabromobisphenol S; transmembrane mitochondrial potential.

Figure 1

Apoptotic alterations in human PBMCs treated with TBBPA and TBBPS in the range from 1 to 50 µg/mL (n = 4) for 24 h (A). The cells were stained with Annexin-FITC and PI. Exemplary dot plots showing apoptotic alterations in human PBMCs unexposed (control) and exposed to TBBPA and TBBPS at 50 μg/mL for 24 h, Q3—live cells, Q2 + Q4—apoptotic cells (B). Statistically different from negative control at * p < 0.05. Statistical analysis was conducted using one-way ANOVA and a posteriori Tukey test.

Figure 1

Apoptotic alterations in human PBMCs treated with TBBPA and TBBPS in the range from 1 to 50 µg/mL (n = 4) for 24 h (A). The cells were stained with Annexin-FITC and PI. Exemplary dot plots showing apoptotic alterations in human PBMCs unexposed (control) and exposed to TBBPA and TBBPS at 50 μg/mL for 24 h, Q3—live cells, Q2 + Q4—apoptotic cells (B). Statistically different from negative control at * p < 0.05. Statistical analysis was conducted using one-way ANOVA and a posteriori Tukey test.

Figure 2

Alterations in cytosolic calcium ion level in human PBMCs treated with TBBPA and TBBPS in the range from 0.01 to 5 µg/mL (n = 4) for 24 h. Statistically different from negative control at * p < 0.05. Statistical analysis was conducted using one-way ANOVA and a posteriori Tukey test.

Figure 3

Alterations in transmembrane mitochondrial potential (ΔΨm) in PBMCs treated with TBBPA and TBBPS in the range from 0.01 to 25 µg/mL (n = 4) for 24 h. Statistically different from negative control at * p < 0.05. Statistical analysis was conducted using one-way ANOVA and a posteriori Tukey test.

Figure 4

Alterations in caspase-8 (A), caspase-9 (B) and caspase-3 (C) activity in PBMCs treated with TBBPA and TBBPS in the range from 1 to 25 µg/mL (n = 4) for 24 h. Statistically different from negative control at * p < 0.05. Statistical analysis was conducted using one-way ANOVA and a posteriori Tukey test.

Figure 5

Changes in the level of 85 kDa PARP1 fragments in PBMCs treated with TBBPA and TBBPS at 25 µg/mL (n = 3) for 24 h. Statistically different from negative control at * p < 0.05. Statistical analysis was conducted using Welch’s test.

Figure 6

Alterations in DNA fragmentation in PBMCs incubated with TBBPA and TBBPS at 25 µg/mL (n = 3) for 24 h. Statistically different from negative control at * p < 0.05. Statistical analysis was conducted using Welch’s test.

Figure 7

Apoptotic alterations in PBMCs treated with 0.2% DMSO (negative control), as well as with TBBPA and TBBPS at 5 µg/mL and 50 µg/mL. PBMCs were stained with Hoechst 33342 and PI. Viable cells (blue fluorescence), early apoptotic cells (intensive bright blue fluorescence), late apoptotic cells (blue/violet fluorescence) and necrotic cells (red fluorescence) [48].

Scheme 1

TBBPS synthesized in a reaction of bisphenol S bromination with liquid bromide in concentrated acetic acid at 80 °C.