Table E1.

Studies on oxidative stress, apoptosis and mitochondrial dysfunction.

Reference	Comments
Tetz et al. (2013). Troubleshooting the dichlorofluorescein assay to avoid artefacts in measurement of toxicant‐stimulated cellular production of reactive oxidant species	TBBPA facilitated conversion of 6‐carboxy‐2′,7′‐dichlorodihydrofluorescein diacetate (carboxy‐H2DCFDA) to the fluorescent dichlorofluorescein (DCF) moiety in the absence of serum. The authors concluded that because TBBPA increased fluorescence in the absence of cells, the increased DCF fluorescence observed with TBBPA in the presence of cells cannot be attributed to cellular ROS and may, instead, be the result of chemical activation of carboxy‐H2DCFDA to the fluorescent DCF moiety. This paper is not informative about TBBPA mode of action, but casts doubt on the reliability of any ROS studies using the DCF assay
Wang et al. (2014). Quest for the binding mode of tetrabromobisphenol A with Calf thymus DNA	Studies of the binding interaction of TBBPA with calf thymus DNA using multispectroscopic and molecular modelling methods. It was concluded that TBBPA might enter the groove of calf thymus DNA, and that hydrogen bonds and hydrophobic forces play a major role in the binding. Study is of little relevance since we do not consider DNA binding in the genotoxicity section and it is not about covalent binding
Krivoshiev et al. (2015). Elucidating toxicological mechanisms of current flame retardants using a bacterial gene profiling assay	Gene expression profiles were monitored using 12 E. coli reporter strains. The bacterial stress‐gene assay demonstrates the induction of a number of stress genes by TBBPA. Clustering suggested that TBBPA results in membrane and protein damage as mode of action with oxidative damage as underlying mechanism
Szychowski et al. (2016). Tetrabromobisphenol A (TBBPA)‐stimulated reactive oxygen species (ROS) production in cell‐free model using the 2′,7′‐dichlorodihydrofluorescein diacetate (H(2)DCFDA) assay‐limitations of method	Study casts doubt on the reliability of ROS studies using the DCF assay, similarly to Tetz et al. (2013)
Choi et al. (2011). Molecular mechanism of tetrabromobisphenol A (TBBPA)‐induced target organ toxicity in Sprague–Dawley male rats	8‐hydroxy‐2′‐deoxyguanosine (8‐OHdG), a biomarker of DNA oxidative damage was measured in the kidney, liver and testes of Sprague–Dawley rats following oral exposure to TBBPA for 30 days (PND18‐48). Expression levels of SOD were significantly increased at 500 mg/kg bw. 500 mg TBBPA/kg bw strongly induced the production of 8‐OhdG in the testis. It was also increased in the kidney at 250 and 500 mg/kg bw
Shao et al. (2017). Mechanism of synergistic DNA damage induced by the hydroquinone metabolite of brominated phenolic environmental pollutants and Cu(II): Formation of DNA‐Cu complex and site‐specific production of hydroxyl Radicals	This paper claims that 2,6‐dibromohydroquinone (2,6‐DBrHQ) is a major metabolite of TBBPA. 2,6‐DBrHQ was reacted with plasmid DNA, or with calf‐thymus DNA, in cell‐free model systems. In the presence, but not absence, of Cu(II), 2,6‐DBrHQ induced double strand breaks and 8‐oxodG formation. It is not clear if this metabolite is formed in mammalian systems and the relevance of these reactions in sub‐cellular systems is unclear
Yu et al. (2019). OPFRs and BFRs induced A549 cell apoptosis by caspase‐dependent mitochondrial pathway	Generation of ROS, mitochondrial membrane potential (MMP) dysfunction, cell apoptosis and overload of intracellular free Ca2+ demonstrated that the cytotoxicity induced by TBBPA in A549 cells (non‐small cell lung cancer) was mediated by oxidative stress. The cell survival rate significantly increased when pretreated with Ac‐DEVD‐CHO, suggesting that the caspase‐3 dependent mitochondrial pathway may have played a primary role in the process of A549 cell apoptosis
Zhang, Wang, et al. (2019). Regulation of TBBPA‐induced oxidative stress on mitochondrial apoptosis in L02 cells through the Nrf2 signalling pathway	At very high concentrations, TBBPA (10, 20 and 40 mM) significantly increased intracellular ROS, malondialdehyde (MDA) and the ratio of oxidised/reduced glutathione (GSSG/GSH) in human hepatocytes (L02 cells). TBBPA also decreased the cell MMP, caused the release of cytochrome c to cytoplasm and promoted the expression of caspase‐9 and caspase‐3, and finally increased the level of apoptosis. The ROS inhibitor N‐acetyl‐L‐cysteine (NAC) relieved the oxidative stress responses, and prevented the decrease of MMP and increase of apoptosis. Moreover, TBBPA promoted the expression of antioxidant genes related to Nrf2
Włuka et al. (2020). Tetrabromobisphenol A, terabromobisphenol S and other bromophenolic flame retardants cause cytotoxic effects and induce oxidative stress in human peripheral blood mononuclear cells (in vitro study)	Exposure of human peripheral blood mononuclear cells (PBMCs) for 1 or 24 h to TBBPA reduced PBMCs viability and ATP level as well as increased reactive oxygen species (including hydroxyl radical) formation. In addition, TBBPA induced lipid peroxidation and caused oxidative damage to proteins in the incubated cells
Barańska, Bukowska, et al. (2022). Determination of Apoptotic Mechanism of Action of Tetrabromobisphenol A and Tetrabromobisphenol S in Human Peripheral Blood Mononuclear Cells: A Comparative Study	The apoptotic potential and mechanism of action of TBBPA in human peripheral blood mononuclear cells (PBMCs) was assessed. TBBPA triggered apoptosis by phosphatidylserine (PS) externalisation on cellular membrane, increasing cytosolic Ca2+ level, decreasing transmembrane mitochondrial potential, activating caspase‐8, −9 and −3, as well as increasing PARP‐1 cleavage, DNA fragmentation and chromatin condensation. TBBPA induced apoptosis mainly by the involvement of the mitochondrial pathway. The accelerated apoptosis of lymphocytes (main population of PBMCs) caused by TBBPA may lead to disorders in the immune system function and possibly be involved in cancer development
Steves et al. (2018). Ubiquitous Flame‐Retardant Toxicants Impair Spermatogenesis in a Human Stem Cell Model	Using a human stem cell‐based model of spermatogenesis, it was shown that TBBPA affected spermatogonia and primary spermatocytes after acute treatment (1 day) at concentrations that are physiologically relevant (25–200 μM). TBBPA exposure impairs cell cycle progression in in vitro cultures but did not affect haploid sperm viability. TBBPA affect spermatogenesis through decreases of the mitochondrial membrane potential and ROS generation, ultimately causing apoptosis
Guo et al. (2022). Tetrabromobisphenol Exposure Impairs Bovine Oocyte Maturation by Inducing Mitochondrial Dysfunction	TBBPA (20, 40 and 80 μM) exposure of bovine cumulus–oocyte complexes (COCs) impaired oocyte maturation because of the abnormality of nuclear and cytoplasmic maturation. TBBPA exposure induced mitochondrial dysfunction, which resulted in oxidative stress and early apoptosis by mediating the expression of superoxide dismutase 2 (SOD2). In addition, mitochondrial dysfunction led to insufficient energy supply to disrupt spindle assembly and chromosome alignment via regulating pyruvate metabolism mediated by pyruvate dehydrogenase kinase 3 (PDK3). The authors suggested that these abnormalities can induce fertilisation failure and subsequent embryonic development arrest
Suh et al. (2017). Tetrabromobisphenol A induces cellular damages in pancreatic β‐cells in vitro	RIN‐m5F cells derived from rat pancreatic b‐cells were exposed to different concentrations of TBBPA for 48 h. TBBPA (≥ 20 μM) significantly decreased cell viability and increased apoptosis, thus, TBBPA‐induced apoptotic cell death. In addition, TBBPA‐induced mitochondrial dysfunction, ROS generation, the formation of certain nitrogen species and elevation of inflammatory cytokine levels in vitro
Zieminska, Lenart, et al. (2017). The Role of Ca2+ Imbalance in the Induction of Acute Oxidative Stress and Cytotoxicity in Cultured Rat Cerebellar Granule Cells Challenged with Tetrabromobisphenol A	The role of calcium transients induced by TBBPA in triggering oxidative stress and cytotoxicity was examined in primary cultures of rat cerebellar granule cells (CGC). In CGC calcium imbalance and oxidative stress both mediate acute cytotoxicity of TBBPA. A causal relationship between these mechanisms depends on the concentration of TBBPA. At a concentration of 10 μM TBBPA, an increase in [Ca2+]i is a primary event triggering oxidative stress, depolarisation of mitochondria and reduction of the neuronal viability. At a concentration of 25 μM TBBPA additional calcium‐independent mechanisms of induction of oxidative stress and cytotoxicity emerge
Cho et al. (2020). Tetrabromobisphenol A‐induced apoptosis in neural stem cells through oxidative stress and mitochondrial dysfunction	The potential involvement of oxidative stress and mitochondrial dysfunction in TBBPA‐mediated neurotoxicity was investigated in neural stem‐like C17.2 cells isolated from neonatal mouse cerebellum and immortalised. TBBPA was more cytotoxic to neural stem cells than to neurons, astrocytes, or fibroblasts isolated from Sprague–Dawley rat cortices. TBBPA‐induced neural stem cell apoptosis was accompanied by increased reactive oxygen species generation and mitochondrial dysfunction. At a molecular level, TBBPA‐induced apoptosis was determined to be mediated by c‐Jun N‐terminal kinase‐p53 pathway activation. According to the authors, these findings suggest that the adverse effects of TBBPA on hippocampal neurogenesis are due to the inhibition of neural stem cell expansion
Zhao et al. (2019). Metabolic perturbation, proliferation and reactive oxygen species jointly contribute to cytotoxicity of human breast cancer cell induced by tetrabromo and tetrachloro bisphenol A	Low micromolar levels (0–10 μM) of TBBPA exposure of MCF‐7 cells (human breast cancer cells) induced cell proliferation and activated the energy metabolism of both glycolysis and amino acid. Higher concentrations of TBBPA (10–50 μM) induce inhibition of cell proliferation in a concentration dependent manner. TBBPA induce the generation of ROS and MDA in accordance with the depletion of GSH, GSH‐Px and SOD activities in MCF‐7 cells at micromolar levels (10–50 μM)
Su et al. (2020). TBBPA stimulated cell migration of endometrial cancer via the contribution of NOX‐generated ROS in lieu of energy metabolism	Low doses of TBBPA (10−12–10−9 mol/L) could promote the migration of endometrial cancer cells. NADPH oxidase (NOX)‐driven ROS instead of energy metabolism was sensitive to TBBPA stimulation. In addition, molecular docking supported a link between TBBPA ligand and NOX receptor
Lee et al. (2019). Tetrabromobisphenol A Induces MMP‐9 Expression via NADPH Oxidase and the activation of ROS, MAPK and Akt Pathways in Human Breast Cancer MCF‐7 Cells	Treatment with TBBPA of human breast cancer MCF‐7 cells significantly induced the expression and promoter activity of matrix metalloproteinase‐9 (MMP‐9). TBBPA‐induced MMP‐9 expression was mediated by the nuclear factor‐kappaB (NF‐κB) and activator protein‐1 (AP‐1) transcription activation as a result of the phosphorylation of the Akt and the mitogen‐activated protein kinase (MAPK) signalling pathways. TBBPA‐induced activation of Akt/MAPK pathways and MMP‐9 expression were attenuated by a specific NADPH oxidase inhibitor (Diphenyleneiodonium chloride (DPI)), and the ROS scavenger (N‐acetyl cysteine (NAC)). These results suggest that TBBPA can induce cancer cell metastasis by releasing MMP‐9 via ROS‐dependent MAPK, and Akt pathways in MCF‐7 cells