Cytotoxicity by endocrine disruptors through effects on ER Ca2+ transporters, aberrations in Ca2+ signalling pathways and ER stress

Abstract

Concerns regarding man-made organic chemicals pervading our ecosystem and having adverse and detrimental effects upon organisms, including man, have now been studied for several decades. Since the 1970s, some environmental pollutants were identified as having endocrine disrupting affects. These endocrine disrupting chemicals (EDC) were initially shown to have estrogenic or anti-estrogenic properties and some were also shown to bind to a variety of hormone receptors. However, since the 1990s it has also been identified that many of these EDC additionally, have the ability of causing abnormal alterations in Ca²⁺ signalling pathways (also commonly involved in hormone signalling), leading to exaggerated elevations in cytosolic [Ca²⁺] levels, that is known to cause activation of a number of cell death pathways. The major emphasis of this review is to present a personal perspective of the evidence for some types of EDC, specifically alkylphenols and brominated flame retardants (BFRs), causing direct effects on Ca²⁺ transporters (mainly the SERCA Ca²⁺ ATPases), culminating in acute cytotoxicity and cell death. Evidence is also presented to indicate that this Ca²⁺ATPase inhibition, which leads to abnormally elevated cytosolic [Ca²⁺], as well as a decreased luminal ER [Ca²⁺], which triggers the ER stress response, are both involved in acute cytotoxicity.

Keywords: Ca2+ homeostasis; Ca2+ transporters; alkylphenols; brominated flame retardants; cell death; endocrine disrupting chemicals.

Fig. 1

Structures and bioaccumulation levels of some common alkylphenols and BFRs. The figure shows the chemical structures of some common alkylphenols and BFRs focussed on in this review. The structures of estradiol and thyroxine hormones are also included as a comparison. The figures given for each chemical comes from measurements made from human blood samples (values given in ng·g⁻¹ lipid weight and equivalent μm concentrations).

Fig. 2

The hyperactivation of key Ca²⁺‐dependent enzymes leading to cell death. The figure illustrates the role of Ca²⁺ release from the ER leading to the exaggerated increase in cytosolic [Ca²⁺], which in turn both hyperactivates key Ca²⁺‐dependent enzymes as well as causing mitochondrial ‘Ca²⁺ overload’ causing activation of apoptosis through the release of cytochrome C. Specific examples of Ca²⁺‐dependent enzymes that are known to contribute to cell death are identified. Calpains, are involved in cytoskeletal protein breakdown and proteolytic modulation of death inducing enzymes [44, 45]; CamKKβ is known to affect AMP‐kinase which leads to mTOR inhibition and autophagic cell death [46]; DNAS1L3 is a Ca²⁺‐dependent DNAase which is involved in DNA fragmentation during apoptosis [47] and PLA₂ is a lipase involved in arachidonic acid production leading to inflammation and cell death [48].

Fig. 3

The effects of EDC on the SERCA Ca²⁺‐ATPase and their correlation with cell death. (A) Shows the correlation between the potency of inducing cell death (as studied in the SH‐SY5Y neuronal cells), presented as the LC₅₀ value (ie concentration which reduces cell viability by 50%, measured by MTT assay) compared with potency of inhibiting the microsomal Ca²⁺ATPase activity from these cells, for a range of BFRs and alkylphenols. Hexabromocyclododecane (HBCD), Tetrabromobisphenol‐A (TBBPA), Decabromodiphenyl ether (DBPE), Dibromobiphenyl (DBBP), Pentabromodiphenyl ether (PBDE), Octabromodiphenyl either (OBDE), Tetrabromobisphenol‐A‐Diallyl ether (TBBPA‐DAE), Nonylphenol (NP) and Bisphenol‐A (BPA). The correlation coefficient between the 2 variables is 0.94. Data taken from [24]. (B) Show the mechanism of the SERCA Ca²⁺ATPase, highlighting the high (E1) and low (E2) Ca²⁺ affinity binding forms. Also highlighted (by the X) is the E2 going to E1 transition step which is postulated to be the major step affected by alkylphenols and BFRs leading to Ca²⁺ATPase inhibition. (C) Shows the elucidated structure of SERCA1 Ca²⁺ATPase in the E2 conformation and from experimental and modelling studies, it highlights the postulated site at which HBCD, TBBPA and nonylphenol binds.

Fig. 4

Overexpression of SERCA Ca²⁺ATPase protects against HBCD‐induced cell death. (A) Shows a fluorescence micrograph of SH‐SY5Y cells transfected with SERCA1‐EGFP. The transfection efficiency for these experiments was determined to be between 35% and 70%. (B) Shows the Ca²⁺ATPase activity of microsomes prepared from SH‐SY5Y cells overexpressing SERCA1‐EGFP or non‐transfected. These membranes were exposed to a range of concentrations of HBCD (0–10 μm) and Ca²⁺ATPase activity measured. The data shows that the Ca²⁺ATPase activity is substantially higher for the SERCA1‐EGFP containing microsomal membranes compared to control membranes, over a range of HBCD concentrations. (Data points are the mean of 3 to 4 determinations ± SD, *P < 0.05). (C) Shows the cell viability (measured by MTT assays) of SH‐SY5Y cells, either expressing SERCA1‐EGFP or control cells, when exposed to a range of HBCD concentrations (0–30 μm for 24 h). Data points are the mean ± SD of 4 to 5 determinations. LC₅₀ values are 4 ± 1 μm for control cells and 15 ± 2 μm for the SERCA1‐EGFP overexpressing cells. (D) Shows the FACs analysis of SH‐SY5Y cells overexpressing SERCA1‐EGFP and exposed to 3 μm HBCD for 24 h. After incubation with propidium iodide (PI), cells which were non‐viable would have high fluorescence intensities (FI) in the red channel and cells which had overexpression of SERCA1‐EGFP, would have high fluorescence intensities in the green channel. The cells were assigned to four distinct populations. The cells that exhibited high levels of SERCA1‐EGFP fluorescence, showed greater levels of viability when exposed to HBCD compared to the population which did not overexpress SERCA1‐EGFP. In the two populations not overexpressing SERCA1‐EGFP, similar levels of viable to non‐viable cells were measured at 3 μm HBCD. The data presented here were adapted from [27].

Fig. 5

ECD‐induced ER Stress and Ca²⁺‐dependent Cell death pathways. The Figure highlights some of the postulated pathways by which elevation of cytosolic [Ca²⁺] and a concomitant decrease in luminal ER [Ca²⁺] concentrations, caused by EDC can lead to apoptosis and autophagy through activation of both Ca²⁺‐dependent mechanisms in the cytosol and the ER stress response.