Thyromimetic actions of tetrabromobisphenol A (TBBPA) in steatotic FaO rat hepatoma cells
Introduction
Brominated flame retardants (BFRs) are used in a variety of commercial products including textiles, paper, plastics, electronic equipment, mobile devices and building materials. BFRs are persistent and lipophilic compounds that may bioaccumulate and are thus regarded as a potential environmental health problem (Darnerud, 2003, de Wit et al., 2010). Tetrabromobisphenol A (2,2-bis(3,5-dibromo-4-hydroxyphenyl) propane, TBBPA) is the most-produced BFR (>200 000 tons year−1) (de Wit et al., 2010). First employed to replace highly persistent BFRs, due to its estimated relatively short half-life in mammals and consequently lower risk of human exposure and toxicity (Kuester et al., 2007), TBBPA has been repeatedly detected in the environment and in human samples (Law et al., 2006, EFSA, 2011, Shaw et al., 2013). This finding is raising particular concern, given the potentially adverse effects of this compound as an endocrine disruptor (Fini et al., 2012).
TBBPA shares structural similarities with Thyroid Hormones (THs) T3 (3,3′,5-L-triiodothyronine) and T4 (3,3′,5,5′-L-tetraiodothyronine) (Kitamura et al., 2005) that are essential for the normal development, growth, differentiation and metabolism of all vertebrates (Zoeller et al., 2002). At the cellular level, THs act through interaction with nuclear Thyroid Receptors (TRs), as well as through TR-independent mechanisms, including mitochondrial uncoupling and activation of rapid cytosolic signal transduction pathways (Senese et al., 2014). There is increasing evidence that TBBPA has the potential to interfere with different aspects of TH physiology: it has been shown to bind transthyretin (Meerts et al., 2000), to have thyroidogenic as well as TH antagonist activity (Kitamura et al., 2005, Terasaki et al., 2011, Fini et al., 2012), to modulate the activity of deiodinases (Butt et al., 2011), to induce mitochondrial uncoupling (Nakagawa et al., 2007), to interfere with complex gene regulation systems involving interaction of corepressor and coactivator proteins with TRs (Levy-Bimbot et al., 2012).
THs play an important role in lipid homeostasis (synthesis, mobilization and degradation), with the liver representing one of their main target tissues (Lanni et al., 2005, Zhu and Cheng, 2010). The direct anti-steatotic effects of THs on the hepatocyte have been investigated in rat hepatoma FaO cells overloaded with lipids by exposure to excess free fatty acids-FFAs (Grasselli et al., 2011a, Grasselli et al., 2014). In this well differentiated liver cell line, defective for functional TRs, both T3 and its derivative T2 (3′,5′-diiodo-L-thyronine) were able to exert lipid lowering effects though TR-independent mechanisms of action, including modulation of transcription of genes involved in lipid metabolism, in particular Peroxisome Proliferator-Activated Receptors (PPARs), and mitochondrial uncoupling (Grasselli et al., 2011a, Grasselli et al., 2014).
In this work, the possible receptor-independent effects of TBBPA on lipid homeostasis were investigated in lipid-loaded (steatotic) FaO cells. Intracellular lipid accumulation was evaluated by determination of triglyceride (TAG) content and lipid droplet (LD) size. Transcription of PPAR isoforms α, β/δ, and γ and of some of their target genes was determined by quantitative (q-PCR). The effects of TBBPA were compared with those of equimolar doses of T3.
Section snippets
Cell culture and treatments
Rat hepatoma cells (FaO) were supplied by European Collection of Cell Cultures (Sigma–Aldrich Corp. Milan, Italy). Cells were grown in Coon’s modified Ham’s F12 supplemented with 10% foetal bovine serum-FBS (Euroclone Milan, Italy), 100 Uml−1 penicillin and 0.1 mg ml−1 streptomycin and maintained at 37 °C in a humidified atmosphere of 95% air and 5% CO2 and used at 70–80% confluence. For treatments cells were incubated in medium supplemented with 0.25% bovine serum albumin (BSA) without FBS. In
Effects of TBBPA on lipid accumulation in steatotic FaO cells
The effect of TBBPA on TAG accumulation in lipid loaded (steatotic) FaO cells was first evaluated (Fig. 1A). Cell exposure to a mixture of FFAs (oleate/palmitate) for 3 h resulted in significant TAG accumulation (+110%; P ⩽ 0.001) as previously demonstrated (Grasselli et al., 2011a, Grasselli et al., 2014). Subsequent exposure to TBBPA (10−8–10−5 M for 24 h) induced a dose-dependent decrease in intracellular TAG content, with maximal effect at both 10−6/10−5 M. Similar effects were observed with
Discussion
The results show that in steatotic FaO hepatoma cells exposure to TBBPA induced a dose-dependent decrease in intracellular TAG content. The maximal effect was observed at 10−6 M, and comparable with that induced by equimolar concentrations of T3 (this work; Grasselli et al., 2014). In steatotic FaO cells, TAG secretion was increased in a likely attempt to eliminate excess lipids; this effect was reversed by both TBBPA and T3, indicating that stimulation of lipid secretion is not involved in the
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