Evaluation of a Screening System for Obesogenic Compounds: Screening of Endocrine Disrupting Compounds and Evaluation of the PPAR Dependency of the Effect

Abstract

Recently the environmental obesogen hypothesis has been formulated, proposing a role for endocrine disrupting compounds (EDCs) in the development of obesity. To evaluate this hypothesis, a screening system for obesogenic compounds is urgently needed. In this study, we suggest a standardised protocol for obesogen screening based on the 3T3-L1 cell line, a well-characterised adipogenesis model, and direct fluorescent measurement using Nile red lipid staining technique. In a first phase, we characterised the assay using the acknowledged obesogens rosiglitazone and tributyltin. Based on the obtained dose-response curves for these model compounds, a lipid accumulation threshold value was calculated to ensure the biological relevance and reliability of statistically significant effects. This threshold based method was combined with the well described strictly standardized mean difference (SSMD) method for classification of non-, weak- or strong obesogenic compounds. In the next step, a range of EDCs, used in personal and household care products (parabens, musks, phthalates and alkylphenol compounds), were tested to further evaluate the obesogenicity screening assay for its discriminative power and sensitivity. Additionally, the peroxisome proliferator activated receptor γ (PPARγ) dependency of the positive compounds was evaluated using PPARγ activation and antagonist experiments. Our results showed the adipogenic potential of all tested parabens, several musks and phthalate compounds and bisphenol A (BPA). PPARγ activation was associated with adipogenesis for parabens, phthalates and BPA, however not required for obesogenic effects induced by Tonalide, indicating the role of other obesogenic mechanisms for this compound.

Alteration of cellular lipids and lipid metabolism markers in RTL-W1 cells exposed to model endocrine disrupters

“…PC -Phosphatidylcholines, TAG -triacylglycerols. accumulation assay and was a weak activator of PPAR␥ receptor (Pereira-Fernandes et al, 2013). However, in-vivo studies have shown an accumulation of liver triglycerides and cholesteryl esters with changes in hepatic FA composition in adult mice exposed to low BPA doses; an activation of lipogenesis and cholesterol biosynthesis were pointed out as the major mechanisms involved (Marmugi et al, 2012).…”

“…Soon after, Marmugi et al (2012) reported the accumulation of triglycerides and cholesteryl esters in the liver of mice exposed to 50 and 500 g BPA/kg/day. Interestingly, micromolar concentrations of BPA and 4-nonylphenol (4-NP) triggered the differentiation of 3T3-L1 cells in adipocytes (Masuno et al, 2002(Masuno et al, , 2003, and BPA induced the expression of adypocite-specific genes (such are FAS and leptin) (Phrakonkham et al, 2008).…”

Endocrine Disruption and Disorders of Energy Metabolism

“…Adipogenic potency increased with the linear length of the alkyl chain [29] and was associated with PPARγ activation [30]. It seems increasingly likely that any ligand for PPARγ will be able to influence adipogenesis and obesity [30]. This poses the question as to whether mixtures of such ligands may also be able to stimulate adipogenesis at lower concentrations than each alone, as already has been shown for the estrogenic effects of EDCs on breast cancer cell growth (see Chapter 10).…”

“…More recently, the alkyl esters of p-hydroxybenzoic acid (parabens) have also been shown to promote adipocyte differentiation in 3T3-L1 cells [29]. Adipogenic potency increased with the linear length of the alkyl chain [29] and was associated with PPARγ activation [30]. It seems increasingly likely that any ligand for PPARγ will be able to influence adipogenesis and obesity [30].…”

Study of the possible migration risks of food contact materials for children under 3 years

“…high priority: 2,6-di-tertbutylbenzoquinone). Furthermore, receptor gene assays were applied to determine the estrogen (ER), androgen (AR), progesterone (PR), glucocorticoid (GR), thyroid beta (TRβ), peroxisome proliferator gamma (PPARγ) and aryl hydrocarbon receptor (AhR) mediated transactivational activity, since these nuclear receptors are involved in several regulating processes in the human body (Zoeller et al 2012;Pereira-Fernandes et al 2013). Based on this series of bioassays, an overall cumulative toxicity scoring was assigned to each identified compound.…”