Bisphenol A environmental exposure and the detrimental effects on human metabolic health: Is it necessary to revise the risk assessment in vulnerable population?

In the last decades, many reports have focused the attention on deleterious effects of novel environmental chemical compounds, including bisphenol A (BPA), on human health. BPA, a common and widely chemical contaminant acting as endocrine disruptor, accumulates in adipose tissue and may affect adipocyte metabolic and inflammatory functions. BPA, at low chronic doses, is now considered as an obesogen compound, and might contribute to the rise of metabolic syndrome, visceral adiposity and diabetes epidemics. The BPA worldwide presence in the environment is responsible for chronic exposure during vulnerable periods, such as foetal and neonatal life. The BPA source of contamination can occur via food, beverage, wastewater, air, dust and soil. BPA, as lipophilic compound, may accumulate into the adipose tissue already during foetal life and may affect adulthood health, through adverse effects on the growth and development of organs and tissues. Thus, based on several studies, it would be crucial to consider further actions aimed to refine risk assessment at least in vulnerable population, such as foetuses, infants and young children, to prevent metabolic diseases and obesity

Human Peripheral Blood Mononuclear Cell Function and Dendritic Cell Differentiation Are Affected by Bisphenol-A Exposure.

Environmental pollutants, including endocrine disruptor chemicals (EDCs), interfere on human health, leading to hormonal, immune and metabolic perturbations. Bisphenol-A (BPA), a main component of polycarbonate plastics, has been receiving increased attention due to its worldwide distribution with a large exposure. In humans, BPA, for its estrogenic activity, may have a role in autoimmunity, inflammatory and allergic diseases. To this aim, we assessed the effect of low BPA doses on functionality of human peripheral blood mononuclear cells (PBMCs), and on in vitro differentiation of dendritic cells from monocytes (mDCs). Fresh peripheral blood samples were obtained from 12 healthy adult volunteers. PBMCs were left unstimulated or were activated with the mitogen phytohemagglutinin (PHA) or the anti-CD3 and anti-CD28 antibodies and incubated in presence or absence of BPA at 0.1 and 1nM concentrations. The immune-modulatory effect of BPA was assessed by evaluating the cell proliferation and the levels of interferon-γ (IFN-γ), interleukin-4 (IL-4), interleukin-10 (IL-10) and interleukin-13 (IL-13) secreted by PBMCs. mDCs were differentiated with IL-4 and GC-CSF with or without BPA and the expression of differentiation/maturation markers (CD11c, CD1a, CD86, HLA-DR) was evaluated by flow cytometry; furthermore, a panel of 27 different cytokines, growth factors and chemokines were assayed in the mDC culture supernatants. PBMCs proliferation significantly increased upon BPA exposure compared to BPA untreated cells. In addition, a significant decrease in IL-10 secretion was observed in PBMCs incubated with BPA, either in unstimulated or mitogen-stimulated cells, and at both 0.1 and 1nM BPA concentrations. Similarly, IL-13 was reduced, mainly in cells activated by antiCD3/CD28. By contrast, no significant changes in IFN-γ and IL-4 production were found in any condition assayed. Finally, BPA at 1nM increased the density of dendritic cells expressing CD1a and concomitantly decreased the expression of HLA-DR and CD86 activation markers. In conclusion, in humans the exposure to BPA causes on PBMCs a significant modulation of proliferative capacity and cytokine production, and on mDCs alteration in differentiation and phenotype. These immune cell alterations suggest that low dose chronic exposure to BPA could be involved in immune deregulation and possibly in the increased susceptibility to develop inflammatory and autoimmune diseases

Low-Dose bisphenol-A impairs adipogenesis and generates dysfunctional 3T3-L1 adipocytes

Environmental endocrine disruptors (EDCs), including bisphenol-A (BPA), have been recently involved in obesity and diabetes by dysregulating adipose tissue function. Our aim was to examine whether prolonged exposure to low doses of BPA could affect adipogenesis and adipocyte metabolic functions. Therefore, 3T3-L1 pre-adipocytes were cultured for three weeks with BPA 1 nM to mimic human environmental exposure. We evaluated BPA effect on cell proliferation, differentiation, gene expression and adipocyte metabolic function. BPA significantly increased pre-adipocyte proliferation (p<0.01). In 3T3-L1 adipocytes differentiated in the presence of BPA, the expression of Peroxisome proliferator-activated receptor gamma (PPARγ), Fatty Acid Binding Protein 4/Adipocyte Protein 2 (FABP4/AP2) and CCAAT/enhancer binding protein (C/EBPα) was increased by 3.5, 1.5 and 3 folds, respectively. Mature adipocytes also showed a significant increase in lipid accumulation (p<0.05) and alterations of insulin action, with significant reduction in insulin-stimulated glucose utilization (p<0.001). Moreover, in mature adipocytes, mRNA levels of Leptin, interleukin-6 (IL6) and interferon-γ (IFNγ) were significantly increased (p<0.05). In conclusion, BPA prolonged exposure at low doses, consistent with those found in the environment, may affect adipocyte differentiation program, enhancing pre-adipocyte proliferation and anticipating the expression of the master genes involved in lipid/glucose metabolism. The resulting adipocytes are hypertrophic, with impaired insulin signaling, reduced glucose utilization and increased pro-inflammatory cytokine expression. Thus, these data supported the hypothesis that BPA exposure, during critical stages of adipose tissue development, may cause adipocyte metabolic dysfunction and inflammation, thereby increasing the risk of developing obesity-related diseases

Bisphenol-A plasma levels are related to inflammatory markers, visceral obesity and insulin-resistance: a cross-sectional study on adult male population.

Background: The current increase of obesity and metabolic syndrome (MS) focuses attention on bisphenol-A (BPA), "obesogen" endocrine disruptor, main plastic component. Aim was to verify the role of BPA in metabolic alterations, insulin resistance, low grade inflammation and visceral obesity. Methods: A cross-sectional study was performed in 76 out of 139 environmentally exposed adult males, unselected Caucasian subjects, enrolled by routine health survey at the "Federico II" University of Naples outpatient facilities. BPA plasma levels (ELISA), metabolic risk factors, homeostasis model assessment of insulin resistance index, plasma monocyte chemoattractant protein 1, interleukin-6 (IL-6) and tumor necrosis factor-alpha were performed. Clinical and biochemical parameters have been compared with BPA and pro-inflammatory cytokines levels. Results: In total 24 subjects out of 76 (32%) presented with waist circumference (WC) >102 cm, 36 (47%) had impaired fasting glucose and 24 (32%) subjects had insulin resistance [11 out 52 (21%) with WC ≤102 cm and 13 out of 24 with WC >102 cm (54%), χ(2) 6.825, p = 0.009]. BPA and pro-inflammatory cytokine levels were significantly higher in subjects with visceral adiposity (WC > 102 cm). BPA correlated with WC, triglycerides, glucose homeostasis and inflammatory markers. At the multivariate analysis WC and IL-6 remained the main predictors of BPA. Conclusions: Detectable BPA plasma levels have been found also in our population. The strictly association between BPA and WC, components of MS, and inflammatory markers, further supports the BPA role in visceral obesity-related low grade chronic inflammation

BPA affects the phenotype of monocyte derived Dendritic Cells (mDCs).

<p>Dendritic Cells (mDCs) (n = 6) were differentiated from PBMCs for 6 days in absence or in presence of BPA at 1nM concentration and then analyzed by flow cytometer. (A) Percentages on total mDCs of cells expressing CD11c, CD86 and HLA-DR, (B) Median of fluorescence intensity of CD11c, CD86 and HLA-DR on mDCs. (C) Examples of staining for CD11c and CD1a on mDCs differentiated with or without BPA. (D) Percentages on total mDCs of cells expressing CD1a. (E) Mixed Lymphocyte Reactions (MLR) (n = 5) were performed with mDCs differentiated and subsequently LPS-matured in presence or absence of BPA, and co-incubated with allogenic non-adherent fractions of PBMCs (1:5 ratio mDCs:NA-cells). Experiments were performed in triplicates and IFN-γ secretion was evaluated after 48 hours. In panels A, B, D and E we reported median and ranges [min max] of the analyzed samples. Asterisks indicate statistically significant differences (*p<0.05) by non-parametric Friedman test, followed by the Wilcoxon test for dependent samples, except for MLR experiments, where paired T-test was applied. In panels A, B, D and E we reported median and ranges [min max] of the analyzed samples. Asterisks indicate statistically significant differences (*p<0.05) by non-parametric Friedman test, followed by the Wilcoxon test for dependent samples.</p

Effect of BPA on 3T3-L1 protein abundance of master differentiation genes.

<p>Protein levels of PPARγ <b>(A)</b>, FABP4/AP2 <b>(B)</b> and GLUT-4 <b>(C)</b> were assayed during adipogenesis at days 4 and 8 by western blot analysis, expressed as Arbitrary Unit (AU). Bars represent the mean ± SD of four independent experiments and blot is representative of four different experiments. Asterisks indicate statistically significant differences (*p<0.05) between days 4 and 8 for PPARγ <b>(A)</b> and day 8 for FABP4/AP2 <b>(B)</b>, without and with BPA incubation, both compared to untreated day 4. Hash (#p<0.05) expresses statistically significant differences between days 4 and 8 for PPARγ <b>(A)</b> and day 8 for FABP4/AP2 <b>(B)</b>, upon BPA incubation compared to controls. No significant differences in GLUT-4 protein expression <b>(C)</b>.</p

BPA effect on insulin transduction pathway.

<p>Insulin signaling was tested in 3T3-L1 pre-adipocytes, before differentiation started (day 0; <b>A-C</b>) and in mature adipocytes (day 8; <b>B-D</b>). ERK1/2 (<b>A-B</b>) and PKB/AKT (<b>C-D</b>) phosphorylation after insulin stimulation were shown in untreated and BPA treated cells. Bars represent the mean ± SD of four independent experiments and blot is representative of four different experiments. Asterisks indicate statistically significant difference (*p<0.05, **p<0.01 and ***p<0.001) in adipocytes cultured upon BPA compared to controls.</p

Pro-inflammatory effect in 3T3-L1 mature adipocytes.

<p>In mature adipocytes, mRNA levels of Leptin <b>(A)</b>, IL6 <b>(B)</b>, IFNγ <b>(C)</b>, TNFα <b>(D)</b> and adiponectin <b>(E)</b> were assayed at day 8, the end of adipogenesis, by Real-time RT-PCR analysis, and expressed as Relative Expression Unit (REU). Bars represent the mean ± SD of four independent experiments. Asterisk indicates statistically significant difference (*p<0.05) between adipocytes cultured upon BPA treatment compared to controls.</p

Effect of BPA on 3T3-L1 proliferation and mRNA gene expression.

<p><b>(A)</b> 3T3-L1 pre-adipocyte were counted and expressed as cells/ml, at days 15, 16, 17 and 18, after 2 weeks of incubation with (BPA) or without (CTR) BPA 1nM, before adipogenesis started. PPARγ <b>(B)</b>, FABP4/AP2 <b>(C)</b> and cEBPα <b>(D)</b> mRNA levels were assayed during adipogenesis at days 0, 4 and 8, by Real-time RT-PCR analysis, expressed as Relative Expression Unit (REU). Bars represent the mean ± SD of four independent experiments. Asterisks indicate statistically significant differences (*p<0.05; **p<0.01; ***p<0.001) at days 4 and 8 compared to untreated day 0 for PPARγ <b>(B)</b>, at day 8 compared to untreated day 4 for FABP4/AP2 <b>(C)</b>, and at day 0 and day 4 compared to untreated day 0 for cEBPα <b>(D)</b>, without or with BPA incubation. Hashes (# p< 0.05; ### p<0.001) express statistically significant differences between day 8 with or without BPA incubation <b>(B and C)</b> and between day 0 and day 4 with or without BPA incubation <b>(D)</b>.</p