Effects of the Commercial Flame Retardant Mixture DE-71 on Cytokine Production by Human Immune Cells

INTRODUCTION:Although production of polybrominated diphenyl ethers (PBDEs) is now banned, release from existing products will continue for many years. The PBDEs are assumed to be neurotoxic and toxic to endocrine organs at low concentrations. Their effect on the immune system has not been investigated thoroughly. We aimed to investigate the influence of DE-71 on cytokine production by peripheral blood mononuclear cells (PBMCs) stimulated with Escherichia Coli lipopolysaccharide (LPS) or phytohaemagglutinin-L (PHA-L). MATERIAL AND METHODS:PBMCs isolated from healthy donors were pre-incubated with DE-71 at various concentrations and subsequently incubated with the monocyte stimulator LPS, or the T-cell activator PHA-L. Interferon (IFN)-γ, interleukin (IL)-1β, IL-2, IL-4, IL-6, IL-8, IL-10, tumor necrosis factor (TNF)-α, IL-17A, and IL-17F were quantified in the supernatants by Luminex kits. RESULTS:At non-cytotoxic concentrations (0.01-10 μg/mL), DE-71 significantly enhanced secretion of IL-1β, IL-6, CXCL8, IL-10, and TNF-α (p<0.001-0.019; n = 6) from LPS-stimulated PBMCs. IFN-γ, TNF-α, IL-17A, and IL-17F (p = <0.001-0.043; n = 6) secretion were enhanced from PHA-L-stimulated PBMCs as well. Secretion of IL-1β, IL-2, IL-10, IL-8 and IL-6 was not significantly affected by DE-71. CONCLUSIONS:We demonstrate an enhancing effect of DE-71 on cytokine production by normal human PBMCs stimulated with LPS or PHA-L ex vivo

The flame retardant DE-71 (a mixture of polybrominated diphenyl ethers) inhibits human differentiated thyroid cell function <i>in vitro</i>

Normal thyroid function is essential for general growth and metabolism, but can be affected by endocrine disrupting chemicals (EDCs). Polybrominated diphenyl ethers (PBDEs) have been used worldwide to reduce flammability in different materials and are suspected to be EDCs. The production of the commercial Penta- and OctaBDE mixtures is banned, but DecaBDEs and existing products may leak PBDEs into the environment. Our aim was to investigate the effect of the PentaBDE mixture DE-71 on human thyroid cells in vitro.Primary human thyroid cells were obtained as paraadenomatous tissue and cultured in monolayers. The influence of DE-71 on cyclic adenosine monophosphate (cAMP) and thyroglobulin (Tg) production was examined in the culture medium by competitive radioimmunoassay and enzyme-linked immunosorbent assay, respectively. Real-time quantitative PCR analysis of thyroid-specific genes was performed on the exposed cell cultures. PBDE concentrations were determined in cellular and supernatant fractions of the cultures.DE-71 inhibited Tg-release from TSH-stimulated thyrocytes. At 50 mg/L DE-71, mean Tg production was reduced by 71.9% (range: 8.5-98.7%), and cAMP by 95.1% (range: 91.5-98.8%) compared to controls). Expression of mRNA encoding Tg, TPO and TSHr were significantly inhibited (p<0.0001, p = 0.0079, and p = 0.0002, respectively). The majority of DE-71 added was found in the cell fraction. No cytotoxicity was found.DE-71 inhibited differentiated thyroid cell functions in a two phase response manner and a concentration-dependent inhibition of Tg and cAMP production, respectively, as well as expression of mRNA encoding Tg, TPO and TSHr. Our findings suggest an inhibiting effect of PBDEs on thyroid cells

Influence of Phthalates on Cytokine Production in Monocytes and Macrophages: A Systematic Review of Experimental Trials

<div><p>Background</p><p>Phthalates are a group of endocrine disrupting chemicals suspected to influence the immune system. The aim of this systematic review is to summarise the present knowledge on the influence of phthalates on monocyte and macrophage production and secretion of cytokines, an influence which could affect both pro- and anti-inflammatory abilities of these cells.</p><p>Strategy and Results</p><p>A systematic search was performed in Medline, Embase and Toxline in June 2013, last updated 3rd of August 2014. Criteria used to select studies were described and published beforehand online on Prospero (<a href="http://www.crd.york.ac.uk/NIHR_PROSPERO" target="_blank">http://www.crd.york.ac.uk/NIHR_PROSPERO</a>, registration number CRD42013004236). <i>In vivo</i>, <i>ex vivo</i> and <i>in vitro</i> studies investigating the influence of phthalates on cytokine mRNA expression and cytokine secretion in animals and humans were included. A total of 11 reports, containing 12 studies, were found eligible for inclusion. In these, a total of four different phthalate diesters, six primary metabolites (phthalate monoesters) and seven different cytokines were investigated. Though all studies varied greatly in study design and species sources, four out of five studies that investigated di-2-ethylhexyl phthalate found an increased tumour necrosis factor-α secretion/production from monocytes or macrophages. A summary of cytokine measurements was not possible since few studies were comparable in study design and due to insufficient reporting of raw data for most of the included studies.</p><p>Conclusion</p><p>Results from this review have suggested that at least one phthalate (di-2-ethylhexyl phthalate) has the ability to enhance tumour necrosis factor-α production/secretion from monocytes/macrophages <i>in vitro</i>, but also observed <i>ex vivo</i>. Influence of other phthalates on other cytokines has only been investigated in few studies. Thus, <i>in vitro</i> studies on primary human monocytes/macrophages as well as more <i>in vivo</i> studies are needed to confirm or dispute these findings.</p></div

Mean of LDH-fluorescence from DE-71 exposed and PHA-L-stimulated PBMCs.

<p>DE-71 was added in six different concentrations to each culture, medium and DMSO in medium served as negative controls in duplicate, n = 2 cultures in triplicates. LDH: lactate dehydrogenase, PHA-L: phytohemagglutinin-L, DMSO: dimethyl sulfoxid, RFU: relative fluorescence unit.</p

Primary and secondary outcomes from individual studies.

<p>Footnote: BBzP: Butylbenzyl phthalate, DEHP: Di-2-ethylhexyl phthalate, DiNP: Di-iso-nonyl phthalate, DnBP: Di-n-butyl phthalate, IL: Interleukin, LDH: lactate dehydrogenase, MEHP: Mono-(2-ethylhexyl) phthalate, ND: not done, PI: Propidium Iodide, RAW 264 cell line: mouse leukemic monocyte-macrophage cell line, SD: standard deviation, SEM: standard error of the mean, THP-1 cell line: acute monocytic cell line, TNF: Tumour necrosis factor.</p><p>Primary and secondary outcomes from individual studies.</p

Median and range of DMSO controls from PHA-L-stimulated PBMCs (n = 6 cultures in duplicates).

<p>Median and range of DMSO controls from PHA-L-stimulated PBMCs (n = 6 cultures in duplicates).</p

Mean of LDH-fluorescence from DE-71 exposed and LPS-stimulated PBMCs.

<p>DE-71 was added in six different concentrations to each culture and medium, and DMSO in medium served as negative controls in duplicate, n = 2 cultures in triplicates. LDH: lactate dehydrogenase, LPS: lipopolysaccharide, DMSO: dimethyl sulfoxid, RFU: relative fluorescence unit.</p

Median and range of DMSO controls from LPS-stimulated PBMCs (n = 6 cultures in duplicates).

<p>Median and range of DMSO controls from LPS-stimulated PBMCs (n = 6 cultures in duplicates).</p

PBDE measurements in stock solution, culture media and cells, mean of double determinations.

<p>PBDE measurements in stock solution, culture media and cells, mean of double determinations.</p