Mono-2-ethylhexylphthalate (MEHP) induces TNF-α release and macrophage differentiation through different signalling pathways in RAW264.7 cells

Bølling, Anette Kocbach; Øvrevik, Johan; Samuelsen, Jan Tore; Holme, Jørn A.; Rakkestad, Kirsten Eline; Mathisen, Gro Haarklou; Paulsen, Ragnhild E.; Korsnes, Mónica Suárez; Becher, Rune

doi:10.1016/j.toxlet.2011.11.016

Cited by 55 publications

(28 citation statements)

References 57 publications

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“…These markers are associated with antigen processing and presentation and cytokine–cytokine receptor interaction in immune cells. Similar increases in inflammatory marker expression have been reported after DEHP and MEHP treatment in different monocyte/macrophage models, such as the macrophage RAW264.7 cell line, 37 human acute monocytic leukemia THP-1 cell line 38 and rat alveolar macrophages. 39 Moreover, MEHP increases IL-5 and IL-10 levels in cultures of local lymph node cells, suggesting that phthalates activate the adaptive immune system.…”

Section: Discussionsupporting

confidence: 77%

In utero exposure to the endocrine disruptor di-(2-ethylhexyl) phthalate promotes local adipose and systemic inflammation in adult male offspring

2014

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Background:Di-(2-ethylhexyl) phthalate (DEHP) is a plasticizer used to increase the flexibility of polyvinyl chloride. DEHP and its active metabolite mono-(2-ethylhexyl) phthalate are detected in many biological fluids during fetal and postnatal life. In rodent models, in utero DEHP exposure has been shown to alter sexual organ development, decrease testosterone and aldosterone production, increase body and epididymal adipose tissue weight, and raise serum lipids and glucose levels in male offspring.Objectives:The objective of this study is to characterize the effects of in utero DEHP exposure on adipose tissue development and function in male offspring.Methods:Sprague–Dawley pregnant dams were gavaged 1, 20, 50 or 300 mg DEHP per kg per day from gestational day 14 until birth.Results:Global gene expression analyses of postnatal day 60 male offspring that were exposed in utero to 300 mg DEHP per kg per day revealed increased expression of immune response and inflammation markers, and increased expression of differentiation pathway genes in the epididymal whole-adipose tissue and isolated stromal vascular fraction. C-reactive protein and tumor necrosis factor (TNF) serum levels were increased in the 300 mg DEHP in utero-exposed offspring. TNF levels in adipose tissue homogenates were increased in the 50 and 300 mg DEHP in utero-exposed offspring. Immunofluorescence studies revealed focal macrophage infiltration in whole-adipose tissue confirmed by increased CD163 tissue content.Conclusions:In utero DEHP exposure promotes local adipose tissue inflammation and chronic low-grade systemic inflammation. Moreover, evidence is presented, suggesting that DEHP increases the differentiation capacity of the pre-adipocytes of male offspring without affecting total body weight.

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Section: Discussionsupporting

confidence: 77%

In utero exposure to the endocrine disruptor di-(2-ethylhexyl) phthalate promotes local adipose and systemic inflammation in adult male offspring

2014

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“…Although numerous caveats have been reported with the DCF assay, including effects of medium composition, serum, heme, heme proteins, metalloporphyrins and bovine serum albumin (Chen, et al, 2010), interactions with chemical toxicants have received nominal attention. Specifically relevant for the present study, reports of MEHP-stimulated and TBBPA-stimulated cellular generation of ROS using the DCF assay did not discuss cell-free assay controls (Bolling, et al, 2012; Fan, et al, 2010; Zhao, et al, 2012, Reistad, et al, 2005)). In contrast, recent reports discuss the impact of various experimental conditions on the accuracy of the DCF assay for assessment of x-radiation-stimulated and UVA-stimulated ROS generation.…”

Section: Discussionmentioning

confidence: 99%

“…Because culture media with or without serum and physiologic salt solutions have been used in reports of the DCF assay (Bolling, et al, 2012; Fan, et al, 2010; Zhao, et al, 2012) we evaluated interactions between MEHP and DCF in RPMI medium and HBSS buffer. Furthermore, we evaluated the effects of serum on MEHP-stimulated DCF fluorescence in RPMI medium but not in HBSS because serum is a common supplement of medium but not salt solutions like HBSS.…”

Section: Methodsmentioning

confidence: 99%

“…MEHP is an active metabolite of the plasticizer di-(2-ethylhexyl) phthalate (DEHP) (Koch, et al, 2006) and TBBPA is a brominated flame retardant (Talsness, et al, 2009). Several reports indicate the use of H 2 DCFDA or carboxy- H 2 DCFDA in either PBS or culture medium to measure MEHP-stimulated ROS production in cells (Bolling, et al, 2012; Fan, et al, 2010; Zhao, et al, 2012). However, these MEHP publications do not indicate whether proper cell-free controls were run to determine potential experimental confounders due to the assay solution.…”

Section: Introductionmentioning

confidence: 99%

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Troubleshooting the dichlorofluorescein assay to avoid artifacts in measurement of toxicant-stimulated cellular production of reactive oxidant species

Tetz

Kamau

Cheng

et al. 2013

Journal of Pharmacological and Toxicological Methods

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Introduction The dichlorofluorescein (DCF) assay is a popular method for measuring cellular reactive oxidant species (ROS). Although caveats have been reported with the DCF assay and other compounds, the potential for artifactual results due to cell-free interactions between the DCF compound and toxicants has hardly been explored. We evaluated the utility of the DCF assay for measuring ROS generation by the toxicants mono-(2-ethylhexyl) phthalate (MEHP), and tetrabromobisphenol A (TBBPA). Methods DCF fluorescence was measured spectrofluorometrically after a 1-h incubation of toxicants with 6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (carboxy-H2DCFDA). MEHP was incubated with carboxy-H2DCFDA in cell-free solutions of Hank’s buffered salt solution (HBSS), or in Royal Park Memorial Institute (RPMI) medium with or without fetal bovine serum. TBBPA was incubated with carboxy-H2DCFDA in cell-free HBSS and with human trophoblast cells (HTR8/SVneo cells). Results MEHP did not increase fluorescence in solutions of carboxy-H2DCFDA in HBSS or RPMI medium without serum. However, MEHP (90 and 180 μM) increased DCF fluorescence in cell-free RPMI medium containing serum. Furthermore, serum-free and cell-free HBSS solutions containing 25 μM TBBPA exhibited concentration-dependent increased fluorescence with 5–100 μM carboxy-H2DCFDA (p<0.05), but not 1 μM carboxy-H2DCFDA. In addition, we observed increased fluorescence in HTR8/SVneo cell cultures exposed to TBBPA (0.5–25 μM) (p<0.05), as we had observed in cell-free buffer. Discussion MEHP demonstrated an interaction with serum in cell-free generation of DCF fluorescence, whereas TBBPA facilitated conversion of carboxy-H2DCFDA to the fluorescent DCF moiety in the absence of serum. Because TBBPA increased fluorescence in the absence of cells, the increased DCF fluorescence observed with TBBPA in the presence of cells cannot be attributed to cellular ROS and may, instead, be the result of chemical activation of carboxy-H2DCFDA to the fluorescent DCF moiety. These data illustrate the importance of including cell-free controls when using the DCF assay to study toxicant-stimulated cellular production of ROS.

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“…As illustrated in Figure 1B, immunofluorescence revealed a barely detectable immunostaining of CD68 in CAFs (Figure 1B, d) and NHDFs (Figure 1B, g) meanwhile no expression can be detected for CD163 and F4/80 markers in CAFs (Figures 1B, e and f) and in NHDFs (Figures 1B, h and i), ruling out that the cells prepared from breast cancer tissue are not macrophages. The RAW264.7 cells, a partially differentiated macrophage-like monocytic cell line [31], was used as positive control, which expresses strongly CD68 (Figure 1B, a) and F4/80 (Figure 1B, c) markers with a moderate expression of CD163 marker (Figure 1B, b). In agreement with the present data, previous studies reported that fibroblasts isolated from normal skin, normal breast, and breast tumor tissue clearly expressed CD68 protein at levels comparable to macrophages [32, 33].…”

Section: Resultsmentioning

confidence: 99%

Stromal fibroblasts present in breast carcinomas promote tumor growth and angiogenesis through adrenomedullin secretion

et al. 2017

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Tumor- or cancer-associated fibroblasts (TAFs or CAFs) are active players in tumorigenesis and exhibit distinct angiogenic and tumorigenic properties. Adrenomedullin (AM), a multifunctional peptide plays an important role in angiogenesis and tumor growth through its receptors calcitonin receptor-like receptor/receptor activity modifying protein-2 and -3 (CLR/RAMP2 and CLR/RAMP3). We show that AM and AM receptors mRNAs are highly expressed in CAFs prepared from invasive breast carcinoma when compared to normal fibroblasts. Immunostaining demonstrates the presence of immunoreactive AM and AM receptors in the CAFs (n = 9). The proliferation of CAFs is decreased by anti-AM antibody (αAM) and anti-AM receptors antibody (αAMR) treatment, suggesting that AM may function as a potent autocrine/paracrine growth factor. Systemic administration of αAMR reduced neovascularization of in vivo Matrigel plugs containing CAFs as demonstrated by reduced numbers of the vessel structures, suggesting that AM is one of the CAFs-derived factors responsible for endothelial cell-like and pericytes recruitment to built a neovascularization. We show that MCF-7 admixed with CAFs generated tumors of greater volume significantly different from the MCF-7 xenografts in nude mice due in part to the induced angiogenesis. αAMR and AM22-52 therapies significantly suppressed the growth of CAFs/MCF-7 tumors. Histological examination of tumors treated with AM22-52 and aAMR showed evidence of disruption of tumor vasculature with depletion of vascular endothelial cells, induced apoptosis and decrease of tumor cell proliferation. Our findings highlight the importance of CAFs-derived AM pathway in growth of breast carcinoma and in neovascularization by supplying and amplifying signals that are essential for pathologic angiogenesis.

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Mono-2-ethylhexylphthalate (MEHP) induces TNF-α release and macrophage differentiation through different signalling pathways in RAW264.7 cells

Cited by 55 publications

References 57 publications

In utero exposure to the endocrine disruptor di-(2-ethylhexyl) phthalate promotes local adipose and systemic inflammation in adult male offspring

In utero exposure to the endocrine disruptor di-(2-ethylhexyl) phthalate promotes local adipose and systemic inflammation in adult male offspring

Troubleshooting the dichlorofluorescein assay to avoid artifacts in measurement of toxicant-stimulated cellular production of reactive oxidant species

Stromal fibroblasts present in breast carcinomas promote tumor growth and angiogenesis through adrenomedullin secretion

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