Jennifer J. Schlezinger

Aromatic hydrocarbon receptor-driven Bax gene expression is required for premature ovarian failure caused by biohazardous environmental chemicals

Polycyclic aromatic hydrocarbons (PAHs) are toxic chemicals released into the environment by fossil fuel combustion. Moreover, a primary route of human exposure to PAHs is tobacco smoke. Oocyte destruction and ovarian failure occur in PAH-treated mice, and cigarette smoking causes early menopause in women. In many cells, PAHs activate the aromatic hydrocarbon receptor (Ahr), a member of the Per-Arnt-Sim family of transcription factors. The Ahr is also activated by dioxin, one of the most intensively studied environmental contaminants. Here we show that an exposure of mice to PAHs induces the expression of Bax in oocytes, followed by apoptosis. Ovarian damage caused by PAHs is prevented by Ahr or Bax inactivation. Oocytes microinjected with a Bax promoter–reporter construct show Ahr-dependent transcriptional activation after PAH, but not dioxin, treatment, consistent with findings that dioxin is not cytotoxic to oocytes. This difference in the action of PAHs versus dioxin is conveyed by a single base pair flanking each Ahr response element in the Bax promoter. Oocytes in human ovarian biopsies grafted into immunodeficient mice also accumulate Bax and undergo apoptosis after PAH exposure in vivo. Thus, Ahr-driven Bax transcription is a novel and evolutionarily conserved cell-death signaling pathway responsible for environmental toxicant-induced ovarian failure.

A role for the aryl hydrocarbon receptor in mammary gland tumorigenesis

Abstract The aryl hydrocarbon receptor (AhR) is an evolutionarily conserved transcription factor bound and activated by ubiquitous environmental pollutants. Historically, the AhR has been studied for its transcriptional regulation of genes encoding cytochrome P450 enzymes, which metabolize many of these chemicals into mutagenic and toxic intermediates. However, recent studies demonstrate that the AhR plays an important role in the biology of several cell types in the absence of environmental chemicals. Here, this paradigm shift is discussed in the context of a putative role for the AhR in mammary gland tumorigenesis. Data demonstrating high levels of constitutively active AhR in mammary tumors are summarized. Particular focus is placed on the likelihood that the AhR contributes to ongoing mammary tumor cell growth and on the possibility that the AhR inhibits apoptosis while promoting transition to an invasive, metastatic phenotype. A working model is proposed that may help explain the sometimes contradictory outcomes observed after AhR manipulation and that serves as a blueprint for the design of therapeutics which target the AhR in breast cancer. The theme that malignant cells reveal the functions for which the AhR has been evolutionarily conserved is presented throughout this discussion.

Ligand binding and activation of PPARγ by Firemaster® 550: effects on adipogenesis and osteogenesis in vitro.

Background: The use of alternative flame retardants has increased since the phase out of pentabromodiphenyl ethers (pentaBDEs). One alternative, Firemaster® 550 (FM550), induces obesity in rats. Triphenyl phosphate (TPP), a component of FM550, has a structure similar to that of organotins, which are obesogenic in rodents. Objectives: We tested the hypothesis that components of FM550 are biologically active peroxisome proliferator-activated receptor γ (PPARγ) ligands and estimated indoor exposure to TPP. Methods: FM550 and its components were assessed for ligand binding to and activation of human PPARγ. Solvent mapping was used to model TPP in the PPARγ binding site. Adipocyte and osteoblast differentiation were assessed in bone marrow multipotent mesenchymal stromal cell models. We estimated exposure of children to TPP using a screening-level indoor exposure model and house dust concentrations determined previously. Results: FM550 bound human PPARγ, and binding appeared to be driven primarily by TPP. Solvent mapping revealed that TPP interacted with binding hot spots within the PPARγ ligand binding domain. FM550 and its organophosphate components increased human PPARγ1 transcriptional activity in a Cos7 reporter assay and induced lipid accumulation and perilipin protein expression in BMS2 cells. FM550 and TPP diverted osteogenic differentiation toward adipogenesis in primary mouse bone marrow cultures. Our estimates suggest that dust ingestion is the major route of exposure of children to TPP. Conclusions: Our findings suggest that FM550 components bind and activate PPARγ. In addition, in vitro exposure initiated adipocyte differentiation and antagonized osteogenesis. TPP likely is a major contributor to these biological actions. Given that TPP is ubiquitous in house dust, further studies are warranted to investigate the health effects of FM550. Citation: Pillai HK, Fang M, Beglov D, Kozakov D, Vajda S, Stapleton HM, Webster TF, Schlezinger JJ. 2014. Ligand binding and activation of PPARγ by Firemaster® 550: effects on adipogenesis and osteogenesis in vitro. Environ Health Perspect 122:1225–1232; http://dx.doi.org/10.1289/ehp.1408111

Peroxisome Proliferator-Activated Receptor γ-Mediated NF-κB Activation and Apoptosis in Pre-B Cells

The role of peroxisome proliferator-activated receptor γ (PPARγ) in adipocyte physiology has been exploited for the treatment of diabetes. The expression of PPARγ in lymphoid organs and its modulation of macrophage inflammatory responses, T cell proliferation and cytokine production, and B cell proliferation also implicate it in immune regulation. Despite significant human exposure to PPARγ agonists, little is known about the consequences of PPARγ activation in the developing immune system. Here, well-characterized models of B lymphopoiesis were used to investigate the effects of PPARγ ligands on nontransformed pro/pre-B (BU-11) and transformed immature B (WEHI-231) cell development. Treatment of BU-11, WEHI-231, or primary bone marrow B cells with PPARγ agonists (ciglitazone and GW347845X) resulted in rapid apoptosis. A role for PPARγ and its dimerization partner, retinoid X receptor (RXR)α, in death signaling was supported by 1) the expression of RXRα mRNA and cytosolic PPARγ protein, 2) agonist-induced binding of PPARγ to a PPRE, and 3) synergistic increases in apoptosis following cotreatment with PPARγ agonists and 9-cis-retinoic acid, an RXRα agonist. PPARγ agonists activated NF-κB (p50, Rel A, c-Rel) binding to the upstream κB regulatory element site of c-myc. Only doses of agonists that induced apoptosis stimulated NF-κB-DNA binding. Cotreatment with 9-cis-retinoic acid and PPARγ agonists decreased the dose required to activate NF-κB. These data suggest that activation of PPARγ-RXR initiates a potent apoptotic signaling cascade in B cells, potentially through NF-κB activation. These results have implications for the nominal role of the PPARγ in B cell development and for the use of PPARγ agonists as immunomodulatory therapeutics.

Aryl hydrocarbon receptor (AhR) agonists suppress interleukin-6 expression by bone marrow stromal cells: an immunotoxicology study

BackgroundBone marrow stromal cells produce cytokines required for the normal growth and development of all eight hematopoietic cell lineages. Aberrant cytokine production by stromal cells contributes to blood cell dyscrasias. Consequently, factors that alter stromal cell cytokine production may significantly compromise the development of normal blood cells. We have shown that environmental chemicals, such as aromatic hydrocarbon receptor (AhR) agonists, suppress B lymphopoiesis by modulating bone marrow stromal cell function. Here, we extend these studies to evaluate the potential for two prototypic AhR agonists, 7,12-dimethylbenz [a]anthracene (DMBA) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), to alter stromal cell cytokine responses.MethodsBone marrow stromal cells were treated with AhR agonists and bacterial lipopolysaccharide (LPS) to mimic innate inflammatory cytokine responses and to study the effects of AhR ligands on those responses. Steady state cytokine RNA levels were screened by RNAse protection assays (RPA) and quantified by real-time PCR. Cytokine (IL-6) protein production was measured by ELISA. NF-κB EMSAs were used to study IL-6 transcriptional regulation.ResultsRPAs indicated that AhR+ bone marrow stromal cells consistently up-regulated genes encoding IL-6 and LIF in response to LPS, presumably through activation of Toll-like receptor 4. Pre-treatment with low doses of DMBA or TCDD selectively abrogated IL-6 gene induction but had no effect on LIF mRNA. Real-time-PCR indicated a significant inhibition of IL-6 mRNA by AhR ligands within 1 hour of LPS challenge which was reflected in a profound down-regulation of IL-6 protein induction, with DMBA and TCDD suppressing IL-6 levels as much as 65% and 88%, respectively. This potent inhibitory effect persisted for at least 72 hours. EMSAs measuring NF-κB binding to IL-6 promoter sequences, an event known to induce IL-6 transcription, indicated a significant decrease in the LPS-mediated induction of DNA-binding RelA/p50 and c-Rel/p50 heterodimers in the presence of DMBA.ConclusionsCommon environmental AhR agonists can suppress the response to bacterial lipopolysaccharide, a model for innate inflammatory responses, through down-regulation of IL-6, a cytokine critical to the growth of several hematopoietic cell subsets, including early B cells. This suppression occurs at least at the level of IL-6 gene transcription and may be regulated by NF-κB.

Identification of Cinnabarinic Acid as a Novel Endogenous Aryl Hydrocarbon Receptor Ligand That Drives IL-22 Production

The aryl hydrocarbon receptor (AHR) binds to environmental toxicants including synthetic halogenated aromatic hydrocarbons and is involved in a diverse array of biological processes. Recently, the AHR was shown to control host immunity by affecting the balance between inflammatory T cells that produce IL-17 (Th17) and IL-22 versus regulatory T cells (Treg) involved in tolerance. While environmental AHR ligands can mediate this effect, endogenous ligands are likely to be more relevant in host immune responses. We investigated downstream metabolites of tryptophan as potential AHR ligands because (1) tryptophan metabolites have been implicated in regulating the balance between Th17 and Treg cells and (2) many of the AHR ligands identified thus far are derivatives of tryptophan. We characterized the ability of tryptophan metabolites to bind and activate the AHR and to increase IL-22 production in human T cells. We report that the tryptophan metabolite, cinnabarinic acid (CA), is an AHR ligand that stimulates the differentiation of human and mouse T cells producing IL-22. We compare the IL-22-stimulating activity of CA to that of other tryptophan metabolites and define stimulation conditions that lead to CA production from immune cells. Our findings link tryptophan metabolism to AHR activation and define a novel endogenous AHR agonist with potentially broad biological functions.

Organotins are potent activators of PPARγ and adipocyte differentiation in bone marrow multipotent mesenchymal stromal cells.

Adipocyte differentiation in bone marrow is potentially deleterious to both bone integrity and lymphopoiesis. Here, we examine the hypothesis that organotins, common environmental contaminants that are dual ligands for peroxisome proliferator-activated receptor (PPAR) γ and its heterodimerization partner retinoid X receptor (RXR), are potent activators of bone marrow adipogenesis. A C57Bl/6-derived bone marrow multipotent mesenchymal stromal cell (MSC) line, BMS2, was treated with rosiglitazone, a PPARγ agonist, bexarotene, an RXR agonist, or a series of organotins. Rosiglitazone and bexarotene potently activated adipocyte differentiation; however, bexarotene had a maximal efficacy of only 20% of that induced by rosiglitazone. Organotins (tributyltin [TBT], triphenyltin, and dibutyltin) also stimulated adipocyte differentiation (EC₅₀ of 10-20 nM) but with submaximal, structure-dependent efficacy. In coexposures, both bexarotene and TBT enhanced rosiglitazone-induced adipogenesis. To investigate the contribution of PPARγ to TBT-induced adipogenesis, we examined expression of PPARγ2, as well as its transcriptional target FABP4. TBT-induced PPARγ2 and FABP4 protein expression with an efficacy intermediate between rosiglitazone and bexarotene, similar to lipid accumulation. A PPARγ antagonist and PPARγ-specific small hairpin RNA suppressed TBT-induced differentiation, although to a lesser extent than rosiglitazone-induced differentiation, suggesting that TBT may engage alternate pathways. TBT and bexarotene, but not rosiglitazone, also induced the expression of TGM2 (an RXR target) and ABCA1 (a liver X receptor target). The results show that an environmental contaminant, acting with the same potency as a therapeutic drug, induces PPARγ-dependent adipocyte differentiation in bone marrow MSCs. Activation of multiple nuclear receptor pathways by organotins may have significant implications for bone physiology.

Rodent Thyroid, Liver, and Fetal Testis Toxicity of the Monoester Metabolite of Bis-(2-Ethylhexyl) Tetrabromophthalate (TBPH), a Novel Brominated Flame Retardant Present in Indoor Dust

Background: Bis-(2-ethylhexyl) tetrabromophthalate (TBPH) is widely used as a replacement for polybrominated diphenyl ethers (PBDEs) in commercial flame retardant mixtures such as Firemaster 550. It is also used in a commercial mixture called DP 45. Mono-(2-ethyhexyl) tetrabromophthalate (TBMEHP) is a potentially toxic metabolite. Objectives: We used in vitro and rodent in vivo models to evaluate human exposure and the potential metabolism and toxicity of TBPH. Methods: Dust collected from homes, offices, and cars was measured for TBPH by gas chromatography followed by mass spectrometry. Pregnant rats were gavaged with TBMEHP (200 or 500 mg/kg) or corn oil on gestational days 18 and 19, and dams and fetuses were evaluated histologically for toxicity. We also assessed TBMEHP for deiodinase inhibition using rat liver microsomes and for peroxisome proliferator-activated receptor (PPAR) α and γ activation using murine FAO cells and NIH 3T3 L1 cells. Results: TBPH concentrations in dust from office buildings (median, 410 ng/g) were higher than in main living areas in homes (median, 150 ng/g). TBPH was metabolized by purified porcine esterases to TBMEHP. Two days of TBMEHP exposure in the rat produced maternal hypothyroidism with markedly decreased serum T3 (3,3´,5-triiodo-l-thyronine), maternal hepatotoxicity, and increased multinucleated germ cells (MNGs) in fetal testes without antiandrogenic effects. In vitro, TBMEHP inhibited deiodinase activity, induced adipocyte differentiation in NIH 3T3 L1 cells, and activated PPARα- and PPARγ-mediated gene transcription in NIH 3T3 L1 cells and FAO cells, respectively. Conclusions: TBPH a) is present in dust from indoor environments (implying human exposure) and b) can be metabolized by porcine esterases to TBMEHP, which c) elicited maternal thyrotoxic and hepatotoxic effects and d) induced MNGs in the fetal testes in a rat model. In mouse NIH 3T3 L1 preadipocyte cells, TBMEHP inhibited rat hepatic microsome deiodinase activity and was an agonist for PPARs in murine FAO and NIH 3T3 L1 cells.

Generalized concentration addition predicts joint effects of aryl hydrocarbon receptor agonists with partial agonists and competitive antagonists

Background Predicting the expected outcome of a combination exposure is critical to risk assessment. The toxic equivalency factor (TEF) approach used for analyzing joint effects of dioxin-like chemicals is a special case of the method of concentration addition. However, the TEF method assumes that individual agents are full aryl hydrocarbon receptor (AhR) agonists with parallel dose–response curves, whereas many mixtures include partial agonists. Objectives We assessed the ability of generalized concentration addition (GCA) to predict effects of combinations of full AhR agonists with partial agonists or competitive antagonists. Methods We measured activation of AhR-dependent gene expression in H1G1.1c3 cells after application of binary combinations of AhR ligands. A full agonist (2,3,7,8-tetrachlorodibenzo-p-dioxin or 2,3,7,8-tetrachlorodibenzofuran) was combined with either a full agonist (3,3′,4,4′,5-pentachlorobiphenyl), a partial agonist (2,3,3′,4,4′-pentachlorobiphenyl or galangin), or an antagonist (3,3′-diindolylmethane). Combination effects were modeled by the TEF and GCA approaches, and goodness of fit of the modeled response surface to the experimental data was assessed using a nonparametric statistical test. Results The GCA and TEF models fit the experimental data equally well for a mixture of two full agonists. In all other cases, GCA fit the experimental data significantly better than the TEF model. Conclusions The TEF model overpredicts effects of AhR ligands at the highest concentration combinations. At lower concentrations, the difference between GCA and TEF approaches depends on the efficacy of the partial agonist. GCA represents a more accurate definition of additivity for mixtures that include partial agonist or competitive antagonist ligands.

Environmental and Endogenous Peroxisome Proliferator-Activated Receptor γ Agonists Induce Bone Marrow B Cell Growth Arrest and Apoptosis: Interactions between Mono(2-ethylhexyl)phthalate, 9-cis-Retinoic Acid, and 15-Deoxy-Δ12,14-prostaglandin J2

The common commercial use of phthalate esters has resulted in significant human exposure to these bioactive compounds. The facts that phthalate ester metabolites, like endogenous PGs, are peroxisome proliferator-activated receptor (PPAR) agonists, and that PPARγ agonists induce lymphocyte apoptosis suggest that phthalate esters are immunosuppressants that could act together with PGs to modulate early B cell development. In this study we examined the effects of a metabolite of one environmental phthalate, mono(2-ethylhexyl)phthalate (MEHP), and 15-deoxy-Δ12,14-PGJ2 (15d-PGJ2), on developing B cells. MEHP inhibited [3H]thymidine incorporation by primary murine bone marrow B cells and a nontransformed murine pro/pre-B cell line (BU-11). Cotreatment with a retinoid X receptor α ligand, 9-cis-retinoic acid, decreased [3H]thymidine incorporation synergistically, thereby implicating activation of a PPARγ-retinoid X receptor α complex. These results were similar to those obtained with the natural PPARγ ligand 15d-PGJ2. At moderate MEHP concentrations (25 or 100 μM for primary pro-B cells and a pro/pre-B cell line, respectively), inhibition of [3H]thymidine incorporation resulted primarily from apoptosis induction, whereas at lower concentrations, the inhibition probably reflected growth arrest without apoptosis. Cotreatment of bone marrow B cells with 15d-PGJ2 and MEHP significantly enhanced the inhibition of [3H]thymidine incorporation seen with MEHP alone, potentially mimicking exposure in the bone marrow microenvironment where PG concentrations are high. Finally, MEHP- and 15d-PGJ2-induced death does not result from a decrease in NF-κB activation. These data demonstrate that environmental phthalates can cooperate with an endogenous ligand, 15d-PGJ2, to inhibit proliferation of and induce apoptosis in developing bone marrow B cells, potentially via PPARγ activation.