Exposure to the anticonvulsant valproic acid (VPA) during the first trimester of pregnancy is associated with an increased risk of congenital malformations including heart defects, craniofacial abnormalities, skeletal and limb defects, and, most frequently, neural tube defects (NTDs). The mechanisms by which VPA induces teratogenic effects are not fully understood, although previous studies support a role for oxidative stress. To investigate the effects of VPA on early development, a wholeembryo culture model was used to evaluate the protective effects of antioxidants, measure intracellular reactive oxygen species (ROS) levels, and assess markers of oxidative damage and apoptosis. Furthermore, in vivo teratological evaluations of antioxidant protection were also completed. VPA (0.60 mM in embryo culture, 400 mg/kg in vivo) induced significant decreases in embryonic growth and increases in NTDs. Of the antioxidants tested, catalase provided partial protection against VPA-mediated reductions in morphological and developmental growth parameters in both whole-embryo culture and in vivo systems. VPA exposure resulted in an increase in ROS staining in the head region, as assessed by whole-mount staining with 5-(and-6)-chloromethyl-2Ј,7Ј-dichlorodihydrofluorescein diacetate. Markers of embryonic oxidative damage including 8-hydroxyguanosine, 4-hydroxynonenal adducts, and 3-nitrotyrosine were not affected by VPA treatment. Increased ROS levels were correlated with increased staining for apoptotic markers, as assessed by Western blotting and immunohistochemistry. Addition of catalase to the medium attenuated VPA-induced increases in ROS formation and apoptosis. These studies identify regions of the embryo susceptible to ROS and apoptosis induced by VPA, thus establishing a possible molecular pathway by which VPA exerts teratogenicity.

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Polybrominated diphenyl ethers (PBDEs) are a class of brominated flame retardants that were extensively used in commercial products. PBDEs are ubiquitous environmental contaminants that are both lipophilic and bioaccumulative. Effects of PBDEs on adipogenesis were studied in the 3T3-L1 preadipocyte cell model in the presence and absence of a known adipogenic agent, dexamethasone (DEX). A PBDE mixture designed to mimic body burden of North Americans was tested, in addition to the technical mixture DE-71 and the individual congener BDE-47. The mixture, DE-71, and BDE-47 all induced adipocyte differentiation as assessed by markers for terminal differentiation [fatty acid binding protein 4 (aP2) and perilipin] and lipid accumulation. Characterization of the differentiation process in response to PBDEs indicated that adipogenesis induced by a minimally effective dose of DEX was enhanced by these PBDEs. Moreover, C/EBPα, PPARγ, and LXRα were induced late in the differentiation process. Taken together, these data indicate that adipocyte differentiation is induced by PBDEs; they act in the absence of glucocorticoid and enhance glucocorticoid-mediated adipogenesis.

No abstract

Firemaster® 550 (FM550) is a chemical mixture currently used as an additive flame retardant in commercial products, and is comprised of 2-ethylhexyl-2,3,4,5-tertrabromobenzoate (TBB), bis(2-ethylhexyl) tetrabromophthalate (TBPH), triphenyl phosphate (TPP), and isopropylated triphenyl phosphate (IPTP). Animal and in vitro studies suggest that FM550, TPP and IPTP may have adipogenic effects and may exert these effects through PPARγ activation. Using murine 3T3-L1 preadipocytes, we investigated the detailed expression of transcription factors and adipogenic markers in response to FM550 and its components. Further we investigated the mechanism of action of the peroxisome proliferator-activated receptor gamma (PPARγ) on downstream targets of the receptor by focussing on the mature adipocyte marker, adipocyte protein 2 (aP2). In addition, we set to elucidate the components responsible for the adipogenic effects seen in the FM550 mixture. We show that FM550 and its components TPP, IPTP, and TBPH, but not TBB induced lipid accumulation in a dose-dependent manner. Interestingly, despite displaying enhanced lipid accumulation, TBPH did not alter the mRNA or protein expression of terminal differentiation markers. In contrast, FM550, TPP, and IPTP treatment enhanced lipid accumulation, and mRNA and protein expression of terminal differentiation markers. To further delineate the mechanisms of action of FM550 and its components we focussed on aP2 promoter activity. For this purpose we used the enhancer region of the mouse aP2 promoter using a 584-bp reporter construct containing an active PPRE located 5.4 kb away from the transcription start site of aP2. Exposure to FM550, IPTP, and TPP significantly increased PPARγ mediated aP2 enhancer activity. Furthermore, we show that TPP- and IPTP-dependent upregulation of aP2 was significantly inhibited by the selective PPARγ antagonist GW9662. In addition, chromatin immunoprecipitation experiments showed that IPTP and TPP treatment led to the recruitment of PPARγ to the regulatory region of aP2.