Administration of ammonium salts of perfluorooctanoate (PFOA) to rats results in peroxisome proliferation and benign liver tumors, events associated with activation of the nuclear receptor (NR) peroxisome proliferator-activated receptor-alpha (PPARalpha). Due to its fatty acid structure, PFOA may activate other NRs, such as PPARbeta, PPARgamma, liver X receptor (LXR), or retinoid X receptor (RXR). In this study, the activation of human, mouse, and rat PPARalpha, PPARbeta, PPARgamma, LXRbeta, and RXRalpha by PFOA (including its linear and branched isomers) and perfluorooctane sulfonate (PFOS) was investigated and compared to several structural classes of natural fatty acids and appropriate positive control ligands. An NR ligand-binding domain/Gal4 DNA-binding domain chimeric reporter system was used. Human, mouse, and rat PPARalpha were activated by PFOA isomers and PFOS. PPARbeta was less sensitive to the agents tested, with only PFOA affecting the mouse receptor. PFOA and PFOS also activated human, mouse, and rat PPARgamma, although the maximum induction of PPARgamma was much less than that seen with rosiglitazone, suggesting that PFOA and PFOS are partial agonists of this receptor. Neither LXRbeta nor the common heterodimerization partner RXRalpha was activated by PFOA in any species examined. Taken together, these data show that of the NRs studied, PPARalpha is the most likely target of PFOA and PFOS, although PPARgamma is also activated to some extent. Compared to naturally occurring long-chain fatty acids, e.g. linoleic and alpha-linolenic acids, these perfluorinated fatty acid analogs were more selective and less potent in their activation of the NRs.

Selenium is an essential micronutrient that suppresses the redox-sensitive transcription factor NF-B-dependent proinflammatory gene expression. To understand the molecular mechanisms underlying the anti-inflammatory property of selenium, we examined the activity of a key kinase of the NF-B cascade, IB-kinase ␤ (IKK␤) subunit, as a function of cellular selenium status in murine primary bone marrow-derived macrophages and RAW264.7 macrophage-like cell line. In vitro kinase assays revealed that selenium supplementation decreased the activity of IKK␤ in lipopolysaccharide (LPS)-treated macrophages. Stimulation by LPS of seleniumsupplemented macrophages resulted in a time-dependent increase in 15-deoxy-⌬ 12,14 -prostaglandin J 2 (15d-PGJ 2 ) formation, an endogenous inhibitor of IKK␤ activity. Further analysis revealed that inhibition of IKK␤ activity in seleniumsupplemented cells correlated with the Michael addition product of 15d-PGJ 2 with Cys-179 of IKK␤, while the formation of such an adduct was significantly decreased in the selenium-deficient macrophages. In addition, anti-inflammatory activities of selenium were also mediated by the 15d-PGJ 2 -dependent activation of the peroxisome proliferator-activated nuclear receptor-␥ in macrophages. Experiments using specific cyclooxygenase (COX) inhibitors and genetic knockdown approaches indicated that COX-1, and not the COX-2 pathway, was responsible for the increased synthesis of 15d-PGJ 2 in selenium-supplemented macrophages. Taken together, our results suggest that selenium supplementation increases the production of 15d-PGJ 2 as an adaptive response to protect cells against oxidative stress-induced pro-inflammatory gene expression. More specifically, modification of protein thiols by 15d-PGJ 2 represents a previously undescribed code for redox regulation of gene expression by selenium.

Administration of phthalates is known to cause toxicity and liver cancer in rodents through the activation of peroxisome proliferator-activated receptors (PPARs), and the monoesters appear to be the active metabolites that function as ligands of PPARs. There is evidence that PPARs exhibit significant species differences in response to ligand activation. In this study, the activation of mouse and human PPARalpha, PPARbeta, and PPARgamma by a broad class of phthalate monoesters was investigated using a trans-activation assay, functional analysis of PPARalpha target gene expression, and a PPARgamma-mediated differentiation assay. These studies demonstrated a range in the ability of various phthalate monoesters to activate PPARalpha, with the mouse PPARalpha generally being activated at lower concentrations and exhibiting a greater response than human PPARalpha. Similarly, a range in the trans-activation of mouse PPARbeta by phthalate monoesters was also observed, but this effect was not found with human PPARbeta. A number of phthalate monoesters activated both mouse and human PPARgamma, with similar sensitivity being exhibited by both receptors. These studies show that the potency and efficacy of phthalate monoesters for the activation of PPARalpha and PPARgamma increase with increasing side-chain length. These studies also show that mouse PPARalpha and PPARbeta are generally activated at lower concentrations of phthalate monoesters than human PPARalpha and PPARbeta, and that both mouse and human PPARgamma exhibit similar sensitivity to phthalate monoesters. Lastly, there is a good relationship between the relative ability of phthalate monoesters to trans-activate PPARalpha and PPARgamma, and the relative induction of PPARalpha target gene mRNA and PPARgamma-mediated adipocyte differentiation, respectively.

2-Arachidonyl glycerol (2-AG) is an endogenous arachidonic acid derivative that binds cannabinoid receptors CB1 and CB2 and is hence termed an endocannabinoid. 2-AG also modulates a variety of immunological responses, including expression of the autocrine/paracrine T cell growth factor interleukin (IL)-2. The objective of the present studies was to determine the mechanism responsible for IL-2 suppression by 2-AG. Because of the labile properties of 2-AG, 2-AG ether, a nonhydrolyzable analog of 2-AG, was also used. Both 2-AG and 2-AG ether suppressed IL-2 expression independently of CB1 and CB2, as demonstrated in leukocytes derived from CB1/CB2-null mice. Moreover, we demonstrated that both 2-AG and 2-AG ether treatment activated peroxisome proliferator-activated receptor ␥ (PPAR␥), as evidenced by forced differentiation of 3T3-L1 cells into adipocytes, induction of aP2 mRNA levels, and activation of a PPAR␥-specific luciferase reporter in transiently transfected 3T3-L1 cells. Consequently, the putative role of PPAR␥ in IL-2 suppression by 2-AG and 2-AG ether was examined in Jurkat T cells. Concordant with PPAR␥ involvement, the PPAR␥-specific antagonist 2-chloro-5-nitro-N-(4-pyridyl)-benzamide (T0070907) blocked 2-AG-and 2-AG ether-mediated IL-2 suppression. Likewise, 2-AG suppressed the transcriptional activity of two transcription factors crucial for IL-2 expression, nuclear factor of activated T cells and nuclear factor B, in the absence but not in the presence of T0070907. 2-AG treatment also induced PPAR␥ binding to a PPAR response element in activated Jurkat T cells. Together, the aforementioned studies identify PPAR␥ as a novel intracellular target of 2-AG, which mediates the suppression of IL-2 by 2-AG in a manner that is independent of CB1 and/or CB2.

The inducible isoform of nitric oxide synthase (iNOS) is implicated in atherosclerosis, malignancy, rheumatoid arthritis, tissue and reperfusion injuries. A key determinant of the pro-oxidant versus protective effects of NO is the underlying redox status of the tissue. Selenoproteins, such as glutathione peroxidases (GPxs) and thioredoxin reductases, are key components of cellular defence and promote optimal antioxidant/oxidant balance. In this study, we have investigated the relationship between Se status, iNOS expression and NO production in Se-deficient and Se-supplemented RAW 264.7 macrophage cell lines. The cellular GPx activity, a measure of Se status, was 17-fold lower in Se-deficient RAW 264.7 cells and the total cellular oxidative tone, as assessed by flow cytometry with 2',7'-dichlorodihydrofluorescein diacetate, was higher in the Se-deficient cells than the Se-supplemented cells. Upon lipopolysaccharide (LPS) stimulation of these cells in culture, we found significantly higher iNOS transcript and protein expression levels with an increase in NO production in Se-deficient RAW 264.7 cells than the Se-supplemented cells. Electrophoretic mobility-shift assays, nuclear factor-kappaB (NF-kappaB)-luciferase reporter assays and Western blot analyses indicate that the increased expression of iNOS in Se deficiency could be due to an increased activation and consequent nuclear localization of the redox-sensitive transcription factor NF-kappaB. These results suggest an inverse relationship between cellular Se status and iNOS expression in LPS-stimulated RAW 264.7 cells and provide evidence for the beneficial effects of dietary Se supplementation in the prevention and/or treatment of oxidative-stress-mediated inflammatory diseases.