Mono(2-ethylhexyl)phthalate and mono-n-butyl phthalate activation of peroxisome proliferator activated-receptors alpha and gamma in breast

The phthalates di(2-ethylhexyl)phthalate (DEHP) and di-n-butyl phthalate (DBP) are environmental contaminants with significant human exposures. Both compounds are known reproductive toxins in rodents and DEHP also induces rodent hepatocarcinogenesis in a process believed to be mediated via the peroxisome proliferator-activated receptor alpha (PPARalpha). DEHP and DBP are metabolised to their respective monoesters, mono-(2-ethylhexyl)phthalate (MEHP) and mono-n-butyl phthalate (MBP), which are the active metabolites. MEHP also activates another member of the PPAR subfamily, PPARgamma. The effects of PPARalpha and PPARgamma activation in human breast cells appears to be opposing; PPARalpha activators in breast cells cause an increase in proliferation, while PPARgamma activation in breast cells is associated with differentiation and an inhibition of cell proliferation. Further to this the activation of the PPARs is cell and ligand specific, suggesting the importance of examining the effect of MEHP and MBP on the activation of PPARalpha, PPARbeta and PPARgamma in human breast. We used the common model of human breast cancer MCF-7 and examined the ability of MEHP and MBP to activate human PPARs in this system. The ability of MBP and MEHP to block PPAR responses was also assessed. We found that both human PPARalpha and PPARgamma were activated by MEHP whereas MEHP could not activate PPARbeta. MBP was unable to activate any PPAR isoforms in this breast model, despite being a weak peroxisome proliferator in liver, although MBP was an antagonist for both PPARgamma and PPARbeta. Our results suggest that the toxicological consequences of MEHP in the breast could be complex given the opposing effects of PPARalpha and PPARgamma in human breast cells.     

Effects of phthalate ester derivatives including oxidized metabolites on coactivator recruiting by PPARalpha and PPARgamma

Phthalate esters (PEs), a group of environmental chemicals, affect biological systems via endocrine and lipid metabolism modulations. These effects are believed to be mediated in part by peroxisome proliferator-activated receptors (PPARs). Evaluations of PE activities as ligands toward PPARs have been investigated in many studies on their primary metabolites, monoesters. However, the activities of various other metabolites, including oxidized derivatives, remain to be determined. Here, we have evaluated the PPAR ligand activities of these PE derivatives by in vitro coactivator recruiting assay. Mono(2-ethyl-5-hydroxyhexyl)phthalate, the most abundant metabolite of di-(2-ethylhexyl)phthalate (DEHP), was less active than mono(2-ethylhexyl)phthalate (MEHP) as a PPAR ligand. Other derivatives oxidized at the alkyl group and benzene ring of DEHP, MEHP, dibutyl phthalate and its monoester were also investigated and some affected PPAR activities. Unexpectedly, MEHP as well as its further oxidized metabolite did not show clear activity for PPARalpha, although MEHP is believed to interact with PPARalpha. This might imply indirect PPAR-mediated mechanisms that lead to observed biological effects such as peroxisome proliferation.     

Role of PPARalpha in mediating the effects of phthalates and metabolites in the liver

Phthalate esters belong to a large class of compounds known as peroxisome proliferators (PP). PP include chemicals that activate different subtypes of the peroxisome proliferator-activated receptor (PPAR) family. The ability of phthalate esters and their metabolites to activate responses through different PPAR subtypes is not fully characterized. We investigated the ability of two phthalate esters di-(2-ethylhexyl) phthalate (DEHP) and di-n-butyl phthalate (DBP) and selected metabolites to activate PPAR (alpha, beta/delta, gamma) using a transient transfection assay. The monoester of DEHP, mono-(2-ethylhexyl) phthalate (MEHP) activated all three subtypes of PPAR, but preferentially activated PPARalpha. A second metabolite of DEHP, 2-ethylhexanoic acid (2-EHXA) was a weaker activator of all three subtypes. DBP, but not the primary metabolite mono-n-butyl phthalate weakly activated all three PPAR subtypes. MEHP and DBP but not DEHP and MBP interacted directly with human PPARalpha and PPARgamma as determined by scintillation proximity assays. Both DEHP and DBP activated expression of PP-inducible gene products in wild-type but not PPARalpha-null mice suggesting that both of these phthalates exert their effects by activation of PPARalpha in vivo. The preferential activation of PPARalpha by phthalate ester metabolites suggests that these phthalates mediate their toxic effects in rodent liver in a manner indistinguishable from other PP.     

Activation of mouse and human peroxisome proliferator-activated receptors (alpha, beta/delta, gamma) by perfluorooctanoic acid and perfluorooctane sulfonate.

This study evaluates the potential for perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) to activate peroxisome proliferator-activated receptors (PPARs), using a transient transfection cell assay. Cos-1 cells were cultured in Dulbecco's Minimal Essential Medium (DMEM) with fetal bovine serum in 96-well plates and transfected with mouse or human PPARalpha, beta/delta, or gamma reporter plasmids. Transfected cells were exposed to PFOA (0.5-100 microM), PFOS (1-250 microM), positive controls (i.e., known agonists and antagonists), and negative controls (i.e., DMEM, 0.1% water, and 0.1% dimethyl sulfoxide). Following treatment for 24 h, activity was measured using the Luciferase reporter assay. In this assay, PFOA had more transactivity than PFOS with both the mouse and human PPAR isoforms. PFOA significantly increased mouse and human PPARalpha and mouse PPARbeta/delta activity relative to vehicle. PFOS significantly increased activation of mouse PPARalpha and PPARbeta/delta isoforms. No significant activation of mouse or human PPARgamma was observed with PFOA or PFOS. The PPARalpha antagonist, MK-886, significantly suppressed PFOA and PFOS activity of mouse and human PPARalpha. The PPARgamma antagonist, GW9662, significantly suppressed PFOA activity on the human isoform. In conclusion, this study characterized the dose response and differential activation of mouse and human PPARalpha, beta/delta, gamma by PFOA and PFOS. While this model allows opportunities to compare potential activation by perfluoroalkyl acids, it only evaluates the interaction and activation of the PPAR reporter constructs and is not necessarily predictive of a toxicological response in vivo.     

Therapeutic significance of peroxisome proliferator-activated receptor modulators in diabetes

Peroxisome proliferator-activated receptors (PPARs) are a group of three nuclear receptor isoforms, identified and encoded by different genes: PPARalpha, PPARdelta and PPARgamma. Each subtype of PPAR appears to be differently expressed in a tissue-specific manner due to its binding to a specific consensus DNA sequence of peroxisome proliferator response elements (PPREs). PPARalpha plays a significant role in the regulation of nutrient metabolism, including fatty acid oxidation, gluconeogenesis and amino acid metabolism. PPARdelta is expressed ubiquitously and has been found to be effective in controlling dyslipidemia and cardiovascular diseases, while PPARgamma isotype is mainly expressed in adipose tissue where it stimulates adipogenesis and lipogenesis. Thus PPARs have emerged as potential molecular targets for the design and synthesis of a different class of compounds, considering the conformation of receptors for the treatment of human metabolic disorders. This review concerns the therapeutic importance of PPARs in diabetes drug development.     

Teratogenic phthalate esters and metabolites activate the nuclear receptors PPARs and induce differentiation of F9 cells

Industrial plasticizers such as phthalates can induce peroxisome proliferation. Some phthalates such as di-2-ethyl-hexyl-phthalate (DEHP) and its metabolites mono-2-ethyl-hexyl-phthalate and 2-ethyl-hexanoic acid are also known teratogens. Recently, we introduced two in vitro test systems consisting of F9 teratocarcinoma cell differentiation and activation of peroxisome proliferator-activated receptor (PPAR)-ligand-binding domain in Chinese hamster ovary-reporter cells for the detection of teratogenic compounds related to the antiepileptic drug valproic acid. We now applied these methods to the class of phthalate esters and their metabolites by testing 2 diphthalate esters and 19 monophthalate esters in vitro. In the F9 cell assay only five compounds, mono-2-ethyl-hexyl-phthalate, mono-1-methyl-heptyl-phthalate, mono-benzyl-phthalate, benzyl-butyl-phthalate, and 2-ethyl-hexanoic acid were found to induce F9 cell differentiation. The other test compounds were not able to induce differentiation of F9 cells. Three compounds (mono-methyl-phthalate, mono-ethyl-phthalate, and mono-2,2-dimethyl-1-phenyl-propyl-phthalate, and phthalic acid di-methyl-ester were found not to interact with any PPARs. All other phthalate esters activated PPARs. Most compounds activated PPARalpha and PPARgamma. Interestingly PPARgamma in most cases was activated stronger than PPARalpha. Only the five test compounds, mono-2-ethyl-hexyl-phthatate, mono-1-methyl-heptyl-phthalate, mono-benzyl-phthalate, benzyl-butyl-phthalate, and 2-ethyl-hexanoic acid activated PPARdelta and interacted with a specific PPARdelta-response element. These are the same compounds that induced F9 cell differentiation and three of them are known teratogenic compounds. It is concluded that phthatate esters are acting like hormones by activating PPARs. The combination of F9 cell differentiation assay and PPARdelta activation assay detected possible teratogenic phthalate-ester and derivatives. Therefore the test systems seem useful for a screening test system in the early development of new plasticizers.