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.     

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.     

Urinary and amniotic fluid levels of phthalate monoesters in rats after the oral administration of di(2-ethylhexyl) phthalate and di-n-butyl phthalate

Two studies were designed to examine amniotic fluid and maternal urine concentrations of the di(2-ethylhexyl) phthalate (DEHP) metabolite mono(2-ethylhexyl) phthalate (MEHP) and the di-n-butyl phthalate (DBP) metabolite monobutyl phthalate (MBP) after administration of DEHP and DBP during pregnancy. In the first study, pregnant Sprague-Dawley rats were administered 0, 11, 33, 100, or 300 mg DEHP/kg/day by oral gavage starting on gestational day (GD) 7. In the second study, DBP was administered by oral gavage to pregnant Sprague-Dawley rats at doses of 0, 100, or 250 mg/kg/day starting on GD 13. Maternal urine and amniotic fluid were collected and analyzed to determine the free and glucuronidated levels of MEHP and MBP. In urine, MEHP and MBP were mostly glucuronidated. By contrast, free MEHP and free MBP predominated in amniotic fluid. Statistically significant correlations were found between maternal DEHP dose and total maternal urinary MEHP (p=0.0117), and between maternal DEHP dose and total amniotic fluid MEHP levels (p=0.0021). Total maternal urinary MEHP and total amniotic fluid MEHP levels were correlated (Pearson correlation coefficient=0.968). Statistically significant differences were found in amniotic MBP levels between animals within the same DBP dose treatment group (p<0.0001) and between animals in different dose treatment groups (p<0.0001). Amniotic fluid MBP levels increased with increasing DBP doses, and high variability in maternal urinary levels of MBP between rats was observed. Although no firm conclusions could be drawn from the urinary MBP data, the MEHP results suggest that maternal urinary MEHP levels may be useful surrogate markers for fetal exposure to DEHP.     

Effects of di-(2-ethylhexyl)-phthalate (DEHP) and its metabolites on fatty acid homeostasis regulating proteins in rat placental HRP-1 trophoblast cells.

Di-(2-ethylhexyl)-phthalate (DEHP) is a widely used plasticizer and ubiquitous environmental contaminant. The potential health hazards, including teratogenicity, from exposure to DEHP may be related to the role of DEHP or its metabolites in the trans-activation of peroxisome proliferator-activated receptors (PPARs). Fetal essential fatty acid (EFA) homeostasis is controlled by directional transfer across the placenta through a highly regulated process, including PPAR activation. Using HRP-1 rat trophoblastic cells, the effects of DEHP and two of its metabolites, mono-(2-ethylhexyl)-phthalate (MEHP) and 2-ethylhexanoic acid (EHA), on the mRNA and protein expression of the three known PPAR isoforms (alpha, beta, and gamma), fatty acid transport protein 1 (FATP1), plasma membrane fatty acid binding protein (FABPpm), and the heart cytoplasmic fatty acid binding protein (HFABP) were investigated. This study also investigated the functional effects of exposure on the uptake and transport of six long chain fatty acids (LCFAs): arachidonic acid (AA), docosahexaenoic acid (DHA), linoleic acid (LA), alpha-linolenic acid (ALA), oleic acid (OA), and stearic acid (SA). In the presence of DEHP, MEHP, and EHA, the expression of PPARalpha, PPARgamma, FATP1, and HFABP were up-regulated in a dose- and time- dependent manner, while PPARbeta and FABPpm demonstrated variable expression. The uptake rates of EFAs (AA, DHA, LA, ALA) increased significantly upon exposure, and the transport of AA (omega-6) and DHA (omega-3) were directionally induced. These results suggest that DEHP, MEHP, and EHA can influence EFA transfer across HRP-1 cells, implying that these compounds may alter placental EFA homeostasis and potentially result in abnormal fetal development.     

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.     

Effects of di(2-ethylhexyl) phthalate, a widely used peroxisome proliferator and plasticizer, on cell growth in the human keratinocyte cell line NCTC 2544

Phthalate esters are a widely used class of water-insoluble organic chemicals. The adverse effects of di(2-ethylhexyl) phthalate (DEHP) were chiefly studied in animals, while their potential toxicity to humans has not been properly evaluated. It was hypothesized that the effect of DEHP on human cells depends on the concentration, and this study examined the effects of different concentrations of DEHP on cell growth in cultured human keratinocytes NCTC 2544, together with the possible involvement of peroxisome proliferator-activated receptors (PPARs) in mediating the effects. After exposure to DEHP, the number of NCTC 2544 cells in the monolayer decreased in a concentration-dependent and time-dependent manner, whereas the cells that were detached from the monolayer increased, and died via necrosis. The decrease of cell growth was confirmed by the inhibition of pErk1, pErk2, and changes in the c-myc protein content. With regard to PPARs, the PPARbeta protein content increased, whereas PPARalpha decreased. To demonstrate the involvement of PPARbeta in inhibiting cell growth, the use of an antisense oligonucleotide against this receptor revealed the prevention of DEHP-induced cell growth inhibition. In addition, the treatment of keratinocytes with a specific ligand of PPARbeta (L165041) showed a concentration-dependent inhibition of cell growth, as with DEHP. In conclusion, the effect of DEHP on human keratinocytes is concentration dependent, and this effect is mediated via PPARs.     

Effect of phthalate esters on energy coupling and succinate oxidation in rat liver mitochondria

Isolated rat liver mitochondria were exposed to mono- and di-n-butyl phthalate (MBP and DBP) and mono- and di(2-ethylhexyl)phthalate (MEHP and DEHP) and examined for effects on mitochondrial energy-dependent processes, including oxidative phosphorylation and active K+ uptake. Additional studies on the effects of these phthalate esters on succinate oxidation and on mitochondrial membrane integrity are also included. DBP and MEHP stimulated succinate state 4 respiration, impaired K+-valinomycin induced swelling with succinate, ascorbate, or ATP as the energy sources, and inhibited succinate state 3 respiration and succinate cytochrome c reductase activity. MEHP was found to act as a non-competitive inhibitor of succinate dehydrogenase activity, with an apparent Ki = 2.4 X 10(-4) M. At concentrations which uncouple energy linked reactions, MEHP and DBP produced only slight energy-independent swelling and release of soluble proteins from isolated mitochondria. MBP caused only slight stimulation of state 4 respiration and impairment of K+-valinomycin induced swelling with each of the 3 energy sources, however, of the 4 phthalate esters, it produced the greatest energy-independent swelling and led to the greatest release of soluble mitochondrial proteins. DEHP had no apparent effect on any of these processes except for slight impairment of ATP-dependent K+-valinomycin induced swelling. It is concluded that phthalate ester toxicity in liver mitochondria is due to uncoupling of energy linked reactions and/or inhibition of succinate dehydrogenase activity. Uncoupling by MBP may involve disruption of mitochondrial membrane integrity, while uncoupling by DBP and MEHP is probably due to an increase in membrane permeability to H+ and other small ions.     

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.     

Quantitative evaluation of alternative mechanisms of blood disposition of di(n-butyl) phthalate and mono(n-butyl) phthalate in rats.

Phthalate esters are ubiquitous, low-level environmental contaminants that induce testicular toxicity in laboratory animals. The diester is rapidly metabolized in the gut to the monoester, which causes the testicular toxicity. Several physiologically based pharmacokinetic (PBPK) model structures have been evaluated for di(2-ethylhexyl) phthalate (DEHP) and mono(2-ethylhexyl) phthalate (MEHP). The objective of this study was to test these PBPK models for a less lipophilic phthalate diester, di(n-butyl) phthalate (DBP), and monoester, mono(n-butyl) phthalate (MBP). Alternate models describing enterohepatic circulation, diffusion-limitation, tissue pH gradients (pH trapping), and a simpler, flow-limited model were evaluated. A combined diffusion-limited and pH trapping model was also tested. MBP tissue:blood partition coefficients were similar when determined either experimentally by a nonvolatile, vial equilibration technique or algorithmically. All other parameters were obtained from the literature or estimated from MBP blood concentrations following intravenous or oral exposure to DBP or MBP. A flow-limited model was unable to predict MBP blood levels, whereas each alternative model had statistically better predictions. The combined diffusion-limited and pH trapping model was the best overall, having the highest log-likelihood function value. This result is consistent with a previous finding that the pH trapping model was the best model for describing DEHP and MEHP blood dosimetry, though it was necessary to extend the model to include diffusion-limitation. The application of the pH trapping model is a step toward developing a generic model structure for all phthalate esters, though more work is required before a generic structure can be identified with confidence. Development of a PBPK model structure applicable to all phthalate esters would support more realistic assessments of risk to human health from exposure to one or more members of this class of compounds.     

PPARalpha and PPARbeta are differentially affected by ethanol and the ethanol metabolite acetaldehyde in the MCF-7 breast cancer cell line.

The activity and/or the level of the peroxisome proliferator-activated receptors (PPARs) in liver and oligodendrocytes are regulated by ethanol. Despite the association between ethanol consumption and breast cancer risk, and the increasing evidence for an involvement of PPARs in some cancers, there have been no studies on the effect of ethanol or its metabolite acetaldehyde on PPARs in breast cancer. Using the MCF-7 breast cancer cell line, we examined the relationship between ethanol and its metabolite acetaldehyde on PPARalpha and PPARbeta transactivation. Ethanol (20 mM) reduced the potency of the PPARbeta ligand GW0742, evident by a rightward shift in the GW0742 dose-response curve, whereas for PPARalpha activation by GW7647, ethanol mediated its effects primarily through reducing efficacy as evidenced by a reduction in maximal response. Using the enzyme inhibitors 4-methylpyrazole and cyanamide and the metabolite acetaldehyde, we showed that PPARalpha and PPARbeta are differentially modulated by ethanol and acetaldehyde. While acetaldehyde is responsible for the inhibition of PPARalpha ligand inhibition with a concentration that inhibits 50% of activity (IC50) of 111 nM, acetaldehyde has no effect on PPARbeta or its ligand activation. Instead, inhibition of PPARbeta transactivation is mediated directly by ethanol. The differential effect of ethanol and acetaldehyde on PPARalpha and PPARbeta further underscores the differences between these receptors and may indicate the relevance of PPARs in the effects of ethanol in the human breast.     

Mono-2-ethyhexyl phthalate advancing the progression of prostate cancer through activating the hedgehog pathway in LNCaP cells

Hedgehog (Hh) pathway plays a critical role in the progression of prostate cancer (PCa), the most commonly diagnosed non-cutaneous cancer in male adults. Studies showed that di-n-butyl phthalate (DBP) could interference with the Hh pathway. Di-2-ethylhexyl phthalate (DEHP), the congener of DBP, is the major plasticizer used in plastic materials that are inevitably exposed by patients with PCa. The aim of this in vitro study was to investigate whether mono-2-ethyhexyl phthalate (MEHP, the active metabolite of DEHP) could activate the Hh pathway of LNCaP cells. Results showed that the expression of the critical gene of Hh pathway PTCH and androgen-regulated gene KLK3 was significantly decreased on 3, 6 and 9 days with Hh pathway inhibitor cyclopamine's treatment. MEHP notably up-regulated the expression of PTCH with a dose–response relationship in the presence of cyclopamine, which indicate that MEHP might target on the downstream components of Hh pathway and advance the progression of PCa through activating the Hh pathway.     

Induction of apoptosis by mono(2-ethylhexyl)phthalate (MEHP) in U937 cells

Treatment of U937 cells with mono(2-ethylhexyl)phthalate (MEHP) for 20 h led to a dose-dependent loss of cell viability, assessed by propidium iodide (PI) staining with fluorescent activated cell sorting (FACS) analysis. The cytotoxic behavior of MEHP is attributed to the induction of apoptosis. MEHP induced activation of caspase-3, internucleosomal DNA fragmentation and the morphological features of nuclear apoptosis. Analysis with LightCycler quantitative RT-PCR demonstrated the decrease of bcl-2 and increase of bax mRNA levels. Peroxisome proliferator-activated receptor (PPAR) gamma antagonists, bisphenol A diglycidyl ether (BADGE) and GW9662, significantly inhibited the MEHP-induced caspase-3 activity and apoptotic nuclear morphological changes. Furthermore, a PPARgamma ligand, rosiglitazone synergized the MEHP-induced caspase-3 activity. These results suggest that MEHP can induce apoptosis in U937 cells through modulation of the balance of bcl-2/bax in part by PPARgamma activation.     

Mono-2-ethylhexyl phthalate, a metabolite of di-(2-ethylhexyl) phthalate, causally linked to testicular atrophy in rats

Acute testicular atrophy results when appropriate dosages of di-(2-ethylhexyl) phthalate (DEHP) or its hydrolysis product mono-2-ethylhexyl phthalate (MEHP) are given to male rats. Events thought to be involved in this pathological effect also occur in cultures of testicular cells in vitro, but require MEHP rather than DEHP. Primary cultures of hepatocytes, Sertoli cells, and Leydig cells were incubated with 14C-labeled MEHP [8 microM] for up to 24 hr. No significant reduction in viability was produced under these conditions. In contrast to the hepatocytes, which extensively metabolized MEHP to a variety of products in 1 hr, the testicular cell cultures were apparently unable to metabolize MEHP (beyond a slight hydrolysis to phthalic acid by Sertoli cells) in 18-24 hr. MEHP was efficiently taken up by hepatocytes, but much less so by testicular cells. These results, combined with related observations from the literature, support the hypothesis that MEHP itself is the metabolite of DEHP responsible for testicular atrophy in rats.     

Mono(2-ethylhexyl)phthalate exhibits genotoxic effects in human lymphocytes and mucosal cells of the upper aerodigestive tract in the comet assay

Phthalic acid esters such as di(2-ethylhexyl)phthalate (DEHP) are widely used as plasticizers in PVC products manufactured for commercial, medical, and consumer purposes. Humans are exposed to phthalates originating, e.g., from blood storage bags, tubing materials, and from food-wrapping. While xenoestrogenic and chronic toxic effects of phthalates have been extensively discussed, there is little data on genotoxic effects in human cells. The alkaline comet assay was used to detect single-strand breaks and alkali labile sites of DNA after incubation of human nasal mucosal cells (n = 11) and peripheral lymphocytes (n = 11) with mono(2-ethylhexyl)phthalate (MEHP), the principal hydrolysis product of DEHP. MEHP showed a dose-dependent enhancement of DNA migration both in human mucosal cells and in lymphocytes. This effect indicates a genotoxic potential of MEHP in human mucosal cells. It confirms previous data obtained on the effect of MEHP on lymphocytes.