Effects of Di-(2-Ethylhexyl)-Phthalate (DEHP) and Its Metabolites on Fatty Acid Homeostasis Regulating Proteins in Rat Placental HRP-1 Trophoblast Cells

Yan Xu, Thomas J. Cook and Gregory T. Knipp1

Department of Pharmaceutics, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, 160 Frelinghuysen Road, Piscataway, New Jersey 08854

1 To whom correspondence should be addressed at Department of Pharmaceutics, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, 160 Frelinghuysen Road, Piscataway, NJ 08854–8022. Fax: (732) 445-3134. E-mail: gknipp{at}rci.rutgers.edu.

Received September 29, 2004; accepted December 15, 2004


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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}, ß, 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 PPAR{alpha}, PPAR{gamma}, FATP1, and HFABP were up-regulated in a dose- and time- dependent manner, while PPARß 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.

Key Words: phthalates; rat; placenta; HRP-1; fatty acid; transport; PPAR.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Di-(2-ethylhexyl)-phthalate (DEHP) is a commonly used phthalate ester plasticizer in polyvinylchloride (PVC) formulations, which have a variety of applications from food packaging to medical devices (Kavlock et al., 2002Go). DEHP readily leaches from plastic surfaces into the environment and is a ubiquitous contaminant in water, food, and soil (Kavlock et al., 2002Go). In most species, DEHP is rapidly hydrolyzed to mono-(2-ethylhexyl)-phthalate (MEHP) and 2-ethylhexanoic acid (EHA) (Albro and Lavenhar, 1989Go). Although daily human exposure to DEHP is estimated only in the range from 5.8 to 19 µg/kg/day in the general population (Kavlock et al., 2002Go), exposure in the medical settings, via plastic tubing, IV bags, etc., may increase up to 167.9 mg/day (Kavlock et al., 2002Go). This is of particular concern to neonates, who, upon exposure to DEHP-containing medical devices, may have a higher incidence of disorders including hypospadism and neurological defects (Latini, 2000Go).

Human exposure to DEHP may begin in the mother's womb, where DEHP has been demonstrated to readily cross the placenta and accumulate in the fetus (Kihlstrom, 1983Go; Singh et al., 1975Go). While the pathological consequences of DEHP exposure in human are uncertain, DEHP is an established reproductive and developmental toxicant and hepatocarcinogen in rodents, where respiratory distress and developmental disorders in fetal/neonatal testis, liver, and other major organs have been observed (Cammack et al., 2003Go; Kavlock et al., 2002Go; Magliozzi et al., 2003Go; Moore et al., 2001Go). DEHP, MEHP, and EHA belong to a diverse class of peroxisome proliferator chemicals (PPCs). Administration of PPCs to rodents results in a pleiotropic response characterized by peroxisome proliferation as well as numerous alterations in gene translation of proteins involved in essential fatty acid (EFA) transport, metabolism, and lipid homeostasis (Lemberger et al., 1996Go; Simpson, 1997Go). Such effects have been identified to be mediated by the peroxisome proliferator-activated receptors (PPARs), established regulators of cellular EFA homeostasis (Lemberger et al., 1996Go). Three PPAR isoforms ({alpha}, ß, and {gamma}) have been identified in various tissues including placenta (Lemberger et al., 1996Go; Wang et al., 2002Go). They regulate the transcription of target genes by binding to PPAR response elements (PPRE) as heterodimers with retinoic X receptors (Bocher et al., 2002Go; Lemberger et al., 1996Go). PPAR{alpha} has been demonstrated to play a role in regulating lipid catabolism, whereas PPAR{gamma} controls adipocyte differentiation and lipid storage (Bocher et al., 2002Go; Lemberger et al., 1996Go). Although PPARß is less understood, it might be a mediator in the control of brain lipid metabolism, fatty acid induced adipogenesis, and atherogenic inflammation (Bocher et al., 2002Go).

Fatty acids, especially EFAs and their long chain polyunsaturated fatty acids derivatives (LCPUFAs), are involved in energy storage and serve as obligatory constituents of biological membranes and precursors of intra- and intercellular signaling molecules in the body (Innis, 2003Go; Uauy et al., 1999Go). Mammals are unable to synthesize EFAs, and therefore, the fetus relies on maternal dietary intake of EFAs and their directional placental transfer to obtain sufficient quantities for normal development (Dutta-Roy, 2000Go; Haggarty, 2002Go). An active, directional mechanism mediated by several distinct fatty acid transporters has been suggested to contribute to this directionality, which should compensate for the limited transfer capacity by simple free diffusion (Dutta-Roy, 2000Go; Haggarty, 2002Go). These fatty acid transfer-conferring proteins have been found in the rat and human placentas as well as in vitro trophoblast models (Dutta-Roy, 2000Go; Haggarty, 2002Go; Knipp et al., 1999Go, 2000Go), and include fatty acid transport protein 1 (FATP1), plasma membrane fatty acid binding protein (FABPpm), and heart cytoplasmic fatty acid binding protein (HFABP).

It is not clear how fetal exposure to DEHP and its metabolites leads to subsequent health hazards. Therefore, the present study was conducted to assess if the teratogenicity of these xenobiotics is related to placental handling of EFAs via PPAR trans-activation. The HRP-1 rat trophoblast model was utilized, which exhibits characteristics resembling those of the trophoblasts in the labyrinthine zone (rat placenta transport barrier) (Soares et al., 1987Go). HRP-1 cells have been previously demonstrated to form a monolayer of viable, polarized, fully differentiated cells and to be a useful in vitro model of the placental barrier to study transport of nutrients, including fatty acids (Shi et al., 1997Go), glutamate (Novak et al., 2001Go), and glucose (Das et al., 1998Go). In this study, the effects of DEHP, MEHP, and EHA on the expression of the three PPAR isoforms ({alpha}, ß, and {gamma}), FATP1, FABPpm, and HFABP were investigated, as well as the functional influences of these compounds on the uptake and transport of long chain fatty acids.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Reagents.
1-14C-Arachidonic acid (55mci/mmol, AA), 1-14C-linoleic acid (55mCi/mmol, LA), and 1-14C-{alpha}-linolenic acid (55mCi/mmol, ALA) were obtained from American Radiolabeled Chemical Co. (St. Louis, MO). 1-14C-Docosahexaenoic acid (56 mCi/mmol, DHA), 1-14C-stearic acid (56 mCi/mmol, SA), 1-14C-oleic acid (54 mCi/mmol, OA), and 3H-mannitol (26.3 mCi/mmol) were purchased from Moravek Biochemicals (Brea, CA). Unlabeled fatty acids, DEHP, EHA, fenofibrate, dimethyl sulfoxide (DMSO), and TRIzol Reagents for RNA extraction were obtained from Sigma Chemical Company (St. Louis, MO). MEHP was purchased from TCI America (Portland, OR). Reverse transcriptase-polymerase chain reaction (RT-PCR) kits and the 100bp ladder were obtained from Invitrogen Life Technologies (Gaithersburg, MD). Reagents for polyacrylamide gel electrophoresis and immunoblotting supplies were purchased from Bio-Rad (Hercules, CA). Polyclonal antibodies specific for PPAR{alpha}, ß, and {gamma}, FATP1, and HFABP were purchased from Santa Cruz Biotechnology (San Francisco, CA), while the polyclonal antibody to FABPpm was generously provided by Dr. Joseph Mattingly, University of Missouri, Kansas City, MO. The BCA protein assay kits and the enhanced chemiluminescent detection reagent kits were obtained from Pierce Chemical Company (Rockford, IL). Transwell® chambers (12-well cluster plates, polycarbonate, 0.4 µm pore size) and 24-well tissue culture plates were purchased from Corning Costar Corp (Cambridge, MA). Unless otherwise noted, all other chemicals and reagents were purchased from Fisher Scientific (Pittsburgh, PA) and used as received.

Cell culture.
The HRP-1 trophoblastic cell line was a generous gift from Dr. Michael J. Soares (University of Kansas Medical Center, Kansas City, KS) and was cultured as described previously (Knipp et al., 2000Go). The 24-well culture plates and polycarbonate Transwell clusters were coated with type I rat tail collagen (Bio-Rad) at 5 µg/cm2 on the same day of cell seeding. For the expression studies, the HRP-1 trophoblastic cells were seeded at 5 x 104 cells/cm2 in T-25 cm2 flasks. For the uptake studies, the HRP-1 trophoblastic cells were seeded at 2.5 x 104 cells/cm2 in 24-well tissue culture plates. For the transport studies, the cells were seeded at 7.5 x 104 cells/cm2 on Transwell 12-well-clusters. The medium was aspirated and replaced with fresh medium every day. The expression/uptake or the transport studies were performed when the cells came to 90% to 95% confluence. All cells used in these studies were between passages 15 and 25.

Time- and dose-dependent exposure to xenobiotics.
Confluent HRP-1 cells were treated with DEHP, MEHP, and EHA diluted 1000-fold in assay buffer from a stock solution in DMSO at the indicated concentrations and harvested at the respective time periods for total RNA isolation and/or whole protein extraction as well as for the uptake and transport studies. Cells treated with 0.1% DMSO were used as negative control.

For the time-dependent study, HRP-1 cells were treated with 50 µM of DEHP, MEHP, and EHA and harvested at 2, 4, 8, 12, and 24 h. For the dose-dependent study, HRP-1 cells were treated with DEHP, MEHP, and EHA at the concentrations of 25, 50, 100, and 200 µM and harvested at 4 h and 12 h for mRNA and protein expression assays, respectively.

Semiquantitative RT-PCR.
Total RNA isolation, RT-PCR, and gel electrophoresis were performed as described previously (Wang et al., 2002Go) with optimized conditions and normalized to ß-actin expression using gene-specific primers (Table 1). PCR reaction products were electrophoretically separated with a 1.5% agarose gel. Ethidium bromide stained bands were visualized, and the resulting densitometry analysis was performed using a NucleoTech 920 Image detection system (NucleoTech Corporation, San Mateo, CA). The molecular weight for each band was determined in reference to a 100 bp ladder.


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TABLE 1 Gene-Specific Forward and Reverse Primer Sequences

 
Protein extraction.
Whole cell protein was solubilized and homogenized with the modified radioimmunoprecipitation (RIPA) assay buffer (50 mM Tris-Cl buffer (pH 7.4), 150 mM NaCl, 0.1% SDS, 1 mM EDTA, 1% Triton X-100, 0.5 mM PMSF, 2 mM DTT, 0.1% protease inhibitor cocktail) at 4°C. After centrifugation at 14,000 rpm for 20 min at 4°C, supernatant lysates were collected, and the protein concentration was measured using the BCA assay reagent according to the manufacturer's protocol.

Protein analysis.
Western and immunoblotting analyses were performed to check the specificity of the antibodies and to quantitate protein expression levels according to the manufacturer's protocol (Bio-Rad), respectively. Briefly, 30 µg of protein was loaded per lane or per well. In Western blotting experiments, the protein lysates were separated in 8 to 15% SDS polyacrymlamide gels under reducing conditions and transferred to nitrocellulose membrane (Millipore, Bedford, MA). For immunoblotting, the protein lysates were loaded into the well and allowed to sit for 10 min; the lysate was then transferred to the nitrocellulose membrane under vacuum. The resulting membranes were handled in the same manner by first blocking with 5% nonfat milk and then incubating overnight with the respective primary antibody (1:500 to 1:2000 with respect to each antibody, as described previously (Knipp et al., 2000Go; Wang et al., 2002Go). The membranes were then washed 3 x 7 min with TBS/T (1x TBS containing 0.05% Tween 20) and then incubated for 1 h with a 1:5000 dilution of the appropriate HRP-conjugated secondary antibodies (Sigma Aldrich). The specific protein bands were visualized using chemiluminescence detection with the Pierce Femto Signal Western Reagent kit and recorded by the NucleoTech 920 Image detection system (NucleoTech Corporation, San Mateo, CA). To normalize the signal for these proteins, ß-actin levels were probed as internal controls.

Uptake assays.
Both the uptake and transport studies were performed in Hank's balanced salt solution (HBSS, Mediatech Inc.) containing 136.7 mM NaCl, 4.167 mM NaHCO3, 0.385 mM Na2HPO4, 0.441 mM KH2PO4, 0.952 mM CaCl2, 5.36 mM KCl, 0.812 mM MgSO4, 5.5 mM D-glucose) supplemented with 10 mM HEPES (pH 7.4). The stop buffer was prepared with 0.1% bovine serum albumin (BSA, Sigma Chemical Co.) and 200 µM phloretin (Sigma Chemical Co.) diluted in uptake/transport buffer (Xu et al., in press). The uptake and transport assay buffers were freshly prepared and comprised of HBSS with 200 µM of different LCFA (fatty acid: BSA molar ratio = 1:1) containing 0.1 µCi/ml of the appropriate 14C-radiolabeled LCFA, as previously described (Campbell et al., 1997Go; Xu et al., in press). The uptake study was performed for 15 min and stopped by rapidly washing the cells three times with ice-cold stop buffer (750 µl/well) followed by one rinse with ice-cold PBS (750 µl/well). The cells were then solubilized with 0.2 N NaOH (250 µl/well) and neutralized with 0.2 N HCL (250 µl/well). An aliquot of 425 µl was removed for scintillation counting, and a 25 µl aliquot for the BCA protein assay.

Transport Assays
Confirming monolayer integrity.
HRP-1 cell monolayer integrity was evaluated with trans-epithelial electrical resistance (TEER) measurements and mannitol permeability studies. The TEER values of the cell monolayers were determined by electrical resistance measurements at 37°C, using the Epithelial Voltohmmeter (EVOM, World Precision Instruments, Sarasota, FL) and corrected by the values obtained across the blank collagen-coated Transwell. Permeability studies of mannitol were performed in the influx (i.e., apical to basolateral) and efflux (i.e., basolateral to apical) directions to check the cell monolayer integrity.

Long chain fatty acid transport assays.
Transport studies were performed in the influx (A->B, AB) and secretory efflux (B->A, BA) directions with the respective 14C-labeled fatty acid and 3H-mannitol, the latter as a marker for monolayer integrity as previously described (Xu et al., in press). For all of the studies, the apical and basolateral test solution volumes were 0.5 and 1.5 ml, respectively, and maintained at 37°C. Each cell monolayer used in the studies was rinsed and equilibrated for 30 min with the transport buffer. Following the addition of the donor solution, samples from the receiver side were removed at 15, 30, 45, 60, 75, 90, and 120 min and replaced with an equal volume of prewarmed transport buffer. The apical and basolateral sampling volumes were 50 µl and 150 µl, respectively, and all sample concentrations were corrected for dilution factors. At the end of the study, 50 µl of the donor side sample was collected to determine mass balance.

Data analysis.
All experiments were repeated a minimum of three times. Data generated from RT-PCR and slot blot were quantitated by densitometry (Gel ExpertTM software program, NucleoTech Corp., San Mateo, CA) and normalized to ß-actin expression.

The apparent permeability coefficients (Papp) were calculated using Equation 1:

(1)
Where is the slope of the linear region of amount of compound in receiver compartment versus time plot, VD is the volume of the donor side, MD0 is the starting donor amount, Mcells is the amount of compound retained by the membrane/cells, and A is the surface area of the exposed cell monolayer (Youdim et al., 2003Go).

The permeability coefficients for the cell monolayer, Pmono, were calculated using Equation 2:

(2)
Where Papp is the apparent permeability coefficient for the collagen-coated polycarbonate membranes in the presence of HRP-1 cell monolayer, and Pblank is the apparent permeability coefficient for the collagen-coated polycarbonate membranes only (Xu et al., in press). After obtaining the influx monolayer permeability, Pmono,influx, and the efflux monolayer permeability, Pmono,efflux, the permeability influx ratios, R, were determined using Equation 3:

(3)

All the data were presented as means ± standard deviation (SD) of at least three replicates. Statistical significance relative to vehicle controls, shown in each figure, was determined using a one-way ANOVA followed by the Student t-test, where p values <0.05 were considered to be statistically significant.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Expression of PPAR Isoforms and Fatty Acid Transferring Protein in HRP-1 Cells
The basal expressions of each target gene and protein were confirmed by RT-PCR (Fig. 1, Panel A) and Western blot analyses (Fig. 1, Panel B), respectively, where specific bands at the expected PCR products size and molecular mass were observed for PPAR{alpha}, ß, and {gamma}, FATP1, FABPpm, and HFABP in HRP-1 cells. In order to normalize both mRNA and protein expression across different samples, ß-actin was used as an internal control. Changes in mRNA level at 4 h and protein expression at 12 h upon xenobiotic exposure versus control groups were similar to those shown in the semiquantitative RT-PCR and immunublotting assays (see below). In the presence of DEHP and its metabolites, the expression of PPAR{alpha}, PPAR{gamma}, FATP1, and HFABP were up-regulated, while PPARß and FABPpm levels did not appear to be significantly altered.



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FIG. 1. Representative RT-PCR gel electrophoresis (Panel A) and Western blot (Panel B) results. Confluent HRP-1 cells were treated with 50 µM of DEHP, MEHP, and EHA diluted in assay buffer from a 1000-fold stock in DMSO and harvested at 4 and 12 h for total RNA and whole cell protein isolation, respectively. Expression of PPAR{alpha}, PPARß, PPAR{gamma}, FATP1, FABPpm, and HFABP mRNA and protein normalized to the ß-actin internal control was assayed using semiquantitative RT-PCR and Western blot as described under "Materials and Methods." The signals exhibited the expected PCR product sizes and molecular masses, as previously described (Bocher et al., 2002Go; Knipp et al., 2000Go; Yokoyama et al., 2003Go).

 
Effects of DEHP, MEHP, and EHA on the Expression of PPAR{alpha}, ß, and {gamma}
The time-dependent changes in the mRNA levels of PPAR{alpha} were induced dramatically upon exposure to each of the studied compounds versus controls at the 2 h and 4 h (p < 0.001 for all) time points. EHA was the strongest inducer compared to MEHP and DEHP, with the highest induction level of 2.4-fold at 4 h (p < 0.01) (Fig. 2, Panel A1). PPAR{alpha} expression was not significantly altered from 8 to 24 h (Fig. 2, Panel A1). In the dose-dependent study, a pronounced increase in PPAR{alpha} versus controls was observed at 50 µM for each compound (p < 0.05) and to a lesser extent at 100 µM (p < 0.05), with little or no difference at 200 µM (Fig. 3, Panel A1). At the lowest concentration studied (25 µM), only EHA induced PPAR{alpha} expression (p < 0.01), whereas DEHP and MEHP had no statistically significant effects at this concentration.



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FIG. 2. Time-dependent expression of PPAR{alpha} (Panel A1, A2), PPARß (Panel B1, B2), PPAR{gamma} (Panel C1, C2) mRNA, and protein induced by DEHP and its metabolites. Confluent HRP-1 cells were treated with 50 µM of DEHP, MEHP, and EHA from a 1000-fold stock in DMSO and harvested at the indicated time. Cells treated with 0.01% DMSO in media were used as control. Total RNA and whole cell protein were isolated, and expression of PPAR{alpha}, PPARß, and PPAR{gamma} mRNA and protein normalized to the ß-actin internal control were assayed using semiquantitative RT-PCR and immublotting as described under "Materials and Methods." Data shown were relative arbitrary units (R.A.U.) with a value of 100 for the respective DMSO controls at each specific time period (means ± SD, n = 3 or 4). Statistics at TOP of each panel: overall ANOVA for each time period; Within-figure numbers: Student t-test analysis relative to controls.

 


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FIG. 3. Dose dependent expression of PPAR{alpha} (Panel A1, A2), PPARß (Panel B1, B2), PPAR{gamma} (Panel C1, C2) mRNA, and protein induced by DEHP and its metabolites. Confluent HRP-1 cells were treated with DEHP, MEHP, and EHA diluted from a 1000-fold stock in DMSO at the concentrations of 25 µM, 50 µM, 100 µM, and 200 µM and harvested at 4 and 12 h for mRNA and protein expression study, respectively. Cells treated with 0.1% DMSO were used as control. Immediately after treatments, total RNA and whole cell protein were isolated, and expression of PPAR{alpha}, PPARß, and PPAR{gamma} mRNA and protein normalized to the ß-actin internal control was assayed using semi-quantitative RT-PCR and immublotting as described under "Materials and Methods." Data shown were relative arbitrary units (R.A.U.) with a value of 100 for the respective DMSO controls at each specific time and concentration point (means ± SD, n = 3 or 4). Statistics at TOP of each panel: overall ANOVA for each time period; Within-figure numbers: Student t-test analysis relative to controls.

 
PPAR{gamma} mRNA expression was significantly increased by exposure to the xenobiotics up to 4 h, but the expression was not altered at later periods (Fig. 2, Panel C1). At 4 h, DEHP markedly up-regulated the expression of PPAR{gamma} at only 25 µM (p < 0.05). In contrast, a relatively higher concentration of 50 µM was needed for MEHP and EHA to achieve a similar level of induction (Fig. 3, Panel C1).

Consistent with the mRNA studies, protein expression of PPAR{alpha} and PPAR{gamma} increased with xenobiotic exposure when contrasted with the control cells, reaching statistical significance at 50 µM concentration of each reagent at 8 h (for PPAR{gamma}, p < 0.001) (Fig. 2, Panel C2) and 12 h (for PPAR{alpha}, p = 0.005) (Fig. 2, Panel A2). Similar to the dose-dependent induction patterns observed in the mRNA experiments, PPAR{alpha} and PPAR{gamma} protein expression were highest at 50 or 100 µM (Fig. 2 and 3, Panel A2 and C2).

The mRNA expression of PPARß was significantly induced at 2 and 4 h (p < 0.05) (Fig. 2, Panel B1) at the concentration of 50 µM for MEHP and DEHP, respectively. However, the induction did not result in a concurrent statistically significant increase in protein expression (Fig. 2, Panel B2) with one exception. At 12 h with 100 µM MEHP, PPARß protein expression had significantly increased versus controls (1.4-fold induction, p < 0.05) (Fig. 3, Panel B2). Thus, despite the increase observed at several time/dose points, PPARß seemed to be largely unchanged.

Effects of DEHP, MEHP, and EHA on the Expression of FATP1, FABPpm, and HFABP
As illustrated in Panel A1 of Figure 4, exposure to 50 µM of DEHP and its metabolites had dramatic effects on the mRNA expression of FATP1 at 4 h, and to a slightly less extent at 8 h (Fig. 4, Panel A1). The initial induction time points were generally later than those observed for PPAR{alpha} and PPAR{gamma} (2 or 4 h). The peak FATP1 mRNA levels noted for DEHP (2.6-fold, p < 0.001) and MEHP (2.6-fold, p < 0.01) at 4 h were higher in contrast to EHA (1.75-fold, p < 0.05). FATP1 mRNA expression declined at 24 h upon exposure to 50 µM EHA (p < 0.05), which was similar to the effects observed with PPAR{alpha} and PPAR{gamma} (Fig. 2, Panel A1 and C1). FATP1 protein expression reached statistically significant induction levels at 12 and 24 h (p < 0.05), although levels appeared to trend upwards since 4 h. Obvious dose-dependent changes in response to the xenobiotics were determined, with peak levels at 50 and 100 µM for both mRNA (p < 0.05) (Fig. 5, panel A1) and protein (p < 0.05) (Fig. 5, panel A2) expressions of FATP1.



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FIG. 4. Time-dependent expression of FATP1 (Panel A1, A2), FABPpm (Panel B1, B2), HFABP (Panel C1, C2) mRNA, and protein induced by DEHP and its metabolites. Confluent HRP-1 cells were treated with 50 µM of DEHP, MEHP, and EHA diluted from a 1000-fold stock in DMSO and harvested at the indicated period of time. Cells treated with 0.1% DMSO were used as control. Total RNA and whole cell protein were isolated, and expression of FATP1, FABPpm, and HFABP mRNA and protein normalized to the ß-actin internal control was assayed using semiquantitative RT-PCR and immublotting as described under "Materials and Methods." Data shown were relative arbitrary units (R.A.U.) with a value of 100 for the respective DMSO controls at each specific time period (means ± SD, n = 3 or 4). Statistics at TOP of each panel: overall ANOVA for each time period; Within-figure numbers: Student t-test analysis relative to controls.

 


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FIG. 5. Dose-dependent expression of FATP1 (Panel A1, A2), FABPpm (Panel B1, B2), and HFABP (Panel C1, C2) mRNA induced by DEHP and its metabolites. Confluent HRP-1 cells were treated with DEHP, MEHP, and EHA diluted from a 1000-fold stock in DMSO at the concentrations of 25 µM, 50 µM, 100 µM, and 200 µM and harvested at 4 and 12 h for mRNA and protein expression study, respectively. Cells treated with 0.01% DMSO were used as control. Total RNA and whole cell protein were isolated, and expression of FATP1, FABPpm, and HFABP mRNA and protein normalized to the ß-actin internal control was assayed using semiquantitative RT-PCR and immublotting as described under "Materials and Methods." Data shown were relative arbitrary units (R.A.U.) with a value of 100 for the respective untreated DMSO controls at each specific time and concentration point. (means ± SD, n = 3 or 4). Statistics at TOP of each panel: overall ANOVA for each time period; Within-figure numbers: Student t-test analysis relative to controls.

 
The expression patterns observed for HFABP were similar to those observed with FATP1 in the presence of the xenobiotics, with the magnitude of induction being lower (Figs. 4 and 5, Panel C1 and C2). HFABP mRNA expression increased about 20% to 60% at 4 and 8 h (p < 0.05), while no notable differences were witnessed at 2 or 24 h (Fig. 4, Panel C1 and C2). Accordingly, a significant change of protein expression was observed from 8 to 24 h (p < 0.05). The effects of DEHP on HFABP expression increased steadily up to 24 h. MEHP and EHA exposure led to peak HFABP protein levels at 12 and 8 h, respectively. The dose-dependent effects of DEHP, MEHP, and EHA on HFABP mRNA and protein expression demonstrated peak induction levels at 50 µM or 100 µM (p < 0.05) (Fig. 5, Panel C1 and C2). In contrast to FATP1 and HFABP, the expression of FABPpm, another plasma membrane fatty acid transporter, was not altered in a statistically significant manner in any of the studies conducted with DEHP and its metabolites (Figs. 4 and 5, Panel B1 and B2).

Effects of DEHP, MEHP, and EHA on the Uptake of Long Chain Fatty Acid
To confirm that a concurrent functional change was occurring with the observed mRNA and protein changes, functional uptake/transport studies were performed after 24-h exposure to 50 µM DEHP, MEHP, and EHA, respectively. In addition, 100 µM of fenofibrate, a well-established PPAR{alpha} agonist (Guay, 2002Go) was used as a positive control, and 0.01% DMSO as negative control. Several representative LCFAs including AA (20:4, {omega}-6), DHA (22:6, {omega}-3), LA (18:2, {omega}-6), ALA (18:3, {omega}-3), OA (18:1, {omega}-9) and SA (18:0, saturated) were selected for these studies. Time-dependent uptake studies revealed that each of the respective LCFAs demonstrated linear uptake kinetics until 30 min, which led to the selection of 15 min for the subsequent uptake studies (data not shown). The physiological, nonesterified fatty-acid-to-albumin ratio found in the human maternal plasma at term is about 1.3:1, and one BSA molecule has the capacity to binding up to six LCFA molecules (Benassayag et al., 1997Go). The subsequent experiments were performed with a 1:1 molar ratio of fatty acid to BSA.

Consistent with previous reports (Campbell et al., 1997Go; Dutta-Roy, 2000Go), the uptake rates of the polyunsaturated fatty acids (LA and ALA) were higher than those of the saturated (SA) and monounsaturated (OA) fatty acids of the same chain length (p < 0.05 for all comparisons) (Fig. 6). For the {omega}-6 series of AA and LA, the uptakes rates decreased significantly (p < 0.01) by 65.5% when the chain length increased from C18 (LA) to C20 (AA). A similar trend was observed for the {omega}-3 series, where the uptakes rates of DHA (C22) decreased 63% (p < 0.01) compared with ALA (C18) (Fig. 6). In addition, the uptake rate of AA was statistically higher than that of DHA (p < 0.01), with a similar phenomenon observed for LA versus ALA (p < 0.05) (Fig. 6). These results suggest that the {omega}-6 family (AA, LA) demonstrated higher uptake rates than {omega}-3 family (DHA, ALA) in these untreated samples.



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FIG. 6. Effects of DEHP and its metabolites on long chain fatty acid uptake. Confluent HRP-1 cells were treated with 50 µM of DEHP, MEHP, and EHA from a 1000-fold stock in DMSO and harvested at 24 h. Cells treated with 0.01% DMSO were used as negative control, and cells treated with fenofibrate (100 µM) as positive control. The 15-min uptake of 200 µM of fatty acid (molar ratio of fatty acid: BSA = 1:1) was assayed. Data shown were means ± SD, n = 3 or 4. Statistics at TOP of each panel: overall ANOVA for each time period; Within-figure numbers: Student t-test analysis relative to controls.

 
Figure 6 also demonstrated the effects of DEHP, MEHP, and EHA on the functional uptake of the studied six LCFAs in HRP-1 cells. For the {omega}-6 family, the uptake rates of the polyunsaturated AA were significantly induced under the treatment of each PPC (p < 0.01), with the highest induction of almost two-fold observed for MEHP (p < 0.01). However, LA, the parent fatty acid for AA in the metabolism pathway with a shorter chain length and lesser degree of saturation, was not significantly increased under these treatments relative to the control cells. An opposite trend was observed with chain length in the {omega}-3 series, where DHA was not significantly altered by DEHP, MEHP, or EHA, while ALA was induced 57, 44, and 97% under the treatment of DEHP (p < 0.05), MEHP, and EHA (p < 0.01), respectively. In contrast, there were no obvious differences observed for the saturated SA and monounsaturated ({omega}-9) OA (Fig. 6).

Effects of DEHP, MEHP, and EHA on the Transport of LCFA
TEER values and mannitol permeability were utilized to confirm cell monolayer integrity. In our preliminary studies, TEER increased to a maximum of 50 to 60 {Omega}·cm2 at 4 to 5 days post-seeding and then leveled off (data not shown). Thus, 50 {Omega}·cm2 was chosen as the critical point of cell integrity and selection of the Transwells for subsequent fatty acid transport studies. Mannitol permeability was also investigated in both the influx and efflux directions. To further ensure the cell integrity upon exposure to phthalates, all of our fatty acid transport studies were performed with 14C-fatty acid and 3H-mannitol. The mannitol permeabilities (3–5 x 10–5 cm/s) did not change under the exposure to DEHP, MEHP, and EHA.

AA and DHA were selected as representative of the {omega}-3 and {omega}-6 EFAs. Since they are polyunsaturated EFAs, the possibilities of appearance of metabolites in the donor and receiver chambers were investigated at the end of the transport study by reverse-phase high performance liquid chromatography (HPLC) in our preliminary studies. The fact that no metabolites were found (data not shown) excluded the potential disturbance of EFA metabolism from transport study. In addition, the mass balance matched very well (95 to 99%).

Although the influx (Pmono,influx) and efflux (Pmono,efflux) permeabilities of DHA were higher than those of AA in the untreated control cells, neither of them demonstrated obvious directional transport, with influx permeability ratios (Pmono,influx/Pmono,efflux) of 0.90 ± 0.07 and 1.03 ± 0.39 for AA and DHA, respectively. However, upon treatment with DEHP and its metabolites, the influx permeability ratios of AA and DHA were differentially induced (Fig. 7), although Pmono,influx and Pmono,efflux of AA and DHA were both elevated (data not shown). Under the exposure to the metabolites of MEHP and EHA, the influx ratio of AA was elevated significantly relative to the control cells, with 13.2 (p < 0.01) and 7.9% (p < 0.001) induction, respectively. DEHP treatment also seemed to slightly induce the influx ratio of AA (7.7%), but did not reach statistical significance (Fig. 7, Panel A). However, with DHA, DEHP elicited a significant increase in the influx ratio (76%, p < 0.01) (Fig. 7, Panel B).



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FIG. 7. Effects of DEHP and its metabolites on AA (Panel A) and DHA (Panel B) transport. Confluent HRP-1 cells were treated with 50 µM of DEHP, MEHP, and EHA diluted in assay buffer from a 1000-fold stock in DMSO for 24 h. Cells treated with DMSO alone were used as negative control and cells treated with fenofibrate (100 µM) as positive control. The transport of 200 µM of fatty acid (molar ratio of fatty acid: BSA = 1:1) was assayed over a 2-h period. Data shown are means ± SD, Within-figure numbers: Student t-test analysis relative to controls.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The placenta is a vital organ that regulates the delivery of nutrients (e.g., fatty acids) to the fetus and thus guides proper fetal development. The placenta is also the first fetal-arising organ that can be affected by exposure to environmental xenobiotics (e.g., DEHP and its metabolites). In these studies, the HRP-1 cell line was utilized as an in vitro placental model to investigate the effects of DEHP, MEHP, and EHA on the expression of proteins known to participate in the regulation of cellular fatty acid homeostasis. The HRP-1 trophoblasts were originally derived from explants of day 11–12 normal rat chorioallantoic placental tissues (Soares et al., 1987Go) and mimick the early cytotrophoblast progenitors that are analogous to those found in the labyrinthine zone during the critical stages of organogenesis in the rat (day 9–16, where day 18–60 are critical for human development) (Juchau et al., 1992Go). During this stage, the conceptus as a whole appears to exhibit a high degree of sensitivity to the potentially deleterious effects of exposure to xenobiotics, as well as exposure to altered EFA homeostasis (Bocher et al., 2002Go; Innis, 2003Go; Juchau et al., 1992Go; Uauy et al., 1999Go).

The concentrations of xenobiotics used in this study were in the range of 25 to 200 µM (9.76 to 78.1 µg/ml). This range is higher than what was observed in human maternal plasma at term in health subjects, where the mean DEHP and MEHP concentrations were 1.15 ± 0.81 and 2.05 ± 1.47 µg/ml, respectively (Latini et al., 2003Go). However, patients having a long-term exposure to DEHP-containing devices were reported to have a whole blood DEHP concentration of about 70 to 80 µg/ml, and neonatal exposure to DEHP following exchange transfusion with PVC catheters could increase blood levels to the range of 13.2 to 84.9 µg/ml (Kavlock et al., 2002Go). Furthermore, to correlate toxic effects observed in animals with acceptable levels of exposure for humans, 10-fold increases in DEHP exposure are commonly used for the inter- and intraspecies variability.

Differences in DEHP toxicities have been demonstrated to be organ/tissue and species specific. It is well understood that DEHP produces a range of hepatic effects, which is believed due to the activation of PPAR{alpha} (Kavlock et al., 2002Go). However, the testicular, renal, developmental, and other extrahepatic toxicities exhibited by DEHP have been demonstrated to be independent of PPAR{alpha}, as suggested from the PPAR{alpha} knockout mouse studies (Peters et al., 1997Go). Considering that the metabolites of DEHP, especially MEHP, activate both PPAR{alpha} and PPAR{gamma} in extrahepatic cell lines and/or tissues and is a stronger teratogen in mice (Kavlock et al., 2002Go; Lampen et al., 2003Go; Maloney et al., 1999Go), it is feasible that DEHP acts through its metabolites to activate a PPAR-mediated signaling pathway. In accordance with this concept, our experiments did demonstrate an induction of PPAR{alpha} and PPAR{gamma} in a time- and dose-dependent manner (Figs. 2 and 3, Panel A and C) in HRP-1 cells. Furthermore, a relatively higher dose and longer exposure period of DEHP was needed compared to MEHP and EHA to obtain the same level of gene/protein activation. It should be noted that the responses of PPAR{alpha} and PPAR{gamma} to DEHP, MEHP, and EHA were similar, but not exactly concordant, which suggests different activities of the two isoforms in placental EFA transport/metabolism. PPAR{gamma} knockouts are known to be embryonic lethal between day 9.5 and 10.5 of gestation due to improper placentation (Barak et al., 1999Go), suggesting the importance of this isoform in regulating fetal development.

There was no consistently significant pattern of PPARß expression regulation by DEHP, MEHP, and EHA (Figs. 2 and 3, Panel B), which is in accordance with previous reports (Kavlock et al., 2002Go). However, a recent investigation by Lampen et al. (2003)Go demonstrated that MEHP and EHA activated mouse and human PPARß as well as PPAR{alpha} and PPAR{gamma} in an embryonic stem cell line. Actually, PPARß was also essential in placentation, because its deficiency resulted in frequent embryonic lethality (Barak et al., 2002Go). Further study is needed to elucidate the regulation of PPARß by phthalates and other xenobiotics.

FATP1 and FABPpm are cell surface proteins involved in placental fatty acid uptake and transport (Knipp et al., 1999Go, 2000Go), This study suggests that the mRNA and protein expression of FABPpm did not change upon administration of DEHP and its metabolites (Figs. 4 and 5, Panel B), which is consistent with a lack of PPAR regulation of FABPpm reported in the literature (Motojima et al., 1998Go). Considering that FABPpm is abundantly expressed throughout the rat chorioallantoic placenta, it probably plays a generalized compensatory function in facilitating fetal fatty acid uptake (Knipp et al., 1999Go, 2000Go). Although Motojima et al. (1998)Go reported that DEHP did not activate either FATP1 or FABPpm in mouse liver, this current investigation demonstrated that FATP1 was significantly induced by DEHP, MEHP, and EHA by 1.7- to 2.5-fold. Many factors might contribute to the inconsistency of induction response (e.g., specifies difference, cell line/tissue utilized, experimental conditions), which reflects the complexity of the activation mechanism.

HFABP is a member of the cytoplasmic fatty acid binding protein family, a large family containing small cytoplasmic proteins responsible for the intracellular trafficking of fatty acids, and is believed to play an integral role in the metabolism, transport, and membrane incorporation of fatty acids (Knipp et al., 1999Go; Soares et al., 1987Go). It was suggested that HFABP might participate in the placental transfer of fatty acids to the fetus due to its specific distribution to the labyrinthine zone of the rat placenta (Knipp et al., 2000Go). The expression levels of the HFABP mRNA and protein were up-regulated in a dose- and time-dependent manner when exposed to DEHP, MEHP, and EHA. It should be noted that the maximum fold change in levels observed for HFABP were lower and occurred at a later time when compared to FATP1. These differences might suggest differential fatty acid transport regulation and/or the possible involvement of other unidentified regulators, e.g., PPAR{gamma} co-activator 1 (PGC-1) (Storey, 2003Go). Interestingly, the consistent effects of DEHP and its metabolites on FATP1 and HFABP did suggest coordinated regulation of their expression, which has been shown to occur through PPAR{gamma} transcriptional regulation.

Notably, induction of the mRNA of the majority of these target genes in this study reached peak levels at 2 or 4 h and then diminished to the levels below those of the control cells at 24 h. The highest protein expression levels were observed at 12 or 24 h, consistent with the nature of post-transcriptional events. While this has not been extensively investigated here, we did see some qualitative indications of apoptosis at 12 or 24 h in our study, including shrinkage of the cell membrane and blebbing on the membrane surface under the microscope (data not shown). This may explain the reduction in the mRNA levels at the 24-h time period. It has been previously reported that DEHP and MEHP can cause rat germ cell apoptosis through Fas and other death-associated receptors (Giammona et al., 2002Go; Richburg et al., 2000Go). Further experiments like DNA fragmentation studies may be utilized to confirm these findings. Recently, Yokoyama (Yokoyama et al., 2003Go) further demonstrated that the apoptosis induced by MEHP in U937 cells was in part caused by PPAR{gamma} (not PPAR{alpha}) activation. These findings imply that continued exposure to DEHP and its metabolites might cause concurrent physiological changes through alternate signal transduction pathways.

The up-regulation of FATP1 and HFABP, known mediators of active LCFA uptake/transport, consisted with the significantly increased uptake of polyunsaturated EFAs (AA, DHA, LA, ALA) when compared with saturated SA and monounsaturated OA in the DEHP-, MEHP-, and EHA-treated HRP-1 cells (Fig. 6). The fact that Pmono,influx and Pmono,efflux of AA and DHA were both increased under exposure to DEHP and its metabolites indicates the up-regulation of the fatty acid transport in both the apical and basolateral membranes of HRP-1 cells. This is, again, in accordance with the membrane localization of FATP1, which has been demonstrated to be expressed in the BBM (brush-border membrane) and BPM (basal-plasma membrane) of the human placental cells (Dutta-Roy, 2000Go; Haggarty, 2002Go). The influx ratio of the representative {omega}-3 (i.e., DHA) and {omega}-6 (i.e., AA) EFA were differentially induced upon treatment (Fig. 7). This differential effect of DEHP, MEHP, and EHA on {omega}-3 and {omega}-6 EFA uptake/transport in placental trophoblastic model might be due to the up-regulation of the PPARs and the subsequent change in PPAR-regulated fatty acid transporters. Other mechanisms including the potential regulation effects of DEHP and its metabolites on placental EFA metabolism enzymes via PPAR mediation might play a role (Bocher et al., 2002Go; Lemberger et al., 1996Go).

Overall, the uptake/transport results imply that EFA nutrition supply to the fetus via the placenta might be altered upon maternal exposure to DEHP and its metabolites, and these effects might differ with respect to the degree of saturation and the classification of EFAs. These results may suggest a potential for altering fetal development, since different EFAs have long been suggested to have differential effects on early neonate development (Bocher et al., 2002Go; Innis, 2003Go; Uauy et al., 1999Go). For example, DHA is critical for myelin synthesis and is linked to cognition and other neurological disorders (Innis, 2003Go; Uauy et al., 1999Go). Furthermore, the maternal fatty acid/lipid homeostasis environment might have dramatic changes upon exposure to phthalates at this period (Uauy et al., 1999Go) (e.g., composition and distribution of maternal fatty acids in the blood and the adipose tissue).

In summary, this study demonstrated that the environmental contaminant DEHP and/or its metabolites could alter the expression of PPAR{alpha} and PPAR{gamma} and major fatty acid transferring proteins (FATP1, HFABP), subsequently increasing the uptake/transport of LCFAs in HRP-1 rat trophoblastic cells. Subsequent changes in placental fatty acid homeostasis may lead to adverse fetal development (i.e., birth defects and potentially fetal mortality). Our work provides a valuable model for further investigating the functional and physiological significance of the effects of xenobiotics on EFA homeostasis and proper fetal development, which may eventually provide prevention/correction methods of abnormal fetal EFA-related congenital defects.


    ACKNOWLEDGMENTS
 
This study was supported by a grant from National Alliance for Autism Research.


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