* Department of Veterinary Science and The Center for Molecular Toxicology and Carcinogenesis, The Pennsylvania State University, University Park, Pennsylvania 16802; ExxonMobil Biomedical Sciences Inc., Annandale, New Jersey 08801-0971;
Consultant to Eastman Chemical Company, Rochester, New York 14652-6272; and
Toxicology Consultants Inc., Gibsonia, Pennsylvania 15044
Received July 6, 2004; accepted August 5, 2004
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ABSTRACT |
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Key Words: peroxisome proliferator-activated receptors (PPARs); phthalate monoesters.
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INTRODUCTION |
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Many of the adverse effects, including hepatocarcinogenesis, induced by monoesters of high-molecular-weight phthalates (e.g., C6 side-chains) in rodents are thought to be mediated by peroxisome proliferator-activated receptors (PPARs) (Doull et al., 1999
). PPARs are members of the nuclear receptor superfamily and consist of three isoforms, namely PPAR
, PPARß(
), and PPAR
(Shearer and Hoekstra, 2003
). In response to ligand activation, PPARs heterodimerize with retinoid-X-receptor-
(RXR
), interact with co-activators and peroxisome proliferator-response elements (PPREs) found in the promoter region of target genes, and modulate expression of target genes (Shearer and Hoekstra, 2003
). Specific ligands have been identified for all three PPARs. For example, a broad class of chemicals collectively referred to as peroxisome proliferators (e.g., the fibrate class of hypolipidemic drugs, endogenous and dietary fatty acids, herbicides and phthalate monoesters), can all bind to, or specifically activate, PPAR
(Berger and Moller, 2002
). Each PPAR participates in regulating biological functions through modulation of specific target genes. PPAR
regulates target genes that modulate fatty acid degradation, PPAR
regulates target genes that modulate glucose homeostasis, and PPARß may regulate fatty acid metabolism in skeletal muscle (Berger and Moller, 2002
; Fredenrich and Grimaldi, 2004
; Wang et al., 2003
). There is also evidence that all three PPARs modulate carcinogenesis. PPAR
is required to mediate hepatocarcinogenesis induced by peroxisome proliferators in rodents, and activation of PPAR
has been shown to be anti-carcinogenic in a number of model systems, but the role of PPARß in carcinogenesis is unclear (Gupta et al., 2004
; Harman et al., 2004
; Michalik et al., 2004
; Stephen et al., 2004
).
Previous work by others has shown that several phthalate monoesters (MEHP, MBenP, MButP) activate PPAR and PPAR
, and that lower concentrations are required for activation of mouse PPAR
than human PPAR
(Hurst and Waxman, 2003
; Maloney and Waxman, 1999
). The purpose of the present studies was three-fold: (1) to examine the ability of a broader class of phthalate monoesters and related substances of varying side-chain length and structures, to activate all three PPARs, (2) to determine if there is a species difference in receptor activation for this broader class of phthalate monoesters, and (3) to compare receptor activation observed in a trans-activation assay with PPAR-mediated biological changes, or PPAR-mediated alterations in target gene expression. It is important to point out that the broad class of monoesters examined for these studies represents the majority of commercially important phthalates.
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MATERIALS AND METHODS |
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Cell culture and trans-activation assay. Mouse 3T3-L1 fibroblasts (ATCC, Manassas, VA) were cultured in high-glucose Dulbecco's Minimal Essential Medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Sigma, St Louis, MO), 0.2 mg/ml streptomycin and 200 U/ml penicillin (Gibco, Grand Island, NY). Cells were transfected with plasmid DNA using Lipofectamine reagent (Invitrogen, Carlsbad, CA) and following the manufacturer's recommended procedures, using 3T3-L1 cells at approximately 80% confluence in 10-cm culture dishes. After 6 h, the DNA-Lipofectamine complex was removed, and the cells were maintained overnight in culture medium. Following overnight culture, the transfected 3T3-L1 cells containing chimeric mouse or human PPAR-LBD/Gal4-DBD-(PPAR
ligand binding domain/Gal4DNA binding domain), PPARß-LBD/Gal4-DBD-, or PPAR
-LBD/Gal4-DBD-Gal4 luciferase reporter plasmids were split to multiwell cluster plates. The media was replaced, 4 h after replating, with serum-free DMEM containing phthalate monoesters at concentrations of 3, 10, 30, 100, or 200 µM to evaluate PPAR activation. Solutions of phthalate monoesters and positive controls were prepared fresh on the day the cells were treated. Wy-14,643 (25 µM) was used as a positive control for the activation of PPAR
, tetradecylthioacetic acid (50 µM) was used as a positive control for PPARß activation, and troglitazone (3 µM) was used as a positive control for PPAR
activation. Twenty-four h after the treatment of the transfected 3T3-L1 cells with phthalate monoesters, the cells were lysed at 20°C with passive lysis buffer (Promega, Madison, WI) for 30 min; luciferase activity was measured using the Luciferase reporter assay kit (Promega, Madison, WI) and a Turner TD-20/20 Luminometer (Turner BioSystems, Sunnyvale, CA) and manufacturer's recommended procedures. The protein concentration of the cell lysate was determined using the BCA protein assay kit (Pierce, Rockford, Illinois). Luciferase activity was normalized to the protein concentration of each sample. The fold induction of normalized luciferase activity was calculated relative to DMSO (vehicle)-treated cells, and represents the mean of three independent samples per treatment group.
Cell culture and Northern blot analysis of PPAR-dependent target mRNA induction in hepatoma cell lines. Rat hepatoma FaO cells were cultured in DMEM supplemented with 5% FBS, 100 units/ml of penicillin, and 100 µg/ml of streptomycin. Human hepatoma HepG2 cells were cultured in alpha Minimal Essential Medium (
MEM) supplemented with 5% FBS, 100 units/ml of penicillin, and 100 µg/ml of streptomycin. FaO and HepG2 cells were seeded in 6-well plates at 7 x 105 cells/well and treated for 48 h with either phthalate monoesters, DMSO (vehicle), or Wy-14,643 (100 µM). The test concentrations of phthalate monoesters were either 10 or 100 µM and corresponded to the lower or upper ends of the respective dose response curves obtained from the trans-activation studies for those phthalate monoesters with significant PPAR
activity. These concentrations were also used for phthalate monoesters that did not exhibit significant PPAR
activity for comparative purposes. After treatment with the indicated phthalate monoester, total RNA was isolated using TRIZOL reagent (Invitrogen, Carlsbad, CA) and following the manufacturers recommended procedures. Five micrograms of total RNA were electrophoresed on 0.22 M formaldehyde denaturing agarose gel, transferred to a nylon membrane, and fixed using UV cross-linking. Membranes were hybridized in ULTRAhyb hybridization buffer (Ambion, Austin, TX) with random primed 32P-labeled probes for known PPAR
target genes including acyl-CoA oxidase (ACOX) and cytochrome P450 4A (CYP4A) or GAPDH (loading control) generated from the respective cDNA using Ready-To-Go DNA Labeling Beads (Amersham Biosciences, Piscataway, NJ) and following manufacturer's recommended procedures. Hybridization signals were obtained after scanning with a phosphorimager and were normalized relative to GAPDH. The fold induction of normalized ACOX and CYP4A mRNAs in both FaO and HepG2 cells were calculated relative to DMSO (vehicle)-treated cells and represent the mean of two independent samples per treatment group.
Cell culture and 3T3-L1 cell differentiation assay. Mouse 3T3-L1 fibroblasts were cultured in DMEM containing 10% FBS at 37°C/5% CO2. The fibroblasts were later trypsinized and seeded at approximately 50% confluence in 12-well plates. Adipogenesis was induced 48 h postconfluence, using a standard differentiation assay (Green and Meuth, 1974). This consists of changing the culture medium to DMEM/4% FBS containing 1.0 µg/ml insulin, 1 µM dexamethasone, 100 µM isomethylbutylxanthine, and troglitazone (10 µM) or the indicated phthalate monoester and monitoring subsequent lipid accumulation. Four phthalate monoesters that had significant PPAR
activity based on the trans-activation studies were used, and two phthalate monoesters that had no significant PPAR
activity were used for comparison. The media was replaced with DMEM/4% FBS containing 1.0 µg/ml insulin and troglitazone (10 µM), or the indicated phthalate monoester, every 48 h after the initiation of differentiation. The 3T3-L1 cells were fixed with formalin and stained with Oil Red O (Sigma Chemical Co, St. Louis, MO) 6 days after initiation of adipocyte differentiation. Briefly, cells were washed twice with phosphate buffered saline (PBS) and then fixed with formalin for 1 h at room temperature. The fixed cells were stained with Oil Red O (0.3 %) for 1 h. Cells were washed three times with water, visualized with a Nikon Eclipse microscope, and photographed.
Statistical analysis. Differences between treatments were determined using ANOVA followed by Dunnett's post hoc test (Prism 4.0, GraphPad Software, Inc., San Diego, CA). Significant differences were determined when p 0.05.
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RESULTS |
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DISCUSSION |
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There was a reasonably good association between the hierarchy of potency and species differences in PPAR activity and the induction of ACOX and CYP4A mRNAs in the hepatoma cell lines. For example, MEP and MButP did not effectively trans-activate mouse PPAR
, and no changes in ACOX or CYP4A mRNA were detected in rat hepatoma FaO cells treated with these phthalate monoesters. In contrast, relatively good dose responses in mouse PPAR
trans-activation were found for MIHP, MEHP, MnOP, MINP, and MIDP between 10 and 100 µM, and treatment of rat hepatoma FaO cells with these chemicals at these concentrations caused a dose-dependent induction of ACOX and CYP4A mRNAs. Others have shown that treating stably transfected rat hepatoma FaO cells expressing higher than endogenous levels of mouse PPAR
with 10300 µM MButP or MBenP causes dose-dependent increased expression of ACOX protein, but no significant differences in mRNA encoding ACOX (Hurst and Waxman, 2003
). Therefore, the results from the present studies are similar to previous reports, as no differences in ACOX mRNA levels were detected in response to either MButP or MBenP in rat hepatoma FaO cells. The reason why Hurst and Waxman (2003) detected higher levels of ACOX protein is uncertain, but could be due to higher than endogenous levels of mouse PPAR
present in the cell line used for these studies. While marginally increased trans-activation of human PPAR
was observed at concentrations typically greater than 30 µM for some of the phthalate monoesters including MIHP, MEHP, MnOP, MINP, and MIDP, no changes in the level of mRNA encoding ACOX or CYP4A were found in HepG2 cells cultured in the presence of these chemicals at 10 or 100 µM. However, it is important to point out that, while statistically significant increases in reporter activity were observed for MIHP, MEHP, MnOP, MINP, and MIDP between 10 and 100 µM using the human PPAR
construct, the fold increase was significantly smaller as compared to that observed using the mouse PPAR
construct. Therefore, the lack of increases in PPAR
target gene mRNA in human HepG2 cells as compared to rodent FaO cells is still relatively consistent with the species differences observed between the human and mouse trans-activation assay. This is also consistent with other reports demonstrating the lack of induction of PPAR
target genes in HepG2 cells in response to peroxisome proliferators (Cornu-Chagnon et al., 1995
; Duclos et al., 1997
; Hsu et al., 2001
; Lawrence et al., 2001
; Savas et al., 2003
).
Previous studies have demonstrated significant differences in the response of humans and rodents to phthalates; rodents are generally sensitive, while humans and other primates are more refractory to the PPAR-mediated pathological effects of phthalates (Doull et al., 1999
; Hall et al., 1999
; Kamendulis et al., 2002
; Klaunig et al., 2003
; Kurata et al., 1998
; Pugh et al., 2000
). Several investigators (Barber et al., 1987
; Smith et al., 2000
) have assessed the effects of a range of phthalates on rodent liver. The in vivo data are reasonably consistent with the in vitro PPAR
trans-activation data in at least a qualitative sense; low-molecular-weight phthalates (e.g., <C6 side-chains) have little effect on either liver weight or ACOX induction, whereas these effects are seen with the higher molecular weight species. Under in vivo conditions, the most active phthalates are DEHP, DINP, and DIDP. Interestingly, MnOP, which was the most active PPAR
agonist with the in vitro trans-activation assay of the present study, does not appear to influence PPAR
-dependent processes under in vivo conditions (Lake et al., 1984
; Mann et al., 1985
). This is possibly due to rapid conversion of DnOP under in vivo conditions to lower-molecular-weight metabolites (Albro and Moore, 1974
). Results from the transactivation studies show that the mouse PPAR
ligand-binding domain is generally more sensitive than its human counterpart to phthalate monoesters. Examination of PPAR
target gene expression in a rodent and human liver cell line provides further evidence that rodents are more sensitive than humans to phthalate monoesters. The reason for this apparent species difference is uncertain, but there is evidence that differences in expression levels of liver PPAR
, mutations, or polymorphisms in target gene response elements, or mutant PPAR
isoforms may contribute to this effect (Klaunig et al., 2003
). However, mice that express human PPAR
in liver at similar levels to those reported in humans (in the absence of mouse PPAR
expression) do not exhibit increased hepatocellular proliferation in response to a potent PPAR
agonist (Cheung et al., 2004
). This suggests that there are likely fundamental structural differences in the PPAR
(e.g., ability to recruit co-activators) that mechanistically explain the species differences observed after exposure to PPAR
agonists such as phthalate monoesters.
Results from these studies have some relevance for human risk assessment, since humans are routinely exposed to phthalate monoesters, as shown by the presence of these compounds in human urine and serum (Barr et al., 2003; Blount et al., 2000
; Kato et al., 2004
; Koch et al., 2003
; Silva et al., 2004
; Takatori et al., 2004
). Based on these studies, it is known that the urinary levels of the shorter side-chain phthalate monoesters are found at the higher levels (e.g., as high as
3800 ppb for MEP; 95th percentile) as compared to the longer side-chain phthalate monoesters (e.g.,
500 ppb for MEHP; 95th percentile). Additionally, the concentration of total MEHP detected in urine and serum may be relatively comparable, although serum levels may actually be lower, since the current technology for measuring serum phthalate monoesters is hampered by the presence of these compounds in many of the reagents used for this analysis in addition to the presence of esterases in serum (Takatori et al., 2004
). In a pharmacokinetic study in rats, oral doses of 30, 500, and 1000 mg DEHP/kg were associated with peak blood concentrations of MEHP of 10, 210, and 390 µM, respectively (Kessler et al., 2004
), a concentration range similar to that used in the present studies. Rats administered DEHP orally in repeated administration studies exhibit significantly elevated liver weight and induction of ACOX at doses ranging from approximately 100 to 2000 mg/kg/day (Barber et al., 1987
). These observations, in addition to results obtained from the present studies, indicate that in rodents there is a qualitative relationship between effective concentrations under in vitro and in vivo conditions. Assuming the same relationship pertains to humans, blood concentrations similar to or higher than those that are effective in rodents, would be required to produce effective interactions with PPAR
. However, data from the human population at large indicate that, at least under ambient conditions, blood concentrations are well below this range. Serum concentrations of MEHP in the U.S. population range from 2.8 to 15.2 ng/ml with a geometric mean of 3.9 ng/ml (
14 nM) (Kato et al., 2004
). Thus, at least for DEHP, the average concentration in humans is approximately three orders of magnitude below minimally effective in vitro concentrations capable of activating PPAR
. Maximum serum levels of phthalate monoesters in humans within the general population are about two orders of magnitude below the effective concentrations required to activate PPAR
. For example, the maximum serum concentrations of MEP, MBP, and MEHP in a reference U.S. human population are 73.3 (0.4 µM), 139.0 (0.6 µM) and 34.8 ng/ml (0.1 µM), respectively (Silva et al., 2003
). Further, internal exposure to DEHP/MEHP can be much higher in patients undergoing some specific procedures, and in some cases the blood concentrations could reach
100 µM. Combined, these observations suggest that activation of PPAR
is not likely to be a significant effect in response to phthalate monoester exposure in the general population, but is theoretically possible under certain conditions.
Results from the present studies also demonstrate that some phthalate monoesters can activate mouse PPARß, and that human PPARß appears to be less responsive to this effect. However, not all phthalate monoesters consistently activated PPARß. For example, no increase in PPARß-dependent reporter activity was detected in response to MEP, and the lack of a consistent dose-response curve for MIHP, MEHP, and MINP, within a known concentration range where solubility is not a confounding variable, suggests that these chemicals do not activate this PPAR isoform. In contrast, relatively good dose-response curves for mouse PPARß activation were found for MButP, MBenP, MIHP2, MIDP, and MnOP, and this effect was essentially lacking when the human PPARß isoform was used for transactivation. Additionally, the efficacy of activation was greater for MIHP2, MIDP, and MnOP as compared to the MButP and MBenP, suggesting that the phthalate monoesters with longer side-chains function better as PPARß ligands, similar to that observed for PPAR activation. Since isoC6, isoC10, and normal C8 side-chain monoesters effectively activated mouse PPARß, whereas isoC7, isoC8, and isoC9 side-chain monoesters were inactive, these data suggest that the structure-activity relationships are more subtle as compared to those observed for activation of PPAR
. While others have shown that human PPARß can be activated by MEHP using a co-transfected reporter and PPAR constructs, a direct comparison with a mouse PPARß construct under these conditions was not provided (Lampen et al., 2003
). The reason for the difference between these results and the present studies cannot be determined from this work. The relevance of phthalate monoester exposure and PPARß activation is unclear. While results from the present trans-activation studies suggest that human PPARß would not likely be activated, data from another group suggests that human PPARß can be activated by MEHP (Lampen et al., 2003
). Interestingly, activation of PPARß is associated with both positive and negative biological effects. For example, there are recent reports that intestinal cancer and breast and prostate cancer cell line growth can be enhanced by treating with a PPARß ligand (Gupta et al., 2004
; Stephen et al., 2004
); however the specificity of these effects has not been examined in a null mouse model. In contrast, recent reports have also shown that PPARß ligands can enhance serum levels of HDL cholesterol (Oliver et al., 2001
), promote epithelial cell differentiation (Schmuth et al., 2004
; Westergaard et al., 2001
), and in the absence of PPARß expression, skin and colon carcinogenesis is exacerbated (Harman et al., 2004
; Kim et al., 2004
). These results suggest that ligand activation of PPARß could function to prevent atherosclerosis and epithelial cancers. Therefore, until the specific biological function of PPARß is determined, the relevance of the present results is uncertain. However, based on observations made in the average human population described above, it is unlikely that human exposure would result in tissue concentrations capable of activating PPARß.
The hierarchy of potency observed in the trans-activation studies for the activation of PPAR by the phthalate monoesters was determined to be MnOP > MINP > (MIDP
MIHP) > (MEHP
MBenP
MEHA) > (MIHP2) > (MButP
MEP), with both species exhibiting similar responsiveness; this hierarchy also correlated well with the ability of the various phthalate monoesters to induce PPAR
-dependent adipogenesis in 3T3-L1 fibroblast cell line. The hierarchy of potency and the similarity in species responsiveness for the activation of PPAR
observed in this study is consistent with previous reports (Hurst and Waxman, 2003
; Maloney and Waxman, 1999
). Similar to the biological role of PPARß, the function of PPAR
is unclear, as there are conflicting reports in the literature. PPAR
agonists have been used for years as drugs to reduce blood glucose in type II diabetics, although the use of some agonists was associated with human mortality (Isley, 2003
). Additionally, several studies have suggested that activation of PPAR
potentiates carcinogenic effects, as ligand treatment resulted in exacerbated carcinogenesis in APCmin mice (Lefebvre et al., 1998
; Saez et al., 1998
), and overexpression of an oncogene and a ligand-independent form of PPAR
leads to exacerbated mammary tumorigenesis in bi-transgenic mice (Saez et al., 2004
). In contrast, other reports have shown that PPAR
ligands can significantly reduce the number of aberrant crypt foci and colon polyps induced by azoxymethane in rats, inhibit growth of transplanted tumors in nude mice, and inhibit growth of colon tumor cell lines (reviewed in Michalik et al., 2004
). Additionally, tumor multiplicity is significantly greater in azoxymethane-treated heterozygous PPAR
-null mice, and loss of function mutations are reported in human cases of colorectal cancer (reviewed in (Michalik et al., 2004
)). Therefore, while the present studies suggest that phthalate monoesters can activate both mouse and human PPAR
, further research is needed to determine the biological relevance of PPAR
activation that might occur in response to exposure to phthalate monoesters.
Lastly, while many ligands for PPARs (such as phthalate monoesters) function by activating specific PPARs, it is becoming increasingly clear that many ligands for nuclear receptors can be promiscuous and activate other nuclear receptors. Thus, while results from these studies demonstrate that phthalate monoesters can activate PPARs and that species differences exist in the ability to activate specific PPAR, it is still possible that these chemicals lead to events that are mediated by other nuclear receptors, and/or through events that are not dependent on receptor activation, per se, and this remains to be examined.
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed at Department of Veterinary Science and The Center for Molecular Toxicology and Carcinogenesis, The Pennsylvania State University, University Park, PA 16802. Fax: (814) 863-1696. E-mail: jmp21{at}psu.edu.
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