Phthalate Exposure Changes the Metabolic Profile of Cardiac Muscle Cells

Background: Phthalates are common plasticizers present in medical-grade plastics and other everyday products. They can also act as endocrine-disrupting chemicals and have been linked to the rise in metabolic disorders. However, the effect of phthalates on cardiac metabolism remains largely unknown. Objectives: We examined the effect of di(2-ethylhexyl)phthalate (DEHP) on the metabolic profile of cardiomyocytes because alterations in metabolic processes can lead to cell dysfunction. Methods: Neonatal rat cardiomyocytes were treated with DEHP at a concentration and duration comparable to clinical exposure (50–100 μg/mL, 72 hr). We assessed the effect of DEHP on gene expression using microarray analysis. Physiological responses were examined via fatty acid utilization, oxygen consumption, mitochondrial mass, and Western blot analysis. Results: Exposure to DEHP led to up-regulation of genes associated with fatty acid transport, esterification, mitochondrial import, and β-oxidation. The functional outcome was an increase in myocyte fatty acid–substrate utilization, oxygen consumption, mitochondrial mass, PPARα (peroxisome proliferator-activated receptor α) protein expression, and extracellular acidosis. Treatment with a PPARα agonist (Wy-14643) only partially mimicked the effects observed in DEHP-treated cells. Conclusions: Data suggest that DEHP exposure results in metabolic remodeling of cardiomyocytes, whereby cardiac cells increase their dependence on fatty acids for energy production. This fuel switch may be regulated at both the gene expression and posttranscription levels. Our findings have important clinical implications because chronic dependence on fatty acids is associated with an accumulation in lipid intermediates, lactate, protons, and reactive oxygen species. This dependence can sensitize the heart to ischemic injury and ventricular dysfunction.


Research
An estimated 34% of U.S. adults have metabolic syndrome, which can be a precursor to diabetes, obesity, heart disease, stroke, and cancer (Ervin 2009). Although the role of life style choices in these disorders cannot be ignored, the dramatic rise in metabolic disease suggests that environmental pollutants may also play a role. Endocrine-disrupting chemicals can interfere with endocrine function, resulting in adverse developmental, reproductive, and metabolic effects. Human exposure to endocrine disruptors has been linked to metabolic disturbances (reviewed by Casals-Casas and Desvergne 2011). Endocrine disruptors are known to interact with nuclear receptors, which modulate downstream gene expression by interfering with receptor function and/or transcription factor activity.
Di(2-ethylhexyl)phthalate (DEHP) is a commonly used plasticizer that imparts flexibility to polyvinyl chloride (PVC) products. It has been identified as an endocrine-disrupting chemical (Casals-Casas and Desvergne 2011). Human exposure to DEHP occurs through contact with food packaging, toys, and personal care and medical products. The presence of DEHP in medical products is of particular concern because exposure to DEHP increases dramatically in patients under going multiple medical interventions, such as cardiopulmonary bypass, hemo dialysis, transfusions, and other procedures that require extensive use of tubing in intensive care units. This is because DEHP is not covalently bound to the PVC polymer and is highly hydro phobic, allowing it to leach from plastics when in contact with blood and other lipo philic fluids [Food and Drug Administration (FDA) 2002]. Several animal studies have reported toxic effects of DEHP (reviewed by Carlson 2010), raising concerns about phthalate leaching and human health. Exposure to DEHP results in broad phthalate distribution throughout the body, including the heart (Hillman et al. 1975). Our recent studies revealed that DEHP adversely affects the synchronicity of a cardiac cell network by disrupting connexin-43, the main component of cardiac gap junctions (Gillum et al. 2009). This effect, which may be attributed to modifications in tubulin and kinesin expression, as well as other gene expression modifications, suggested an arrhythmo genic effect of phthalates in vitro (Posnack et al. 2011). We sought to expand these studies to examine DEHP's effect on the metabolic profile of cardiomyocytes because metabolic alterations can point to additional dysfunctions.
To meet high energy demands, cardiac muscle is able to metabolize multiple substrates. Fatty acids (FAs) are the preferred substrate: At least 60% of ATP is generated from FA oxidation (FAO) (reviewed by Lopaschuk et al. 2010). The remaining energy demand is provided by glucose, lactate, and ketone metabolism. Several studies have addressed the effects of phthalates on metabolism (Casals-Casas et al. 2011;Desvergne et al. 2009;Stahlhut et al. 2007;Svensson et al. 2011). In contrast, the effect of DEHP on cardiac FA metabolism remains largely unknown (Itsuki-Yoneda et al. 2007;Reubsaet et al. 1990). We found that DEHP exposure results in an up-regulation of genes associated with FAO. DEHP-induced genetic modifications resulted in increased FA substrate utilization, increased mitochondrial mass, increased oxygen consumption, and extra cellular acidosis. Data suggest that these modifications involve up-regulation of PPARα (peroxisome proliferator-activated receptor α) and its coactivator PGC-1α (peroxisome proliferator-activated receptor γ, coactivator 1α), as well as alternative pathways.

Materials and Methods
All animals were treated humanely and with regard for alleviation of suffering.
Background: Phthalates are common plasticizers present in medical-grade plastics and other everyday products. They can also act as endocrine-disrupting chemicals and have been linked to the rise in metabolic disorders. However, the effect of phthalates on cardiac metabolism remains largely unknown. oBjectives: We examined the effect of di(2-ethylhexyl)phthalate (DEHP) on the metabolic profile of cardio myocytes because alterations in metabolic processes can lead to cell dysfunction. Methods: Neonatal rat cardiomyocytes were treated with DEHP at a concentration and duration comparable to clinical exposure (50-100 μg/mL, 72 hr). We assessed the effect of DEHP on gene expression using microarray analysis. Physiological responses were examined via fatty acid utilization, oxygen consumption, mitochondrial mass, and Western blot analysis. results: Exposure to DEHP led to up-regulation of genes associated with fatty acid transport, esterification, mitochondrial import, and β-oxidation. The functional outcome was an increase in myo cyte fatty acid-substrate utilization, oxygen consumption, mitochondrial mass, PPARα (peroxisome proliferator-activated receptor α) protein expression, and extra cellular acidosis. Treatment with a PPARα agonist (Wy-14643) only partially mimicked the effects observed in DEHP-treated cells. conclusions: Data suggest that DEHP exposure results in metabolic remodeling of cardiomyocytes, whereby cardiac cells increase their dependence on fatty acids for energy production. This fuel switch may be regulated at both the gene expression and post transcription levels. Our findings have important clinical implications because chronic dependence on fatty acids is associated with an accumulation in lipid inter mediates, lactate, protons, and reactive oxygen species. This dependence can sensitize the heart to ischemic injury and ventricular dysfunction.
FA utilization. Seventy-two hours after treatment with DMSO (control) or 50 μg/mL DEHP, cell culture media was changed to a glucose-free media supplemented with 100 μM palmitic acid and 33 μM bovine serum albumin (BSA). An increased rate of FA utilization was detected as a decrease in media concentration over time. FA concentration was measured over 30 hr using a fluorescence-based assay (Free Fatty Acid Quantification Kit; BioVision, Milpitas, CA) in which FAs were converted to coenzyme A (CoA) derivatives for quantification.
Proton production. We prepared standards by adjusting the pH of media samples containing phenol red. Absorbance spectra were acquired for the standards and the cell culture samples using a plate reader. The cell culture samples were then normalized against the standards to identify pH modifications.
Oxygen consumption. Cardiomyocytes were plated on an XF24 cell culture microplate (100,000 cells/well; Seahorse Bioscience, North Billerica, MA). Cells were treated for 72 hr with DMSO or 50 μg/mL DEHP. An XF24 Analyzer (Seahorse Biosciences) was then used to measure oxygen consumption in real time. Prior to XF measurements, cells were incubated for 1 hr at 37°C in unbuffered media supplemented with a) 100 μM palmitic acid with 33 μM BSA, or b) 25 mM glucose. To validate these results independently, cells were loaded with an oxygen-sensitive probe (MitoXpress; Axxora, San Diego, CA) in media supplemented with palmitic acid and BSA. Mineral oil was used to seal the samples and diminish the interference of ambient oxygen. A high rate of cell respiration depletes the sample oxygen concentration quickly, which is detected as an increase in probe signal.
Statistical methods. Student's t-test was used to evaluate the significance of the differences in mean values between two treatment groups. Analysis of variance and Bonferroni's multiple comparison test were used to evaluate values between three or more groups. Values are presented as mean ± SE, with p < 0.05 considered statistically significant.

Gene expression profile of DEHP-treated cardio myocytes.
We studied the effect of DEHP on neonatal rat cardio myocytes at a dose (50-100 μg/mL) comparable to plasticizer presence in neonates under going clinical procedures, such as transfusion or extra corporeal membrane oxygenation (FDA 2002;Gillum et al. 2009). After 72 hr exposure, RNA was harvested from cardiomyocytes for DNA micro array experiments. At a 1.5-fold expression difference cut-off, a total of 814 mRNAs were differentially expressed. Of these genes, 334 were up-regulated and 480 were down-regulated by DEHP treatment.
Gene Ontology (GO) analysis revealed that differentially expressed genes were significantly over represented in 97 categories (p < 0.01). The lipid metabolic process GO category was the most significantly modified (p < 0.00001). Figure 1A shows a polyhierarchical graph specific for GO categories under the metabolic process ontology. We used MeV to identify genes signifi cantly up-or down-regulated within four downstream GO categories ( Figure 1B): cholesterol metabolism, FA metabo lic process, regulation of FA metabolic process, and FA bio synthetic process. The latter two categories are sub divisions of FA metabo lic process; genes that over lapped between categories are displayed only once in the heatmap images. In DEHP-treated samples, most genes associated with FA metabolic processes were up-regulated, whereas most genes associated with bio synthesis were down-regulated ( Figure 1B).
qRT-PCR validation. The mRNA expression of 11 genes associated with FA metabo lism and regulation were validated using qRT-PCR ( Figure 1C). Genes associated with regulation of FA metabolism were up-regulated: PPARα, PGC-1α, and PGC-1β. Genes encoding FA metabolic enzymes were also up-regulated: ACSL1, ACAA2, ACADL, ACADVL, and HADHA. CPT1B, which transports FAs into the mitochondria, was also up-regulated. We also detected an up-regulation in ECH1, an enzyme that functions in the auxiliary step of the β-oxidation pathway, and PDK4, a protein that regulates glucose metabolism.
Gene network analysis. We used IPA and PathVisio to identify biological pathways pertinent to lipid metabolism. A mitochondrial FAO pathway overlaid with mRNA expression fold-change values is shown in Figure 2A. Genes involved in multiple parts of the FAO pathway were up-regulated in DEHP-treated samples. These included triglyceride hydrolysis (PNPLA2), FA esterification (ACSL1), mitochondrial import (CPT1A, CPT1B, and SLC25A20) and β-oxidation (ACADL, ACADVL, HADHA, and HADHB). Conversely, ACSL3, which encodes an acyl-CoA synthetase enzyme involved in FA biosynthesis, was down-regulated.
In addition to FA metabolism and FA elongation, alternative lipid canonical pathways were also altered, including those related to steroid, ketone, arachidonic acid, linoleic acid, and phospholipid metabolism and degradation ( Figure 2B). IPA toxicity analysis also revealed functionally grouped gene sets that are altered in response to a xeno biotic exposure (DEHP). As expected, PPARα activation and FA metabolism genes were up-regulated ( Figure 2C). In comparison, cholesterol biosynthesis was down-regulated. Gene sets associated with detoxification pathways, including glutathione depletion and cytochrome P450, were also up-regulated in DEHP samples.
Toxicity gene sets associated with mitochondrial membrane depolarization and NRF2 (NF-E2-related factor 2)-mediated oxidative stress response were also up-regulated. These are of interest because increased FAO is likely to enhance reactive oxygen species production, and NRF2 up-regulates anti oxidant genes (Gao et al. 2007).

Increased FAO and mitochondrial oxygen consumption.
To determine if gene expression changes resulted in a physiological response, we measured the rate of FA utilization during a 30-hr period by modifying the cell culture media to contain only FAs (glucose free). After 6 hr, DEHP-treated samples    Figure 3A). This effect became more pronounced at later time points. Compared with glucose oxidation, FAO requires a greater amount of oxygen per molecule of ATP produced. Therefore, increased reliance on FA substrates for energy production should also increase oxygen consumption. To further investigate the effect of DEHP on FAO, we measured oxygen consumption rates in cardio myocytes cultured with either 100 μM palmitic acid or 25 mM glucose using an extra cellular flux analyzer.
Oxygen consumption rates were increased in DEHP-treated samples supplemented with palmitic acid, and decreased in DEHPtreated samples supplemented with glucose ( Figure 3B). This decrease in oxygen consumption may signify a lack of substrate flexibility in response to DEHP whereby    glucose utilization is diminished. These data were independently validated by measuring total oxygen consumption using an oxygensensitive probe in the presence of 100 μM palmitic acid ( Figure 3C).

Percent of genes Percent of genes
Increased PPARα expression. The PPARα nuclear receptor acts as a lipid sensor that modifies transcriptional responses to metabolic status. Activation (or inhibition) of PPARα via exogenous compounds can interfere with metabolic homeostasis. Phthalates are known PPARα agonists (Bility et al. 2004). PPARα protein expression was increased in DEHPtreated samples compared with the control, as shown by immuno staining ( Figure 4A) and Western blot analysis ( Figure 4B).

Increased mitochondrial mass.
Activation of PPARα and its transcriptional coactivator protein, PGC-1α, can increase mitochondria bio genesis (Wu et al. 1999). Because both PPARα and PGC-1α are up-regulated in DEHP samples, we examined mitochondrial mass using MitoTracker. As a positive control, PPARα was activated directly via Wy-14643, a specific PPARα agonist (Ren et al. 2010). Assessment of mitochondrial structure showed an increase in mitochondrial mass, particularly in cells treated with a high concentration of DEHP (100 μg/mL) or Wy-14643 ( Figure 5).
Increased proton production. FAO is inversely proportional to glucose oxidation.
By-products of FAO inactivate the pyruvate dehydrogenase complex (PDC), thereby diverting pyruvate to lactate via LDH. Therefore, increased FAO can uncouple glycolysis from glucose oxidation, leading to proton and lactate accumulation ( Figure 6D). To confirm this pathway, we performed pH measure ments on treated cells. After 3 days of treatment, cells exposed to a high concentration of DEHP (100 μg/mL) showed significant extra cellular acidosis ( Figure 6A). Acidosis increased with time. After 6 days of treatment, cell samples treated with 100 μg/mL DEHP reached a pH of 5, whereas cell samples treated with 50 μg/mL DEHP were also more acidic, although to a lesser extent.  However, Wy-14643-treated samples were more basic (pH 7.5) compared with controls. The observed pH changes were not attributed to chemical additives ( Figure 6A). Because pH changes can also reflect cell culture overgrowth or cell death, we examined cell viability to rule out these effects. Wy-14643treated samples had a decrease in cell viability (2.5% of control) and total cell number (20% of control), which may explain the shift to a more basic pH. However, neither cell viability nor total cell number was altered in DEHPtreated samples ( Figure 6B,C). DEHPinduced acidosis was independently verified in extra cellular flux experiments; DEHP samples were more acidic than controls after 1 hr incubation in unbuffered media (data not shown).

Discussion
DEHP is a widely used phthalate found in FDA-approved medical devices, tubing, and bags for intravenous fluids. DEHP toxicity has been reported for multiple organs in animals (Carlson 2010), and human studies have raised concerns pertaining to the male reproductive tract and development (Swan 2008). In addition, public health studies have  correlated phthalate exposure with metabolic dis orders in humans (Stahlhut et al. 2007;Svensson et al. 2011). In contrast, the cardiac effects of DEHP remain largely ignored, and little has been published on the effects of DEHP on cardiac metabolism (Itsuki-Yoneda et al. 2007;Reubsaet et al. 1990). To the best of our knowledge, our study is the first to thoroughly assess the effects of DEHP on substrate metabolism in cardiac cells at clinically relevant doses.
We showed that DEHP treatment of cardio myocytes results in increased expression of genes associated with FA metabolism. Figure 6D illustrates genes of interest for which expression was modified in our experiments (Lopaschuk et al. 2010). Myocyte metabolism of FAs proceeds as follows: FAs enter the myocyte (step 1; Figure 6D) via fatty acid translocase (CD36) and are transported by FA binding protein (FABP). Once inside the cell, FAs under go esterification (step 2). ACSL1, the predominant isoform found in the heart (Durgan et al. 2006), catalyzes the synthesis of acyl-CoA from FAs; cardiac over expression of ACSL can result in severe cardio myopathy due to increased FA uptake and an ensuing accumulation of lipids (Chiu et al. 2001). Acyl-CoA is then converted to acyl-carnitine by CPT1 (step 3), which facilitates entry into the mitochondria. CPT1 activity is tightly controlled by its inhibitor, malonyl-CoA (step 4), which is regulated by AMP-activated kinase (AMPK). AMPK promotes conversion of malonyl-CoA to acetyl-CoA (step 5), thereby alleviating the inhibitory signal on CPT1. Acyl-carnitine is translocated across the mitochondrial inner membrane (step 6) by acylcarnitine translocase (SLC25A20). Once inside the mitochondria, FAs enter the β-oxidation spiral (step 7), whereby acyl-CoA molecules are converted to acetyl-CoA. This conversion is catalyzed by numerous enzymes, including Acadl, Acadvl, Hadha, Hadhb, Decr1, and Acaa2. Each cycle through the β-oxidation spiral produces one molecule each of FADH 2 , NADH, and acetyl-CoA (step 8), and acetyl-CoA is transferred to the citric acid cycle for ATP production. FADH 2 and NADH by-products are then   In DEHP-treated samples, we observed a decrease in oxygen consumption in the presence of glucose, as well as an increase in proton generation. These results confirm that FAO inhibited glucose oxidation, a phenome non known as the "Randle cycle" (Randle et al. 1963). Increased FAO leads to inhibition of PDC, which is a multi enzyme complex that converts pyruvate to acetyl-CoA, thereby linking glycolysis with the citric acid cycle. By-products of FAO increase activity of pyruvate dehydrogenase kinases (PDK), which functions to inactivate the PDC enzyme, pyruvate dehydrogenase (PDH; step 10). Inactivation of PDC prevents the conversion of pyruvate to acetyl-CoA, and instead diverts pyruvate to lactate production via LDH (step 11). Consequently, an increase in FAO and a decrease in PDC activity uncouple glycolysis and glucose oxidation. This scenario, whereby glycolysis is unaltered but pyruvate oxidation is inhibited, can result in an accumulation of protons and lactate. Notably, increased lactate levels have been observed in the muscle of DEHP-treated animals (Martinelli et al. 2006). In the present study, we found that DEHP treatment resulted in an up-regulation in both LDH (microarray analysis; data not shown) and PDK4. This change in gene expression likely explains the extra cellular acidosis observed in DEHP samples. A similar phenome non was observed when adipocytes were exposed to MEHP, which led to increased PDK4 expression and diverted pyruvate away from the citric acid cycle (Ellero-Simatos et al. 2011).
We hypothesize that the described changes in metabolism are partially, but not exclusively, related to up-regulation of PPARα and its coactivator PGC-1α. Both PPARα and PGC-1α act as lipid sensors that modify transcriptional responses to metabolic status (Lopaschuk et al. 2010). PPARα is abundant in metabolically active tissues, such as the heart, and we observed DEHP-induced modifica tions to a number of PPARα target genes ( Figure 6D). The influence of PPARα on metabolism has been elucidated in both gainand loss-of-function experiments. Transgenic mice with cardiac-restrictive PPARα overexpression develop a metabolic phenotype that mimics the diabetic heart, including high FAO rates, low glucose oxidation rates, and gene expression changes including PDK4 up-regulation (Finck et al. 2002). Moreover, hearts over expressing PPARα showed signs of cardio myopathy. Treatment with the PPARα agonist Wy-14643 similarly increased PDK4 expression in both cardiac (Zungu et al. 2009) and skeletal muscle (reviewed by Burri et al. 2010). In comparison, PPARα-null mice have decreased rates of FAO and increased rates of glucose oxidation (Campbell et al. 2002).
Phthalates are known PPARα agonists (Bility et al. 2004), and many of the effects observed in our studies can be attributed to PPARα up-regulation. However, DEHPinduced toxicity has also been reported in multiple organs of PPARα-null mice (Ito et al. 2007;Peters et al. 1997;Ward et al. 1998). The genes modified by DEHP in PPARα-null mice included those involved in xeno biotic metabolism, including targets of constitutive androstane receptor (CAR) or pregnane X receptor (PXR), and cholesterol bio synthesis which is regulated by other transcription factors, including retinoid X receptor (RXR) (Ren et al. 2010). This is of interest to our studies, as our data illustrated differences between DEHP and PPARα agonist treatment (Wy-14643), suggesting involvement of an alternative receptor.
We propose that increased AMPK expression is an additional mechanism by which DEHP can exert its effects on metabolism. AMPK is an energy sensor: It turns on ATPproducing catabolic processes and turns off ATP-consuming anabolic processes. AMPK can be activated by increased FA supply, increased AMP:ATP ratio, and ischemia. In turn, AMPK activation leads to an up-regulation of PPARα and CPT1β as a way to dispose of FAs via β-oxidation (Lopaschuk et al. 2010). The downstream effects of AMPK activation, including mitochondrial bio genesis, have been attributed to PGC-1α (Jager et al. 2007). Increased AMPK activity was also the main mechanism attributed to an insulin-resistant pheno type observed in PDK4-overexpressing hearts, which included increased FAO, decreased glucose oxidation, and increased lactate levels in myocardial tissue (Chambers et al. 2011). Last, AMPK activity is associated with an inhibition of cholesterol synthesis (Henin et al. 1995), a result we observed in DEHP-treated samples. Despite crosstalk between PPAR and AMPK, PPARα agonists modulate AMPK activity independently of the PPAR nuclear receptor (Chanda et al. 2009).
All-in-all, our data suggest that DEHP exposure results in metabolic remodeling of cardio myocytes, whereby cardiac cells increase their dependence on FAs for energy production. This dependence on FAs may be regulated at both the gene expression and post transcription levels. This phenomenon should be examined further in both in vitro adult cardio myocytes and in vivo.

Conclusions
The heart exhibits energy substrate flexibility; however, several conditions are associated with alterations in glucose and/or FA metabolism. Diabetic cardiac dysfunction is partly driven by metabolic abnormalities, chiefly a loss of energy substrate flexibility. The diabetic pheno type consists of a reduced responsiveness to insulin, decreased glucose utilization, and increased dependence on FAs. Chronic dependence on FAs as a fuel source is associated with an accumulation in lipid intermediates, lactate, protons, and reactive oxygen species. Therefore, dependence on FAs can sensitize the heart to ischemic injury and ventricular dysfunction. Diabetic mouse models also display an increase in myo cardial oxygen consumption. The metabolic profile seen in DEHP-treated samples has a striking similarity to that seen in diabetic cardio myopathy (Lopaschuk et al. 2010). It is possible that the adverse metabolic cascade typically initiated by high levels of circulating triglycerides in diabetic patients could also be initiated by chronic phthalate exposure. This parallel to diabetes has important public health implications that should be further addressed.