Toxicology and Applied Pharmacology 236 (2009) 282–292
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Toxicology and Applied Pharmacology
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Identification of potential mechanisms of toxicity after di-(2-ethylhexyl)-phthalate(DEHP) adult exposure in the liver using a systems biology approach
Alexandre Eveillard a, Frédéric Lasserre a, Marie de Tayrac b, Arnaud Polizzi a, Sandrine Claus c, Cécile Canlet d,Laïla Mselli-Lakhal a, Gaëlle Gotardi d, Alain Paris d, Hervé Guillou a, Pascal G.P. Martin a, Thierry Pineau a,⁎a Pôle de Toxicologie Alimentaire, Laboratoire de Pharmacologie et Toxicologie, Institut National de la Recherche Agronomique INRA UR66, 180 Chemin de Tournefeuille,Toulouse Cedex 3, F31027, Franceb CNRS-UMR 6061, Regulation of Transcription and Oncogenesis, IFR140 GFAS, Faculty of Medicine Rennes, Francec Division of Surgery Oncology Reproductive Biology and Anaesthetics Department, Imperial College London, UKd Laboratoire des Xénobiotiques, Institut National de la Recherche Agronomique INRA UMR1089 Toulouse, France
ditives widely used as plasticizers. In addition to deleterious effects on malegenital development, population studies have documented correlations between phthalates exposure andimpacts on reproductive tract development and on the metabolic syndrome in male adults. In this work weinvestigated potential mechanisms underlying the impact of DEHP on adult mouse liver in vivo.A parallel analysis of hepatic transcript and metabolic profiles from adult mice exposed to varying DEHPdoses was performed. Hepatic genes modulated by DEHP are predominantly PPARα targets. However, theinduction of prototypic cytochrome P450 genes strongly supports the activation of additional NR pathways,including Constitutive Androstane Receptor (CAR). Integration of transcriptomic and metabonomic profilesrevealed a correlation between the impacts of DEHP on genes and metabolites related to heme synthesisand to the Rev-erbα pathway that senses endogenous heme level. We further confirmed the combinedimpact of DEHP on the hepatic expression of Alas1, a critical enzyme in heme synthesis and on theexpression of Rev-erbα target genes involved in the cellular clock and in energy metabolism.This work shows that DEHP interferes with hepatic CAR and Rev-erbα pathways which are both involved inthe control of metabolism. The identification of these new hepatic pathways targeted by DEHP couldcontribute to metabolic and endocrine disruption associated with phthalate exposure. Gene expressionprofiles performed on microdissected testis territories displayed a differential responsiveness to DEHP.Altogether, this suggests that impacts of DEHP on adult organs, including testis, could be documented anddeserve further investigations.
Phthalates are a class of synthetic chemicals which are used fornumerous industrial applications (e.g. polyvinyl chloride plasticizersfor food contact or medical devices, personal care products, residentialconstruction and automotive industries). Di-(2-ethylhexyl)-phthalate(DEHP) is the most widely used congener. Humans are exposed tothese chemicals through transcutaneous absorption, inhalation,
medical transfusions and ingestion. Despite their rather rapid turn-over (half life of DEHP b24 h) phthalates and their metabolites areconsistently detected in human body fluids such as plasma, urine,amniotic fluid or breast milk, thus reflecting substantial and constantexposure (Koch et al., 2003; Silva et al., 2004). Cumulative impactscould result from chronic intakes, especially in certain populations ofhighly exposed workers (Pan et al., 2006). More acute exposures havealso been documented (Inoue et al., 2005). The potential public healthrisks associated with phthalates exposure not only include carcino-genesis (for a review, see Ito and Nakajima, 2008) but also metabolicand endocrine disruption.
Recently, several phthalate congeners, including DEHP, wereshown by independent research groups, to be significantly correlatedto the prevalence of key features of the metabolic syndrome(abdominal adiposity, body mass index and insulin resistance), inthe male subpopulation of a health and nutrition survey (NHANES1999–2002), (Stahlhut et al., 2007; Hatch et al., 2008). Thesepopulation studies further supported the concept of environmental
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obesogens acting, in association with excessive caloric intake andinsufficient exercise, as co-factors in the obesity crisis (Grun andBlumberg, 2007). Different studies showed that DEHP and/or itsmajor metabolite MEHP (mono-(2-ethylhexyl) phthalate) modulatethe activities of three nuclear receptors: PPARα (Lapinskas et al.,2005), PPARγ (Feige et al., 2007) and PXR (Hurst and Waxman,2004). Since these receptors regulate energy homeostasis, hormoneand xenobiotics metabolism, some of the endocrine disruptiveeffects of DEHP are likely to be related to its impact on thesenuclear receptors.
In humans, population studies have also established thatdysmorphic disorders of the genital tract, observed in male infants,are significantly associated with prenatal exposure to phthalates(Swan et al., 2005;Marsee et al., 2006). These effects likely result fromthe antiandrogenic (Stroheker et al., 2005) estrogeno-mimeticactivities of DEHP (Lovekamp-Swan and Davis, 2003), but its bindingto the human estrogen receptorα (ERα, Ohashi et al. 2005; Inoue et al.2002), or to the androgen receptor (Parks et al., 2000) is eitherconflicting or negative. However, phthalates disturb androgensbioavailability at critical stages of the development of the fetus(Akingbemi et al., 2004). In rodents, experimental doses of DEHPtrigger endocrine disruptive effects resulting in adverse outcomes onthe male reproductive tract anatomy and physiology (Parks et al.,2000). Despite many studies, the mechanisms underlying the adversedevelopmental effects of DEHP are not yet fully understood.Additionally, inconsistent results have been obtained concerning theadverse effects of phthalate exposure through adulthood on para-meters related to reproductive functions (Modigh et al., 2002; Panet al., 2006; Hauser, 2008).
Through a systems biology approach, we investigated in vivowhether we could identify new NR-related pathways likely to berelated to metabolic disruption in the liver. We used mice exposed tothree distinct DEHP doses (30, 180, 1100 mg/kg/day) for 14 days.Transcriptomic profiling using a nuclear receptor-dedicated macro-array and NMR-derived metabonomic profiling were performed onliver samples. Our data reveal for the first time that genes related toboth CAR and Rev-erbα signaling are sensitive to DEHP. These twopathways are likely to contribute to the adverse metabolic effects ofDEHP. Additionally, a preliminary transcriptomic screening in adulttestis cells exposed to DEHP shows that DEHP also modulates geneexpression in adult tissues other than the liver.
Materials and methods
Animals and maintenance. Male C57BL/6J mice (Charles River, LesOncins, France) were acclimatized for eight weeks, housed inpolycarbonate cages at 22±2 °C on a 12 hour light/dark cycle andallowed free access to water and food. In vivo studies were conductedunder E.U. Guidelines for the use and care of laboratory animals andwere approved by an independent ethics committee.
Experimental design, plasma and organs sampling. Fifteen week-oldmice were randomly divided into four groups (n=6/group). Di-(2-ethylhexyl)-phthalate (DEHP, Sigma, Lyon, France), in solution intocorn oil, was administered for 14 days by daily gavage at 0 (corn oilcontrols), 30, 180 or 1100 mg/kg/day. On day 14, blood was collectedat the retro-orbital sinus with heparin-coated capillaries. Plasma wasprepared by centrifugation (16,000 ×g, 10 min) and kept at−80 °C. Atday 14, liver, kidney and testis were sampled, weighed, snap-frozen inliquid nitrogen and stored at −80 °C.
Transcriptomic analysis. Total RNA was extracted from frozentissues with TRIzol® reagent (Invitrogen), controlled for integrityon an Agilent 2100 Bioanalyzer (Agilent Technologies) and assayedat 260 nm on a Lambda 650 spectrophotometer (Perkin Elmer).Transcriptomic profiles relevant to NR signalling were obtained
using a dedicated cDNA macroarray previously developed andvalidated (Martin et al., 2005) full list of INRArray 01.4 cDNAsavailable at www.inra.fr/internet/Centres/toulouse/pharmacologie/lpt.htm). RNA labelling and INRArray 01.4 hybridization wereperformed as previously described (Martin et al., 2007). Nineteenmacroarrays (6 controls, 4 DEHP 30 mg/kg/day, 4 DEHP 180 mg/kg/day and 5 DEHP 1100 mg/kg/day) hybridized with RNA samplesfrom individual livers passed all quality controls and weresubsequently analyzed. The raw and processed data are availablein the Gene Expression Omnibus (GEO) database (Barrett et al.,2007) under the accession number GSE14629. For real-timequantitative PCR (Q-PCR), total RNA samples (2 μg) were reverse-transcribed using SuperScript™ II reverse transcriptase (Invitrogen).Primers and Taqman probes for TATA-box binding protein (TBP:Mm00446973-m1), Cyp4a14 (Mm00484132-m1), Cyp2b10(Mm00456591-m1) and Cyp3a11 (Mm00731565-m1) wereobtained from Applied Biosystems. Primers for SYBR Green assaysare presented in Supplementary material (Table S1). Real-timeamplifications were performed on an ABI Prism 7000 SDS (AppliedBiosystems). All Q-PCR data were normalized by TBP mRNA levels.Differential gene expression was calculated by the ΔΔCT calculationmethod.
Metabonomic analyses by 1H nuclear magnetic resonance (NMR)spectroscopy. Plasma samples (50–80 μL) were mixed with 500 μLof deuterium oxide (D2O) containing sodium 3-trimethylsilyl-2,2,3,3-d4-propionate (TMSP,1mM) as a chemical shift standard (δ=0 ppm).1H NMR spectra were acquired on a Brucker Avance DRX-600spectrometer operating at 600.13 MHz and equipped with a 5 mmH,C,N inverse triple resonance TXI cryoprobe attached to a cryo-platform. The Carr-Purcell-Meiboom-Gill (CPMG) spin-echo pulsesequence, D-[-90°-(τ-180°-τ)n-FID] (Nicholson et al., 1995) wasapplied to acquire 1H NMR spectra of all plasma samples. For eachsample, 256 free induction decays (FID) were collected into a spectralwidth of 12 ppm in 32 K data points with a relaxation delay of 2 s andan acquisition time of 2.28 s. 1H NMR spectroscopy was performed onaqueous liver extracts prepared from liver samples homogenized in2mL CH3CN/H2O (1/1, v/v) containing 0.1% butyl hydroxytoluene andcentrifuged at 5000 ×g for 10 min at 4 °C. The supernatant wascollected, lyophilized and reconstituted in D2O (600 μL) containingTMSP (1mM). NMR spectrawere acquired using the 1D standard pulsesequence. For each sample 128 FIDwere collected into 32Kdata points.All NMR spectra were data reduced using AMIX (Bruker, Rheinstetten,Germany) to integrate 0.04 ppm-wide regions from δ 10.0 to 0.5 ppm.The regions containing the residual water signal (δ 5.0–4.5) in allspectra and acetonitrile signal (δ 2.06) in liver aqueous extractwere setto zero.
Statistical analysis. Macroarray data filtering, normalization andquality controls were described previously (Dejean et al., 2007).Differential effects were analyzed via ANOVA followed by a Studentt test with a pooled variance estimate (false discovery ratecontrolled at 5% using the Benjamini–Hochberg procedure).Unsupervised (principal component analysis) and supervisedfactorial methods (linear discriminant analysis — LDA) wereemployed to explore the main effects in the macroarray and NMRdata sets, respectively. Both methods reduce the dimension of theinitial data sets by projecting the samples on latent variables whichare linear combinations of the initial gene expression andmetabolite variables, respectively. To explore the relationshipsbetween metabolite and gene expression changes, we used partialleast squares regression-based canonical analysis (O2PLS) whichidentifies the fundamental correlat ions between twomultidimensional data sets through projections on latent variables(Bylesjo et al., 2007). R with various Bioconductor packages, Splus2000 (Insightful Corp., Seattle, WA) with the multidim library
The table reports the fold change of each DEHP-regulated transcript which has been recorded, in the liver, from macroarray analysis using INRArray 01.4 gene set. Significantdifferences were analyzed by one-way ANOVA followed by Student's t test. Resulting P-values were adjusted by the Benjamini–Hochberg procedure to control the FDR at 5%. Geneswith adjusted Pb0.05 and with at least a 1.3-fold change in expression (induction or repression) are presented.
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(www.lsp.ups-tlse.fr/Carlier/Logiciel.html) and Simca-P 11.0 (Umea,Sweden) softwares were used to analyze all data.
Results
Dose-dependent effect of DEHP exposure on hepatic transcriptionalsignature
To gain insights into the impact of DEHP on nuclear receptor-mediated gene expression,we examined the level of expression of a setof 320 selected transcripts relevant to metabolic, endocrine andreproductive disorders (INRArray 01.4 macroarray). Following aninitial filter (signal significantly above background for at least 25% ofthe macroarrays), 246 transcripts were selected for further analysis.Among those, 79 transcripts displayed at least one significantmodulation (FDRb5%) between a DEHP-treated group and the controlgroup (Table 1). A principal components analysis (PCA) of the data (Fig.1A) evidences a dose responsive-sensitivity of liver to DEHP exposure.
Consistent with a dose–response effect, detailed examination ofhepatic DEHP-regulated transcripts identified 10, 32 and 72 mRNAwhichweremodulated byDEHP treatment at 30,180 and 1100mg/kg/day respectively (Table 1). These regulated genes were assigned to 8major biological functions (Table 1). Hepatic fatty acid homeostasis(synthesis, metabolism and transport), followed by xenobioticmetabolism, are primarily modulated by DEHP. These results wereconfirmed by Gene Set Enrichment Analysis (GSEA) which identifiedseveral gene sets related to fatty acid metabolism as the main targetsof DEHP (Supplementary material, Table S2).
Whereas most of these genes were upregulated (maximum 5 to46-fold depending on the dose), some downregulations also occurred(Table 1). These are related to glucose homeostasis (Pklr, Pck1, G6pcGck and Slc2a2/Glut2), steroid hormone metabolism (Hsd11b1,Hsd3b4 and Cyp7b1) and to xenobiotic metabolism (Cyp1a1, Gstp1,Slco1a1). The PCA results in a clear segregation of all control andtreated groups even at the lowest dose (Fig.1A). This demonstrates thecoordinate dose-dependent regulation of these transcripts by DEHPwhich is captured throughmultidimensional analysis evenwhen eachindividual effect is of low amplitude and statistical significance (lowdose).
A number of gene regulations identified by macroarray wereconfirmed by QPCR. We deliberately focused on three cytochromeP450s (Cyp): Cyp4a14, Cyp2b10 and Cyp3a11 which are prototypicdownstream targets of PPARα (Kroetz et al., 1998), ConstitutiveAndrostane Receptor (CAR, (Wei et al., 2000) and Pregnane X Receptor
(PXR, (Goodwin et al., 2002), respectively (Figs. 1B–D). In addition,Cyp3a11 and Cyp2b10 are major contributors to hepatic testosteronemetabolism. Cyp4a14 displayed the highest sensitivity to the low doseof DEHP (21.2-fold increase; Fig. 1B).
Metabonomic study of biochemical end points of DEHP exposure,in plasma and liver
To further characterize functional consequences of the DEHP-induced transcriptional perturbation, we used 1H NMR to record theabundance of lowmolecular weightmetabolites. Through a previouslydescribed variable selection procedure (Dumas et al., 2002), weextracted ten uncorrelated plasma metabolites whose concentrationswere significantly modified by DEHP (Table 2). A linear discriminantanalysis (LDA, Fig. 2A) of these data first evidenced a dose-dependenteffect of DEHP on these metabolites (LD1). DEHP exposure dose-dependently increased glutamine, glycerol and glyceryls anddecreased valine concentrations. LD2 evidenced a differential effectof the high DEHP dose compared to the low and intermediate doses.This was due to a drop in plasma VLDL and triglycerides at the highestdose only (data not shown) which are likely consequences of thewell-known activation of PPARα by DEHP.
Similarly, we analyzed aqueous liver extracts and selected ten polarmetabolites sensitive to DEHP treatment (Table 2). LDA (Fig. 2B)evidenced a dose-dependent separation of DEHP-treated groups alongLD1, due to an increase in glycerophosphocholine and glutamine, and adecrease in tyrosine/phenylalanine, 3-hydroxybutyrate/valine and iso-leucine concentrations (Table 2). LD2 evidenced the increase in two yetunidentifiedmetabolites at the lowand intermediate doses ofDEHPonly.
Integration of transcriptomic and metabonomic DEHP impacts by O2PLS
We used O2PLS to highlight covariations of transcripts andmetabolites in the liver. Groups treated by 180 and 1100 mg/kg/dayDEHPwere clearly separated along the 1st component of the PLS graph(Fig. 3A). The 2nd PLS component revealed an opposition between thecontrol group and the groups treated at the intermediate and highdoses. The transcriptomic and metabonomic variables were projectedon these first two PLS components (Fig. 3B). The closeness of variables,on this graph, reveals their covariation.We focused our interpretationson the two clusters of variables involved in the opposition between thecontrol group and the high DEHP dose group (Fig. 3B. Ellipse 1 forDEHP-induced variables and ellipse 2 for variables decreased byDEHP). Within these ellipses, the variables which displayed the
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strongest coordinated responsiveness to DEHP are found. Ellipse 1(Fig. 3B) is mainly composed of genes involved in xenobiotic (Cyps,Aldh, Gst) and fatty acid metabolism, but also contains amino acidsand genes of miscellaneous functions such as Vnn1 or Alas1.Interestingly, a previous study reported a robust impact of DEHP ontranscripts involved in amino acid metabolism (Currie et al., 2005).Ellipse 2 (Fig. 3B) contains xenobioticmetabolism transcripts (Cyp1a1,Gstp1), mRNAs related to lipid (mainly cholesterol) and bile acidmetabolism (Cyp7b1, Lipc, Abcb11, Elovl3, Hsd11b1, Hsd3b4), acutephase response proteins (Mup2, Serpina3k) and genes involved inglucose homeostasis (Pklr, Pck1, G6Pc, Slc2a2/Glut2).
Impact of DEHP on pathways upstream of heme synthesis
We first focused on DEHP-induced variables (Fig. 3B, Ellipse 1),leaving aside the known PPARα target genes involved in lipidmetabolism and the xenobiotic metabolism genes. We confirmedthe dose-dependent increase in Vnn1 (Fig. 4A) and Alas1 (Fig. 4B)transcripts expressions upon DEHP treatment. Since threonine wasthe amino acid most correlated to Vnn1, we explored the pathwaysthat could link these two variables. The mRNA expression of theenzymes involved in threonine catabolism (Fig. 4C, Sds and Thatranscripts were detected by QPCR in the liver but not Tdh transcript)were unaffected by DEHP administration (data not shown). On theother hand, glycine is catabolized through the aminomethyltransfer-ase enzyme (Amt), a key component of the glycine cleavage system(GCS) which is inhibited by the metabolites of the vanin1 protein
Fig. 1. Effect of DEHP administration on hepatic transcripts levels. Principal component anevidences a clear dose-dependent effect of DEHP on hepatic gene expression (A). The 79 trCyp2b10 (C) and Cyp3a11 (D) mRNAwere determined using Q-PCR normalized to the exprescomparison with the control group.
(Yudkoff et al., 1981). Amt mRNA expression is unaffected by DEHPtreatment (data not shown). However, in accordance with Vnn1regulation, cystamine (δ=2.99 ppm, highlighted on Fig. 3B) isincreased by DEHP treatment, suggesting that glycine degradationthrough the GCS is reduced. Overall, combined threonine increase andVnn1 induction would thus contribute to increase glycine levels.However, NMR spectra analysis did not reveal significant change inglycine levels upon DEHP exposure. Glycine is the co-substrate ofsuccinyl-CoA in the first and rate-limiting step of heme synthesis (Fig.4C) catalyzed by Alas1 which is induced by DEHP (Fig. 4B). Alas-1inductionmay contribute to buffer glycine level under DEHP exposure.Hence, this would lead to an increased heme synthesis.
Impact of DEHP on pathways downstream of heme synthesis
Therefore, we next questioned whether the expression of heme-sensitive genes may also be altered under DEHP exposure. Heme is aligand for Rev-erbα (Raghuram et al., 2007; Yin et al., 2007), a NRwhich, in turn, represses crucial genes involved in the cellular clockand in energy metabolism. Therefore, we first measured changes inthe expression of Rev-erb isoforms. While Rev-erbβ expression wasunaffected (Fig. 5A), Rev-erbα expressionwas increased by DEHP (Fig.5B). Remarkably, among the genes repressed by DEHP, O2PLS (Fig. 3B,Ellipse 2) highlighted Elongase 3 (Elovl3), G6Pc and Pck1 (Table 1) allwell-characterized targets of Rev-erbα (Yin et al., 2007). Weconfirmed the repressive effect of DEHP exposure on these genesthrough QPCR (Figs. 5C–E). To further document a likely link between
alysis (PCA) of liver transcriptional signature (each symbol is an individual sample)anscripts presented in Table 1 were used for PCA. Relative expressions of Cyp4a14 (B),sion of TBP. Values shown are the mean±S.E.M. (n=6 for each group). ⁎Pb0.05 for the
Fig. 2. Linear discriminant analysis (LDA) of NMR data. Ten NMR signals of plasmadataset (A) and aqueous liver extract (B) that contribute the most to the first two axesof the LDAwere used to project all individuals on the first factorial plan. All individualsof each groupwere represented in a polygonwhere 0,1, 2 and 3 correspond respectivelyto 0, 30, 180 and 1100 mg of DEHP/kg/day treated group.
Table 2Summary of themain variations of plasma and aqueous liver extract metabolites duringDEHP treatment evidenced by LDA.
The correlation coefficients of the selected metabolites with the first two lineardiscriminant axis are presented. Variations compared to control samples: ↑, indicatesrelative increase in NMR signal; ↓ relative decrease in NMR signal.
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Alas1 expression and Rev-erb signalling, we also investigated theresponse of Bmal1 transcript to DEHP. This key component of themammalian clock is under the tight control of Rev-erb activity(Raghuram et al., 2007; Yin et al., 2007). We showed that it issignificantly repressed by DEHP (Fig. 5F).
Discussion
From morphogenic to metabolic impacts, exposure to phthalatesmetabolites affects several biological functions in various organs. Inaddition to being considered as potential nongenotoxic carcinogens(Ito and Nakajima, 2008), recent population studies have suggestedthat phthalates are also responsible for: i) key features of themetabolic syndrome (Stahlhut et al., 2007; Hatch et al., 2008) andii) subtle developmental reproductive effects that evoke antiandrogenexposure (Swan et al., 2005).
Systems biology (Urbanczyk-Wochniak et al., 2003) may representa relevant approach to capture such a broad spectrum of impacts, evenat low experimental doses, closer to the ones humanpopulationmightbe exposed to.
CAR canonical target gene responds to DEHP exposure
Many of the DEHP-regulated genes in liver and kidney are well-described PPARα targets (Mandard et al., 2004). Thus, as anticipated(Lapinskas et al., 2005), the hepatic expression profiles we obtaineddisplayed a dose-dependent modulation of the PPARα signalingpathway. In addition, our results support the in vivo recruitment oftwo additional hepatic NR pathways. This was suggested by thedownstream induction of two prototypical target genes of PXR andCAR. Indeed, we observed a DEHP-induced increase in Cyp3a11expression (Table 1, Fig. 1D). Cyp3a11 is a highly sensitive PXR targetgene (Goodwin et al., 2002). It was previously shown that theinduction of hepatic Cyp3a11 by DEHP in SV129 mice is PPARα-independent (Fan et al., 2004). Gene reporter assays in cell lines withmouse and human PXR (Hurst and Waxman, 2004; Mnif et al., 2007)and from DEHP-treated primary human hepatocytes (Mnif et al.,2007) supported the role of PXR in this regulation.
We also measured a more pronounced effect of DEHP on theCyp2b10 mRNA (Table 1, Fig. 1C), a prototypical target of CAR (Wei
et al., 2000). A similar effect of DBP (Di-n-butyl phthalate) wasreported in rat (Wyde et al., 2005). In our study, the effect of DEHP onadult mouse liver is more pronounced (4.5-fold at 180mg/kg/day, 70-fold at 1100 mg/kg/day).
A recent report has highlighted the sex-specific regulation of anumber of growth hormone-sensitive genes inmice liver (Clodfelter etal., 2007). Interestingly, in DEHP-treated mice we observed asignificant upregulation of several genes (including Cyp2b10 andalso Cyp4a14, Cyp2b13, CD36) that were shown to be female-specific.Moreover, we also observed in DEHP-treated mice a decreasedexpression of male-specific genes (such as Cyp7b1, Elov13, Gstp1,Mup2). Since our experiments were performed in male mice, thehypothesis that DEHP exposure disturbs the growth hormonesignaling in the liver and contributes to endocrine disruption mustbe considered.
It has been suggested that phthalates could exert part of theirendocrine disruptor activities by altering PXR-regulated steroidhormone metabolism in humans (Hurst and Waxman, 2004). Ourdata further support endocrine disruption associated with hormonesbioavailability. Since Cyp2b10 is also a critical enzyme in testosteronemetabolism (Wei et al., 2000), DEHP might also alter testosteronelevel by influencing not only the PXR-regulated expression of Cyp3a11but also CAR-regulated expression of Cyp2b10.
PPARα, PXR and CAR are embedded within a tangle of networkswhich control energy homeostasis, particularly in the liver (Konnoet al., 2008; Moreau et al., 2008). These receptors were shown to bekey players in lipid utilization and synthesis as well as in glucose
Fig. 3. Partial least square regression of transcripts and metabolites. Hepatic transcriptomic and aqueous liver extract metabolites datasets were subjected to O2PLS analysis (LV1/2for latent variables 1/2). (A) Projection of the individuals (scores plot) treated with 30 (light grey; n=4), 180 (dark grey; n=6) and 1100 mg of DEHP/kg/day (black; n=5) orvehicle (white; n=6). (B) Loadings plot of metabolites (grey dots) and transcripts (black dots) levels. Magnified view on the most strongly DEHP-upregulated (Ellipse 1) and -downregulated (Ellipse 2) variables.
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metabolism. Consistent with these conclusions, CAR could contributeto the downregulation of its direct negative target Pck1 (Miao et al.,2006) observed in our study (Fig. 5E).
Integrated analysis of transcriptomic and metabonomic data highlightsthe hepatic effect of DEHP on Rev-erbα
PLS analysis highlighted several covariations between transcriptsand metabolites affected by DEHP treatment. Together with genesstrongly induced by DEHP (Fig. 3B ellipse 1) were changes in amino
acids (such as threonine) and two genes related to amino acidmetabolism (Vnn1 and Alas1). We questioned about the biologicalsignificance of this covariation. This led us to postulate that it mightreveal an impact of DEHP on heme homeostasis. Amino acidmetabolism is coupled to heme synthesis (Fig. 4C). Moreovercytochrome p450 require heme for their activity. Finally, Alas1 is therate-limiting enzyme in heme synthesis. Therefore, changes in aminoacids metabolism might be coupled to increased Alas1-mediatedheme synthesis and provide cellular heme for efficient cytochromeP450 activities.
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Alas1 expression is sensitive to several xenobiotics including CARand PXR activators (Maglich et al., 2002; Ueda et al., 2002). In additionAlas1 is regulated by the nutritional status, likely through changes inPPARγ Coactivator 1 α (PGC1α) expression (Handschin et al., 2005).Following DEHP exposure, we did not observe any change in PGC1αexpression (Supplementary material, Fig. S1). Hence, the effect ofDEHP on Alas1 expression does not seem to be a consequence ofchanges in the nutritional status and PGC1α. Overall, the mechanismby which DEHP increases Alas1 expression remains to be defined.
One likely consequence of Alas1-mediated heme increase is achange in the expression of genes sensitive to cellular heme level.Recently, heme has been identified as a ligand for the NR Rev-erbα andβ (Raghuram et al., 2007; Yin et al., 2007). Rev-erbα mediates therepressive effects of heme on the expression of glucose (Pck1 and
Fig. 4. Biological pathways linking threonine, glycine, vanin1 and Alas1. (A) Relative expressioof TBP. Values shown are themean±S.E.M. (n=6 for each group). ⁎Pb0.05 for the comparisoand downstream of heme synthesis.
G6pc) and fatty acidmetabolism (Elovl3) genes and of Bmal1, amasterregulator of the mammalian circadian clock (Raghuram et al., 2007;Yin et al., 2007). Using our NR-dedicated macroarray and QPCR, weobserved a dose-dependent downregulation of the typical Rev-erbαtarget genes Elovl3, G6pc and Pck1 (Table 1, Figs. 5C–E). Strikingly, inthe PLS analysis, these genes formed, together with Slc2a2/Glut2, acluster located in Ellipse 2 at the opposite corner when compared toEllipse 1 (Fig. 3B). This evidences a correlation in the oppositeregulations between these groups of genes andmetabolites. To furtheraddress the putative effect of DEHP on the Rev-erbα pathway, wemeasured by QPCR the expression of Bmal1 whose cyclic transcriptionismainly controlled by Rev-erbα (Preitner et al., 2002). Like other Rev-erbα targets, BmalI expressionwas reduced following DEHP exposure(Fig. 5F). Altogether, these results support the hypothesis that DEHP
ns of Vnn1 and Alas1mRNAwere determined using Q-PCR normalized to the expressionnwith the control group. (B) Proposedmodel of the combined effects of DEHP upstream
Fig. 5. Effects of DEHP on genes related to the Rev-erbα pathway. Relative expressions of Rev-erbβ (A), Rev-erbα (B), Elovl3 (C), G6pc (D), Pck1 (E) and Bmal1 (F) mRNA weredetermined using Q-PCR normalized to the expression of TBP. Values shown are the mean±S.E.M. (n=6 for each group). ⁎Pb0.05 for the comparison with the control group.
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also acts via hepatic Rev-erbα induction coupled with increased hemesynthesis through Alas1 induction to affect hepatic metabolic andcircadian pathways (Fig. 4C). This effect of DEHP on the Rev-erb
pathway is likely to contribute to the regulation of other clock genessuch as Per and cry which has been previously reported after DEHPexposure (Currie et al., 2005).
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Lately, consistent reports have raised genuine concerns over theintrinsic potential of phthalates to exert metabolic disruption. Relyingon in vivo experimentation and a systems biology approach this workhas confirmed previously observed effects of DEHP on two NR-drivenpathways, those involving PPARα and PXR. Moreover, new impacts ofDEHP on NR-driven pathways are also reported in this work. We showthat DEHP impacts on both CAR and Rev-erbαwhich both control notonly xenobiotic detoxification but also energy homeostasis and thecircadian clock respectively. These molecular findings furtherstrengthen the ability of phthalates to behave as metabolic disruptorsand their alleged role as environmental obesogens (Grun andBlumberg, 2007).
In addition to the effects of phthalates on genital tract develop-ment (Swan et al., 2005; Marsee et al., 2006), recent studies haveraised concerns about the effects of phthalate adult exposure onreproductive functions. Two independent studies have reported aninverse association between MEHP and testosterone levels in humansexposed to different extents to DEHP (Pan et al., 2006; Meeker et al., inpress). The effects of DEHP in the liver such as the induction of Cyp3aand Cyp2b could contribute to such an effect. However, an associationbetween MEHP and sperm DNA damage has also been reported(Hauser et al., 2007; Hauser, 2008). A direct role of the liver in thiscontext appears unlikely and other organs could thus be importanttargets of DEHP and its metabolites. We have performed a preliminarygenome-wide gene expression study in microdissected areas of thetestis from adult mice exposed or not to DEHP (Supplementary FilesS1, S2 and S3). This study revealed in particular that the transcriptomeof adult Leydig cells is markedly modulated by DEHP exposure.Further studies are thus required in organs other than the liver tounderstand at the level of the whole organism the mechanismsunderlying the effects of DEHP.
Through a systems biology approach we showed that DEHPinterferes with hepatic CAR and Rev-erbα pathways. Our preliminarydata further suggest an effect of DEHP on the transcriptome ofendocrine cells isolated from the mature testis. The relevance of suchfindings to major public health concerns such as the metabolicsyndrome and male fertility should be further considered.
Acknowledgments
We acknowledge the excellent technical assistance of ColetteBétoulières and Gérard Galy. We are grateful to Dr Talal Al Saati andFlorence Capilla (Histopathology core facility of IFR30, Toulouse) fortheir help in laser-capture microdissection. We thank Dr Jean Mosser,Amandine Etcheverry and Régis Bouvet (Transcriptomic Facility,Ouest-Génopôle, Rennes) for their advices concerning gene expressionprofiling. This work was supported by a grant from ANR (PNRA-PlastImpact program). A.E. was recipient of a fellowship from MESR.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.taap.2009.02.008.
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