The Role Of Peroxisome Proliferator Activated Receptor In The Effects Of Gestation Exposure To Phthalates On Preterm Delivery And Fetal Development

1. Back groundPhthalates Acid Ester (PAEs) are a class of synthetic chemicals which are produced inlarge volumes and used in a wide variety of industrial and common household products andare commonly found in the environment because of its widespread use in the world. Somephthalates can cross the placental barrier and be detected in cord blood. In our previousstudies, phthalates were found in water samples from the Yangtze and Jialing rivers. And9phthalates were detected in cord blood in pregnant women. Nevertheless, the effects ofprenatal exposure to phthalates on preterm delivery and fetal growth are still unknown.Other studies have suggested an association between phthalate exposure and shortergestational age and lower birth weight. but other studies found no significant relationshipbetween prenatal exposure to phthalates and birth outcomes.Our previous studies shown that the MEHP, one metabolite of DEHP, could increasethe expression of peroxisome proliferator activated receptor gamma (PPARγ) in ovariangranulosa cell. Others found phthalates can active PPARs as its ligands. PPARs in placentacan affect the hormone production, expression of protein associated with inflammation likematrix metalloproteinases (MMPs), fat and glucose metabolism. Both of them areassociated with preterm delivery and development. so we presume that the expression ofPPARs in placenta were play a role in the association of phthalates with preterm deliveryand fetal development.In this study we used both epidemiology and experiments research to investigate theassociations of phthalate levels in cord blood with fetal growth and preterm delivery and themechanism of them.2. Material and methods2.1epidemiology experiments research for the relationship of phthalate levels in cord blood with fetal growth and preterm delivery.2.1.1Study population. Two-hundred and seven volunteers were recruited atSouthwest Hospital in Chongqing, Southwest China. None of the participants had a familyor personal history of occupational exposure to phthalates. We administered a questionnaireto the participants after labor to obtain information on socio-demographic characteristics,medical history and lifestyle factors. Delivery characteristics and birth outcomes wereobtained from the perinatal database of Southwest Hospital, including preterm delivery(defined as gestational age less than37weeks), premature rupture of membranes (PROM),chorioamnionitis, intrahepatic cholestasis of pregnancy (ICP), placenta previa,pregnancy-induced hypertension syndrome (PIH, including preeclampsia), gestationaldiabetes mellitus (GDM), abruption placentae, intravenous transfusion history(defined asbeen put on a drip within the last week before labor), gestational age, birth weight, birthlength, head circumference (HC), biparietal diameter (BPD), abdominal circumference (AC)and femur length (FL). Ten millilitre of cord blood were obtained from subjects within10min of delivery,5ml was stored in a heparinized glass container for phthalatesmeasurements and5ml was separated the serum for hormonal assess. The placental samplefrom each pregnant were collected to detect the protein expression of PPARγ.2.1.2Phthalates measurements. Phthalates in cord blood were extracted by liquidextraction and analyzed by GC/MS. Fifteen phthalates were identified and quantitated bytheir characteristic retention time, including Dimethyl phthalate (DMP), Diethyl phthalate(DEP), Diisobutyl phthalate (DIBP), Dibutyl phthalate (DBP), bis (2-methoxyethyl)phthalate (DMEP), bis (4-methyl-2-pentyl) phthalate (BMPP), bis (2-ethoxyethyl) phthalate(DEEP), Diamyl phthalate (DPP), Dihexyl phthalate (DNHP), Benzyl butyl phthalate(BBP), bis (2-n-butoxyethyl) phthalate (DBEP), Dicyclohexyl phthalate (DCHP), bis(2-ethyl hexyl) phthalate (DEHP), Di-n-octyl phthalate (DNOP) and Dinonyl phthalate(DNP).2.1.3Hormonal assess. The matrix metalloproteinases (MMPs) and prostaglandin E2(PGE2) in serum were assessed by ELISA method and the progesterone (P), estradiol (E2)and estriol (E3) were assessed by radioimmunoassay.2.1.4Protein assess. The protein was extract from placenta by RIPA Lysis Buffer andthe expression of PPARγ assessed by Western Blot and BeyoECL Plus. 2.1.5Statistical analyses. All statistical analyses were performed with SPSS forwindows version18.0. Independent sample T-test and Chi-square test were used to analyzethe characteristics of pregnant women in preterm and term delivery group and theiroffspring. We used linear regressions and Independent sample T-test to estimate theassociation of phthalates with expression of PPARγ, hormonal, gestational age and birthoutcomes, and used binary logistic regression and Independent sample T-test to estimate theassociation of preterm delivery with phthalates, expression of PPARγ in placenta and serumhormonal.2.2The role of PPARγ in the adverse effects of DEHP in pregnant rats.2.2.1A total60female Sprague-Dawley rats (6-7weeks old) were randomized into2groups,20in control group which received by gavage corn oil,40in exposure group whichreceived by gavage2g/kg of DEHP for27days. Then the exposure group were equallyrandomized into2group (20per group) and respectively received by gavage2g/kg ofDEHP (exposure group), DEHP2ml/kg+0.5mg/kg GW9662（antagonist of PPARγ）(intervention group) for3days and mate with health male rats. After one week for mating,the pregnant rats were still gavaged corn oil (the control group),2g/kg of DEHP (theexposure group), DEHP2ml/kg+0.5mg/kg GW9662(the intervention group). Themorning in which a vaginal plug was found was designated day1of gestation. Treated ratswere sacrificed between at day20of gestation. The uteri were excised immediately toevaluate the effect of DEHP and GW9662on fetal development. Blood samples werecollected to assess the effect of Bap and DBP on plasma hormonal concentrations. Placentawere isolated for the detection of protein and mRNA expression.2.2.2Hormonal assess. The estrone (E1), testesterone (T), dehydroepi androsteronesulfate (DHEAS),5-androstenedione (ASD) prostaglandin E2(PGE2) and prostaglandinF2a (PGF2a) in serum were assessed by ELISA method and the progesterone (P), estradiol(E2) were assessed by radioimmunoassay.2.2.3Protein and mRNA assess. The protein was extract from placenta by RIPA LysisBuffer and the expression of PPARγ was assessed by Western Blot and BeyoECL Plus. ThemRNA was extract from placenta by Trizol and the expression of PPARγ, PPARα, CYP191,MMP2and MMP9were assessed by Realtime RT-PCR (SYBR Green I).2.2.4Statistical analyses. All statistical analyses were performed with SPSS for windows version18.0. Independent sample T-test and Chi-square test were used to analyzethe effect of DEHP and GW9662.3. Results and discussion3.1Cord blood phthalate levels and preterm delivery.Cord blood levels of14phthalates (except DCHP) were increased in preterm group.Cord blood levels of phthalates (except DNHP, DMEP, DBEP and DCHP) were increased inPROM group which was associated with preterm. But the cord blood levels of13phthalates(except DCHP and DNHP) were also increased in preterm group in non-PROM pregnants.The intravenous transfution history was associated with preterm delivery and3phthalates(DMP, DEEP and BMPP). And14phthalates (except BMPP, DNHP and DCHP) were alsoassociated with preterm delivery in the group which hadn t has the intravenous transfutionhistory. Those indicates that the PROM and the intravenous transfution history may play arole in the between phthalates in cord blood and preterm, but not the key one.3.2Cord blood phthalate levels and birth outcomes.Higher phthalate concentrations in cord blood had decreased birth weight, birth length,abdominal circumference, femur length, biparietal diameter, head circumference, both inmale and female infants, respectively. Majority of these associations disappeared afteradjusting for gestational age or analysis in term group. And the preterm delivery andgestational age reduction were also associated with birth outcomes. These suggested thatthe phthalates in cord blood may have adverse effects on fetal development, which may betotally dependent on the gestational duration reduction and preterm delivery. After adjustingfor gestational age, the sensitive to phthalates and sensitive indicator were differencesbetween the genders. These indicate the gender differencesmay exist in the associationsbetween phthalates exposure and birth outcomes.3.3The associations of protein expression of PPARγ in placenta with phthalates incord blood, preterm, and birth outcomes.3.3.1The associations of protein expression of PPARγ in placenta with phthalates incord blood.Higher phthalate concentrations in cord blood had significantly higher proteinexpression of PPARγ in placenta. And the phthalate levels were positive correlation withprotein expression of PPARγ in placenta. These associations were also existed in term and preterm group, respectively. These indicated that the phthalate levels were directly positivecorrelation with protein expression of PPARγ in placenta.3.3.2The associations of protein expression of PPARγ in placenta with preterm.The protein expression of PPARγ were significantly increased in preterm and PROMgroup. And in non-PROM pregnant, the protein expression of PPARγ were alsosignificantly increased in preterm. These indicated that the protein expression of PPARγmay associated with preterm delivery which may partially dependent on PROM and theprotein expression of PPARγ in placenta may play a role in the associations betweenphthalates in cord blood and preterm delivery.3.3.3The associations of protein expression of PPARγ in placenta with birth outcomes.The protein expression of PPARγ was negative correlated with birth weight, birthlength, abdominal circumference, femur length, biparietal diameter, head circumference,both in male and female infants, respectively. Majority of these associations disappearedwhen analysis in term group. These suggested that, like the phthalates in cord blood, theprotein expression of PPARγ may have adverse effects on fetal development, which may bepartially to dependent on the preterm delivery.3.4The associations of serum hormonal and inflammation-associated protein withphthalates in cord blood, protein expression of PPARγ in placenta, preterm, and birthoutcomes3.4.1The associations of phthalates in cord blood with serum hormonal andinflammation-associated protein.In total pregnant, higher phthalate concentrations in cord blood had significantlyhigher progesterone levels, and lower estriol, estradiol, matrix metalloproteinases andprostaglandin E2levels. But in term and preterm group, respectively, the phthalateconcentrations in cord blood was positive correlated with estriol and estradiol level. Theseindicates that the phthalate concentrations in cord blood was positive correlated with estrins(include estriol and estradiol) and progesterone level, and negative correlated withinflammation-associated protein (include matrix metalloproteinases and prostaglandin E2).3.4.2The associations of protein expression of PPARγ in placenta with serumhormonal and inflammation-associated protein.The protein expression of PPARγ in placenta was negatively associated with prostaglandin E2in term group.3.4.3The associations of serum hormonal and inflammation-associated protein withpreterm delivery and PROM.The preterm group had significantly lower estriol level. And the PROM group had notany significantly change in serum hormonal and inflammation-associated protein.3.4.3The associations of serum hormonal and inflammation-associated protein withbirth outcomes.The estradiol level was negatively related with abdominal circumference, biparietaldiameter and head circumference; and the progesterone level was negatively related withabdominal circumference; and prostaglandin E2was positively related with birth length infemale infants in term group. The estradiol level was negatively related with birth weight;and matrix metalloproteinases was positively related with birth length in male infants interm group. Because the phthalates in cord blood are positive correlated with estriol andnegative correlated with matrix metalloproteinases and prostaglandin E2, and the proteinexpression of PPARγ in placenta was negatively associated with prostaglandin E2. wesupposed that serum hormonal and inflammation-associated protein a role in theassociations of phthalates in cord blood and the protein expression of PPARγ in placentawith preterm.3.5The role of PPARγ in the reproductive toxicity of DEHP in pregnant rats.3.5.1The effect of DEHP and PPARγ on rat growth and fetuses development.The exposure and intervention group had significangt changes in body gains ofpregnant rats versus control group before and after pregnancy. And the pregnancy Rate,viable fetuses rate, fetuses number per pregnant rat and fetal weight were significantlydecreased in both exposure and intervention group. Indicates the exposure dose used in thestudy had serious reproductive toxicity but didn’t affacted the growth of pregnent rats.3.5.2The expression of PPARγ in placenta in DEHP exposed pregnant rats.The protein expression of PPARγ in placenta were significantly increased and themRNA expression of PPARγ and PPARα, another isoform of PPARs, in placenta weresignificantly decreased in both exposure and intervention group. These indicates the DEHPmay up-regulate the protain expression of PPARγ after Transcription, not Transcription level.3.5.3The role of PPARγ in hormonal regulation of DEHP in pregnent rats. The estradiol level was significantly increased and the ratio of testesterone to estradiol,which reflected the active of P450Arom was significantly decreased when exposoed toDEHP. But the mRNA expression of P450Arom was significantly decreased in the exposuregroup. And the ratio of estradiol to estrone, which reflected the active of17β-HSDs wassignificantly increased. When inhibiting the activity of PPARγ by GW9662, the mRNAexpression of P450Arom increased and ratio of estradiol to estrone increased moresignificantly. So wo supposed that the inhibition of P450Arom expression by PPARγ mayplay a role in hormonal regulation of DEHP, but not the key one. The change of17β-HSDsactivity may be more important in hormonal regulation of DEHP. And the DEHP canincrease the estrins level, which may play a role in the association of phthalates in cordblood with preterm and birth outcomes.3.5.4The role of PPARγ in regulation of inflammation-associated protein in DEHPexposed pregnent rats.The mRNA expression of MMP2, MMP9were significantly decreased when exposedto DEHP, and the prostaglandin E2and prostaglandin F2a levels in serum were not changed.When inhibiting the activity of PPARγ by GW9662, the mRNA expression of MMP9wereincreased. These indicated that the PPARγ play a role in regulating the mRNA expression ofMMP9by DEHP.The MMPs and prostaglandin E2were important to fetal development, which werepositively correlated to birth outcomes in our epidemiologic study. Our study suggested thatthe phthalates exposure may down regulate the MMPs and the prostagland in E2partiallythrough PPARγ pathway, which may play a role in the association of phthalates exposureand birth outcomes.4. ConclusionIn our study, there is a strong evidence of the association between phthalate levels incord blood and preterm delivery and birth outcomes. The expression of PPARγ in placentamay play a role in there association through alterating the serum sexual hormone andinflammation-associated protein like MMPs and prostaglandin E2.