Prenatal dexamethasone exposure-induced a gender-difference and sustainable multi-organ damage in offspring rats via serum metabolic profile analysis

Toxicology Letters 316 (2019) 136–146

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Prenatal dexamethasone exposure-induced a gender-difference and sustainable multi-organ damage in offspring rats via serum metabolic profile analysis

T

Guanghui Chena,1, Hao Xiaob,1, Jinzhi Zhanga, Huizhen Zhangd, Bin Lib, Tao Jianga, Yajie Wend, ⁎⁎ ⁎ Yimin Jiangd, Kaili Fud, Dan Xua,c, Yu Guoa,c, Ying Aoa,c, Huichang Bid, , Hui Wanga,c, a

Department of Pharmacology, School of Basic Medical Science of Wuhan University, Wuhan, 430071, China Department of Orthopedic Surgery, Zhongnan Hospital of Wuhan University, Wuhan 430071, China Hubei Provincial Key Laboratory of Developmentally Originated Disorder, Wuhan, 430071, China d School of Pharmaceutical Sciences, Sun Yat-sen University, 132# Waihuandong Road, Guangzhou University City, Guangzhou, 510006, China b c

G R A P H I C A L A B S T R A C T

A R T I C LE I N FO

A B S T R A C T

Keywords: Metabolomics Developmental toxicity Protein breakdown Glycolysis Lipid metabolism

Prenatal dexamethasone exposure (PDE) induces developmental toxicities of multiple organs in offspring, but its serum metabolic profile changes before and after birth are unclear. Here, we employed a LC–MS-based metabolomic approach to detect serum metabolites of PDE offspring rats in utero and adulthood, and explore its change characteristics and toxicological significances. Meanwhile, the bodyweight, serum index related to hepatic and renal function were detected. As compared to healthy control rats, PDE reduced offspring birthweight but caused postnatal catch-up growth accompanied by adult liver and kidney function injury. In utero, the differential metabolites in response to PDE were mainly manifested as enhanced glycolysis, increased protein breakdown and disordered lipid metabolism, and multiple metabolic pathways were changed, which displayed gender differences. In adulthood, PDE offspring showed fewer and inconsistent types of differential metabolites

Abbreviations: PDE, prenatal dexamethasone exposure; IUGR, intrauterine growth retardation; GD, gestational day; PD, postnatal day; PW, postnatal week; ACTH, adrenocorticotrophic hormone; NMR, nuclear magnetic resonance; GC-MS, gas chromatography-mass spectrometry; LC–MS, liquid chromatography-mass spectrometry; OPLS-DA, orthogonal partial least square-discriminate analysis; CA, cholic acid; CDCA, chenodeoxycholic acid; MCA, muricholic acid; TMCA, tauro-muricholic acid; HCA, hyocholic acid; HPLC, high performance liquid chromatography; PE, phosphatidyl ethanolamine; PC, phosphatidylcholine ⁎ Corresponding author at: Department of Pharmacology, School of Basic Medical Science of Wuhan University, Wuhan, 430071, China. ⁎⁎ Corresponding author at: Department of Pharmacology, School of Basic Medical Science of Wuhan University, Wuhan, 430071, China. E-mail addresses: [email protected] (H. Bi), [email protected] (H. Wang). 1 Guanghui Chen and Hao Xiao contributed equally to this work. https://doi.org/10.1016/j.toxlet.2019.09.007 Received 22 August 2018; Received in revised form 1 August 2019; Accepted 8 September 2019 Available online 11 September 2019 0378-4274/ © 2019 Published by Elsevier B.V.

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compared to those in utero, which exhibited significant gender differences. The main differential metabolites induced by PDE included lactic acid, carnitine, cortexolone, bile acid, phosphatidylcholine, uric acid and platelet activating factor, which may participate in dexamethasone multi-organ toxicities and multi-disease susceptibility. In conclusion, PDE could induce a gender-difference and sustainable multi-organ damage in the offspring rats via serum metabolic profile analysis, which will enhance offspring susceptibility to multiple adult diseases.

1. Introduction

toxicological significances. Therefore, the bodyweight, liver and kidney function and serum metabolic profiles based on LC–MS metabolomic techniques were detected between the control and PDE offspring rats in utero and adulthood. This study is beneficial to fully recognize the short and long-term developmental toxicities caused by PDE, and to explore the early warning and therapeutic biomarkers of dexamethasone development toxicities.

Dexamethasone is a synthetic glucocorticoid that penetrates easily through the placenta into the foetal circulation to accelerate foetal lung maturation (Porto et al., 2011; Vogel et al., 2017). Therefore, it is widely used in multiple pregnancy-related diseases including threatened premature birth, neonatal atelectasis, placenta previa and multiple pregnancy, and its clinical efficacy is definite (Crowther et al., 2015). According to the statistical data from World Health Organization (WHO), the estimated global preterm birth rate was 10.6%, equating to an estimated 14.84 million live preterm births (Chawanpaiboon, et al. 2019). Moreover, survey data from 359 institutions in 29 countries indicated that the average rate of prophylactic treatment with synthetic glucocorticoids in preterm infants at gestation weeks 22–36 is 54%, and the utilization rate in some countries is up to 91% (Vogel et al., 2014). However, dexamethasone treatment during pregnancy is a doubleedged sword because it could increase the incidence rate of foetal intrauterine growth retardation (IUGR) (Murphy et al., 2008, 2012), multi-organ dysplasia (Xu et al., 2011; Zhang et al., 2016) and susceptibility to various diseases in adulthood (Sun et al., 2016; Nguyen et al., 2015; Tseng et al., 2016). Nevertheless, the metabolic profile characteristics, early warning and therapeutic targets of dexamethasone developmental toxicity have not been systematically elucidated. Metabolomics mainly applies nuclear magnetic resonance (NMR), gas chromatography-mass spectrometry (GC–MS), and liquid chromatography-mass spectrometry (LC–MS) to qualitatively and quantitatively analyse small molecules in biological samples for revealing the molecular mechanism of biological process or searching potential biomarkers (Wishart, 2016; Ding and Mohan, 2016). Recently, the technique has been used in exploring the mechanisms of IUGR induced by adverse environments during pregnancy (Pedroso et al., 2017). In our previous studies, we found that maternal and foetal serum metabolic profiles were changed in the IUGR offspring rats induced by prenatal caffeine and nicotine exposure, which were mainly due to the intrauterine programming effects of overexposure to maternal glucocorticoids (Kou et al., 2014; Feng et al., 2014; Liu et al., 2012). However, whether dexamethasone exposure during pregnancy could cause the changes of offspring’s serum metabolic profiling before and after birth, the profiling characteristics of the differential metabolites, gender differences, and candidate biomarkers for developmental toxicity are unknown. In the present study, we aimed to investigate the serum metabolic profile characteristics of offspring before and after birth induced by prenatal dexamethasone exposure (PDE), and then to clarify the

2. Materials and methods 2.1. Animals and treatment Specific pathogen-free Wistar rats (No. 2012-2014, Certification No. 42000600002258, License No. SCXK (Hubei)) weighing 209 ± 12 g (females) and 258 ± 17 g (males) were obtained from the Experimental Center of the Hubei Medical Scientific Academy (Wuhan, China). Animal experiments were performed in the Center for Animal Experiment of Wuhan University (Wuhan, China), which is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International). The Committee on the Ethics of Animal Experiments of the Wuhan University School of Medicine approved the protocol (Permit No. 201719). All animal experimental procedures were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals of the Chinese Animal Welfare Committee. To reduce bias in animal experiments, rats were housed and treated by a technician, while different co-authors were in charge of serum sample harvesting and data analysis. Rats were housed in metal cages with wire-mesh floors in an airconditioned room under standard conditions (room temperature: 1822℃; humidity: 40%–60%; light cycle: 12 -h light-dark cycle; 10–15 air changes per hour) and allowed free access to rat chow and tap water. All rats were acclimated one week before treatment, and two female rats were placed together with one male rat overnight in a cage for mating. The appearance of sperm in vaginal smears confirmed mating, and the day of mating was taken as gestational day (GD) 0. From GD9 to GD20, the pregnant rats were injected subcutaneously once a day with 0.2 mg/kg dexamethasone, and the control rats were sham-treated with the same volume of the vehicle (saline solution). On GD20, to execute some of the pregnant rats, inhaled isoflurane was applied to maintain an anaesthesia status. The animals were rapidly sacrificed after their righting reflexes disappeared. Pregnant rats with litter sizes of 12–14 (the female/male ratio was approximately 1:1) were considered qualified (n = 15 pregnant rats for each group). IUGR was diagnosed when the bodyweight of an animal was two standard deviations lower than

Fig. 1. The animal treatment procedures of this research. 137

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14,000 × g at 4 °C for 5 min. Finally, an aliquot (5 μL) of the supernatant was injected for ultra-high performance liquid chromatography electrospray ionization mass spectrometry (UHPLC-ESI-MS) analysis. Hydrophilic interaction liquid chromatography (HILIC) separation was carried out by using an Atlantis Silica HILIC column (3 μm, 2.1 mm i.d. × 100 mm, Waters, Milford, MA, USA) on a Thermo Scientific Dionex Ultimate 3000 UHPLC system. The flow rate was 300 μL/min with column temperature at 40 °C. Binary mobile phases were (A) 5% water in acetonitrile with 10 mM ammonium formate and 0.1% formic acid and (B) 50% water in acetonitrile with 10 mM ammonium formate and 0.1% formic acid. Linear gradient was implemented as follows: 0–1.0 min holding at 100% A, linearly increasing to 100% B at 20 min, then washing column for the next 4.9 min, and equilibrating until 30 min. MS was operated with a Thermo Scientific Q Exactive TM benchtop Orbitrap mass spectrometer equipped with heated ESI source in ESI positive and negative modes (Thermo Scientific, San Jose, CA). The samples were independently examined in both ESI positive and negative modes. Untargeted profiling analyses acquired the data at full scan mode (80–900 m/z) and 70,000 FWHM resolution, followed by top-10 data-dependent MS/MS at 17,500 FWHM resolution. The main parameters for MS/MS included AGC target 1e 5; maximum IT 70 ms; isolation window 2.0 m/z; normalized collision energy 15, 30, and 45 eV; apex trigger 5∼10 s; and dynamic exclusion 10 s. Ionization conditions were optimized and finally operated at spray voltage 3.5/ 2.8 kV (+/−) and heater and capillary temperatures 350 and 325 °C, respectively. Total ion chromatograms and mass spectra from LC-HRMS runs were generated as raw files in Xcalibur (Thermo Scientific, San Jose, CA).

the mean bodyweight of the control group (Simic Klaric et al., 2012), and the IUGR rate was calculated according to the following formula. The foetal blood was collected, combined (one litter per tube), centrifuged and stored at −80 °C until further analysis. IUGR rate per litter (%) = (number per litter IUGR rat foetuses/ the total number of foetal rats per litter) × 100 IUGR rate per group (%) = (the sum of foetal rat IUGR rate per litter/ each group of litter number) ×100 The rest of the pregnant rats were kept until normal delivery to produce the adult offspring. The number of pregnant rats in each group was assigned to 8 (the litter size comprised 12 to 14 at birth, and the female/male ratio was approximately 1:1). After weaning, all offspring were maintained on standard laboratory chow ad libitum. The offspring rats were weighed once every two weeks until postnatal week (PW) 12, and the bodyweight gain rates was calculated according to the following formula. All offspring were sacrificed with inhaled isoflurane at PW12 for serum as before (Fig. 1). Gain rate of bodyweight (%) = [(bodyweight at PWχ–bodyweight at PW1) /bodyweight at PW1] × 100 2.2. Serum sample analysis for liver and kidney function Serum alanine transaminase (ALT), serum aspartate transaminase (AST), serum glutathione S-transferase (GST), serum albumin (ALB), serum creatinine (Scr) and serum urea nitrogen (BUN) were detected using assay kits following the manufacturer's protocol (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) 2.3. Liquid chromatography/mass spectrometry (LC/MS) analysis

2.4. Multivariate data processing and statistical analyses Serum samples were thawed and 20 μL was added to a tube containing 180 μL 67% aqueous acetonitrile. The samples were vortexed for 30 s each and centrifuged at 18,000 × g for 20 min at 4 °C to remove proteins. One millilitre of supernatant was transferred to a clean tube and dried under nitrogen flow at room temperature. The residuals were resuspended using 200 μL 70% ACN/water and then centrifuged at

For data processing, analysis, and identification of metabolites for untargeted profiling analysis, an optimized workflow was carried out for comprehensive metabolite phenotyping of the two groups and discovering differential metabolites. It consisted of background noise subtraction, automated peak detection and integration, peak alignment,

Fig. 2. Fetal birth weight and serum index concentrations of liver and kidney function in adulthood after prenatal dexamethasone exposure. a: fetal bodyweight; b: intrauterine growth retardation (IUGR) rate; c: female bodyweight; d: gain rate of female bodyweight; e: male bodyweight; f: gain rate of male bodyweight; g: serum alanine transaminase (ALT); h: serum aspartate transaminase (AST); i: serum glutathione S-transferase (GST); j: serum albumin (ALB); k: serum creatinine (Scr); l: blood urea nitrogen (BUN). Mean ± S.E.M., n = 12, *P < 0.05, **P < 0.01 vs. control. 138

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A heatmap plot was also constructed from metabolites using MultiExperiment Viewer (http://www.tm4.org/mev). To capture the disturbed metabolic pathway (P < 0.05), MetaboAnalyst was used to create the significant pathways in KEGG pathway database (Xia and Wishart, 2010).

orthogonal partial least square-discriminate analysis (OPLS-DA) analyses, and univariate analysis, which was performed using Thermo Scientific label-free differential analysis bioinformatics software SIEVE 2.2 (Thermo Scientific, San Jose, CA) and SIMCA-P 13.0 (Umetrics, Kinnelon, NJ). Data acquired using positive and negative ionization were mean-centred, Pareto scaled and log-transformed before multivariate statistical analyses. Data obtained using positive ionization and negative were mean-centred and log-transformed before OPLS-DA analyses. Validation of the model was tested using sevenfold internal cross validation and permutation tests 200 times using R programming. To further evaluate the predictive ability of the OPLS-DA model, an external validation procedure was performed (Llorach et al., 2010; Brindle et al., 2002). The LCeMS metabolomics data set was split into a training set and a test set. Approximately 70% of the samples were randomly selected as the training set, and the remaining 30% were treated as the test set. OPLS-DA models were built based on the training set and then blindly predicted the classes of the samples in the test set.

3. Results 3.1. Prenatal and postnatal bodyweight, liver and kidney function changes in adulthood Firstly, pregnant rats were subcutaneously injected with 0.2 mg/kg/ d dexamethasone on GD9-20 and sacrificed by isoflurane anaesthesia to obtain foetal rats on GD20. Compared with the control group, the foetal bodyweights were significantly reduced (P < 0.01, Fig. 2A) and the IUGR rates were increased (P < 0.05, Fig. 2B) by PDE. To understand the effect of PDE on offspring growth and development after birth, we measured the offspring bodyweights between the control and PDE groups every two weeks in the postnatal life. The results showed that the bodyweights of female offspring in the PDE group were close to those of the control group before PW8 and then lower than those of the control (Fig. 2C); the bodyweights of male offspring in the PDE group were higher than those of the control in PW2-6, and then close to the control (Fig. 2E). The bodyweight gain rates of the PDE female and male offspring rats in PW2-12 were significantly higher than those of their respective controls (P < 0.01, Fig. 2D, F). These results suggested that the bodyweights of female and male offspring rats in the PDE group displayed postnatal catch-up growth. Further, to investigate the effects of PDE on liver and kidney function in adult offspring in PW12, we detected the serum indexes, such as ALT, AST, GST, ALB, Scr and BUN. The results indicated that the liver function indexes (serum ALT, AST and GST activities) of female and male offspring were significantly increased by PDE, and the serum ALB concentration was significantly decreased (P < 0.05, P < 0.01, Fig. 2G–J). Meanwhile, the kidney function indexes (serum Scr and BUN concentrations) were significantly

2.5. Statistical analysis and identification of discriminant metabolites SPSS 17.0 (SPSS Science, Inc., Chicago, Illinois) was used to analyse the data. Quantitative data were expressed as the means ± S.E.M. and statistical significance was tested with an independent samples t-test. For enumeration data, such as IUGR rates, the proportion of affected animals per litter was first calculated; subsequently, the IUGR rate was arcsine square-root transformed before t-test evaluations (Vidmar et al., 2011). Statistical significance was defined as P < 0.05. Discriminant metabolic features were identified based on their accurate masses and/or product ion spectra in negative and positive mode. Human Metabolome Database (HMDB) and Metlin were searched to assist metabolite identification. To identify significantly changed metabolites between groups (differentially changed metabolites), significance was set with a false discovery rate (FDR)-corrected q value of q < 0.05, at a significance level of P < 0.05; P(corr) > 0.8 was used as an arbitrary cutoff value to select the potential biomarkers.

Fig. 3. Based on Liquid Chromatography-Mass Spectrometry (LC–MS) metabonomic analysis in fetal serum samples of gestational day (GD) 20 for prenatal dexamethasone exposure (PDE). a–b, orthogonal partial least-squares discriminant analysis (OPLS-DA) score plots displaying discrimination between PDE from their respective control for negative ionization and positive acquisition in female offspring on GD20; c–d, OPLS-DA score plots displaying discrimination between PDE from their respective control for negative ionization and positive acquisition in male offspring on GD20. 139

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negative and positive ionization was 0.922 and 0.692, respectively, indicating a very good predictability of supervised models. In addition, the R2Y of supervised models based on negative ionization and positive ionization was 0.992 and 0.888, respectively, in male offspring (Supplementary Fig. 1C, D) and the Q2 was 0.868 and 0.51, respectively. No misclassification occurred during the cross validation.

increased (P < 0.05, P < 0.01, Fig. 2K–L). These results suggested that PDE induced the liver and kidney function injuries in female and male adult offspring rats. 3.2. Multivariate statistical analyses of serum samples in female and male offspring Two sets of data acquired by LCeMS negative ionization and positive ionization were subjected to multivariate analyses. Score plots of OPLS-DA (Fig. 3A, B) for either negative ionization or positive ionization acquisition demonstrated a clear separation of serum in the female control and PDE groups on GD20. A principal component and an orthogonal component were used to construct the OPLS-DA model. The R2Y of supervised models based on negative ionization and positive ionization was 0.916 and 0.834, respectively. Seven-fold internal cross validation was performed on the OPLS-DA model. Q2 obtained from the OPLS-DA model derived from negative and positive ionization mode was 0.977 and 0.968, respectively. In addition, the R2Y of supervised models based on negative ionization and positive ionization was 0.981 and 0.991, respectively, in male offspring (Fig. 3C, D) and the Q2 was 0.981 and 0.962, respectively. One or two serum samples were misclassified during cross validation. Misclassification is unlikely to occur if a supervised model has an excellent predictability with Q2 higher than 0.8. Similarly, OPLS-DA models were applied to investigate the metabolome differences for negative ionization and positive ionization in offspring PDE serum vs. control serum in PW12. OPLS-DA models were able to separate female offspring PDE serum vs. control serum at PW12 (Supplementary Fig. 1A, B). OPLS-DA models derived from both negative and positive ionization had a good fit to the data. The R2Y of these supervised models was 0.974 and 0.934, respectively. Q2 calculated from 7-fold cross-validation for OPLS-DA models derived from

3.3. Identification and classification of differential metabolites of serum samples in female and male offspring on GD20 By analysing the s-plot of OPLS-DA, we identified 28 differential metabolites (Supplementary Table 1) (｜p [1] < 0.05, ｜p (corr) [1] > 0.80) that distinguished PDE female rats from control rats on GD20. Compared with controls, PDE rats were characterized by higher levels of 3’-sialyllactose, pantothenic acid, uric acid, N-lactoyl-phenylalanine, hydroxyphenyllactic acid, theophylline, ergothioneine, 5-Lglutamyl-L-alanine, isobutyryl carnitine, 2-methylbutyroyl carnitine, hexanoyl carnitine, acetyl carnitine, cholic acid (CA), chenodeoxycholic acid (CDCA), and muricholic acid (MCA), as well as lower levels of phosphatidyl ethanolamine (PE), 7Z,10Z,13Z,16Z,19Z-docosapentaenoic acid, sphingosine-1-phosphate, enantio-platelet activating factor (enantio-PAF), lyso-PAF, cortexolone, oleamide, sphingosine-1-phosphate, linoelaidic acid, cholesta-4,6-dien-3-one, sphinganine, prolyl-hydroxyproline, asymmetric dimethylarginine, thymoquinone, CA, CDCA, and tauro-muricholic acid (TMCA). A heat map of these metabolites is presented in Fig. 4. Furthermore, we identified 24 differential metabolites (Supplementary Table 2) that distinguished male foetal rats from controls. Compared with controls, PDE rats were characterized by higher levels of 3’-sialyllactose, pantothenic acid, uric acid, N-lactoyl-phenylalanine, theophylline, ergothioneine, 5-l-glutamyl-l-alanine, isobutyryl carnitine, CA, CDCA, muricholic acid (MCA), 2-methylbutyroyl carnitine, hexanoyl carnitine

Fig. 4. The heat map of the differential metabolites in female fetal rat serum of prenatal dexamethasone exposure relate to female control. 140

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and acetyl carnitine, as well as lower levels of butanoyl-platelet activating factor, enantio-platelet activating factor, PE, phosphatidylcholine (PC), lysoPE, cortexolone, histidine, and L-monomethyl arginine (L-NMMA). A heat map of these metabolites is presented in Fig. 5. Then, we classified these differential metabolites. In females: ①glucose metabolism: 3'-sialyllactose acid and hydroxyphenyllactic acid were increased; ②amino acid metabolism: N-lactoyl-phenylalanine, 5-L-glutamyl-L-alanine, isobutyryl-carnitine, 2-methylbutyryl-carnitine, hexanoyl-carnitine and acetyl-carnitine were increased, while prolyl-hydroxyproline and asymmetric dimethylarginine were decreased; ③lipid metabolism: CA, CDCA, and MCA were increased, while docosapentaenoic acid, sphingosine-1-phosphate, cortexolone, PE, oleamide, and sphingosine were decreased; ④purine metabolism: uric acid was increased; ⑤others: pantothenic acid was increased, while enanti-PAF and lyso-PAF were decreased. In males: ①glucose metabolism: hydroxyphenyllactic acid was increased; ②amino acids metabolism: N2-acetyl-L-ornithine, 5-L-glutamyl-L-alanine, 2-methylbutyrylcarnitine, hexanoyl-carnitine, sphinganine, phytosphingosine, palmitoyl-L-carnitine and elaidic-carnitine were increased, while L-NMMA and histidine were decreased; ③lipid metabolism: CA, CDCA and TMCA were increased, while PC, PE, Lyso-PE and cortexolone were decreased; ④purine metabolism: uric acid was increased; ⑤others: butanoyl-PAF and enantio-PAF were decreased. To uncover the molecular basis of foetal serum metabolic profiling in IUGR offspring induced by PDE, we used MetaboAnalyst 3.0 to examine the metabolic pathways affected by these differential metabolites. Twelve metabolic pathways were significantly affected in the female offspring (Supplementary Table 5), and 4 differential metabolites were involved in these pathways, with 3 metabolites significantly changed only. Among them, the sphinganine signalling pathway and sphinganine metabolic signalling pathway were key pathways (Fig. 6). Meanwhile, 8 metabolic pathways in the male offspring were significantly affected (Supplementary Table 5), and 5

Fig. 6. The network of the metabolites and the Kyoto Encyclopedia of Genes and Genomes pathways for female fetal rats in gestational day 20.

Fig. 5. The heat map of the differential metabolites in male fetal rat serum of prenatal dexamethasone exposure relate to male control. 141

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(0.2 mg/kg·d) during the second and third trimesters of pregnancy to affirm the effect and gender differences of PDE on offspring serum metabolic profiles before and after birth. In combination with the dose conversion relationship between rats and humans (conversion coefficient is 6.16), prenatal treatment with dexamethasone in rats at 0.2 mg/ kg·d was comparable with that used in humans at 0.03 mg/kg·d. Due to the standard dose of dexamethasone used clinically is 0.05-0.2 mg/kg·d (Moisiadis and Matthews, 2014), the exposure dose of dexamethasone in this study can be achieved in clinical practice. It is known that the gestational period of humans is about 280 days (40 weeks), and the morphological development time of multiple organs accounts for 20% of the whole gestational period (the first 8 weeks of gestational period). However, for rats, the gestational period is about 22 days, and the morphological development time of multiple organs accounts for 80% of the whole gestational period, which extends from gestational day 1 to 18 (O’Rahilly, 1979; Pinkerton and Joad, 2000). Hence, it can be concluded that, compared with human beings, small animals like rats have a longer development period of organ morphology, which also accounts for a larger proportion of gestational period. Therefore, in this study, dexamethasone was administered to pregnant Wistar rats for a longer time than clinical application in humans, accounting for 50% of the pregnancy period, to investigate the development toxicities of multiple organs through analyzing the serum metabolic profiles of the offspring.

differential metabolites were involved in these pathways, with 3 metabolites significantly changed only. Among them, glycerol phospholipid metabolism, the endogenous cannabinoid retrograde signalling pathway and the sphingosine signalling pathway were key pathways (Fig. 7). 3.4. Identification and classification of differential metabolites of serum samples in female and male offspring on PW12 Similarly, 10 and 11 differential metabolites for female and male rats, respectively, were found at PW12 (Supplementary Table 3, 4). Compared with the controls, PDE females were characterized by higher levels of hyocholic acid (HCA), L-ascorbic acid, 4-hydroxybenzenesulfonic acid, methacholine, 2-ethylhexyl phthalate, N2acetyl-l-ornithine, tetracosahexaenoic acid, CDCA, and MCA, as well as lower levels of pentadecanal. PDE males were characterized by higher levels of 3β-hydroxy-12-oxo-5β-chol-9(11)-en-24-oic acid, 3-oxo-4,6choladienoic acid, indoxylsulfuric acid, citric acid, DCA, TUDCA, CA, and CDCA, as well as lower levels of ( ± )12- hydroxy-eicosatetraenoicacid (HETE), PAF, and PC. The heat maps of these metabolites are presented in Figs. 8–9. we further classified these differential metabolites. The functions of these differential metabolites were various, mainly reflecting the abnormalities of glucose metabolism, amino acid metabolism, and lipid metabolism. In females: ①amino acid metabolism: N2-Acetyl-L-ornithine was increased; ② lipid metabolism: CDCA, MCA, HCA, 2ethylhexyl phthalate and tetracosahexaenoic acid were increased; ③others: 4-hydroxybenzenesulfonic acid, L-ascorbic acid and methacholine were increased, while pentadecanal was decreased. In males: ①glucose metabolism: citric acid was increased; ②lipid metabolism: CA, CDCA, DCA and TUDCA were increased, while HETE and PC were decreased; ③others: PAF was decreased. Five metabolic pathways were significantly affected in female offspring (Supplementary Table 6), and 3 differential metabolites were involved in these pathways. Among them, the HIF1 signalling pathway and arginine biosynthesis signalling pathway were key pathways (Fig. 10). Fifteen metabolic pathways were significantly affected in male offspring (Supplementary Table 6), and 8 differential metabolites were involved in these pathways. Among them, glycerol phospholipid metabolism and the retrograde nerve signalling pathway were key pathways (Fig. 11). In summary, many differential metabolites in glucose metabolism, amino acid metabolism, lipid metabolism and purine metabolism were found in both female and male IUGR offspring induced by PDE, and a variety of differential metabolic pathways had also significantly changed, which presented with certain gender differences. Furthermore, we found that the number of differential metabolites of PDE offspring was significantly less in adulthood than in utero, the types of differential metabolites were not consistent with those in utero, and there were significant gender differences. The reason may be mainly related to the catch-up growth and multi-functional compensatory enhancement of IUGR offspring in the early postnatal period, which resulted in some metabolites being close to or exceeding the normal level.

4.2. Key differential metabolites and toxicological significances in the PDE offspring Hyperlactacidemia in utero: Dexamethasone can promote gluconeogenesis and glycogen synthesis and reduce the uptake and utilization of glucose in peripheral tissues, which increases serum glucose sources and reduces glucose consumption (Franko, et al. 2007). In the present study, we found that the levels of hydroxyphenyl lactic acid were

4. Discussion 4.1. Dosing and timing reasonability of dexamethasone in this study Dexamethasone is often used to perinatally treat preterm birth-related diseases. However, prenatal dexamethasone treatment will lead to foetal growth retardation (Murphy et al., 2008; Elfayomy and Almasry, 2014). Our previous study also found that PDE could induce mice IUGR in course-, dose-, and stage-dependent manners (Chen et al., 2018). In the rat model of IUGR caused by PDE, dexamethasone was proved to induce development toxicities of multiple organs, including bone, hippocampus and ovary (Zhang et al., 2016; Dong et al., 2018; Lv et al., 2018a,b). In this study, we treated pregnant rats with dexamethasone

Fig. 7. The network of the metabolites and the Kyoto Encyclopedia of Genes and Genomes pathways for male fetal rats in gestational day 20. 142

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Fig. 8. The heat map of the differential metabolites in female rat serum of prenatal dexamethasone exposure relate to female control in postnatal week 12.

Fig. 9. The heat map of the differential metabolites in male rat serum of prenatal dexamethasone exposure relate to male control in postnatal week 12.

phenylalanine and 5-L-glutamyl-L-alanine in females, N2-acetyl-L-ornithine and 5-L-glutamyl-L-alanine in males) and carnitines (isobutyrylcarnitine, 2-methylbutyryl-carnitine, hexanoyl-carnitine and acetylcarnitine in females, 2-methylbutyryl-carnitine, hexanoyl-carnitine, palmitoyl-L-carnitine and elaidic-carnitine in males) were increased in the foetal serum. Carnitines are required to transport fatty acids from cytoplasm to mitochondria and can accelerate lipid metabolism and prevent accumulation of fatty acids in cells (Marcovina et al., 2013; Liu et al., 2015). Therefore, these findings suggested that the elevated level of carnitine mediates the enhancement of lipid breakdown caused by PDE. Decreased cortexolone in utero: Long-term and high-dose dexamethasone treatment promotes lipolysis, fatty acid oxidation and cholesterol synthesis in adults (Wang, et al. 2013; Lv, et al. 2018). Here, we found that the serum lipid level was significantly reduced by PDE, which was manifested as the decreased level of docosapentaenoic acid, sphingosine-1-phosphate, cortexolone, PE, oleamide and sphingosine in female offspring and a decreased level of PC, PE, Lyso-PE and cortexolone in male offspring. These results suggested that PDE could promote the use of phospholipids and fatty acids in foetuses. Cortexolone is

significantly increased by PDE in female and male foetuses, which is contrary to the inhibitory effect of dexamethasone on glycolysis (reduced hydroxyphenyl lactic levels). Previous studies reported that chronic glucocorticoid exposure could increase vascular resistance between foetal and placental circulation, which leads to foetal hypoxia (Nugent et al., 2013; Cahill and Rennie, 2017). In population studies, the hydroxyphenyl lactic acid levels in IUGR foetal umbilical cord blood and intracerebral areas were also significantly elevated (Holzmann et al., 2012; Andescavage et al., 2015). Animal studies also found that the level of cerebral hydroxyphenyl lactic acid in IUGR newborn piglets significantly increased (Moxon-Lester et al., 2007). In the rat IUGR model induced by prenatal caffeine and nicotine exposure, we found that the levels of hydroxyphenyl lactic in the foetal serum were also increased (Feng et al., 2014; Liu et al., 2012). Therefore, we suspected that the hyperlactacidemia in male and female foetal offspring may attribute to the enhanced glycolysis result from the increased placental vascular resistance and foetal hypoxia induced by PDE. Increased carnitine in utero: Glucocorticoids promote multi-organ protein breakdown and inhibit protein synthesis at high concentrations (Sami, et al. 2015). In the present study, several amino acids (N-lactoyl143

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Fig. 10. The network of the metabolites and the Kyoto Encyclopedia of Genes and Genomes pathways for female rats in postnatal week 12.

the serum total bile acid concentration was increased after birth in low birthweight infants (Boehm et al., 1988, 1990). The bile acid synthesis rate was increased in infants once their mothers received dexamethasone treatment before delivery (Watkins et al., 1975). In vitro, dexamethasone increased bile acid synthesis (including CA and CDCA) by promoting the expression of CYP8B1, a key rate-limiting enzyme in bile acid synthesis (Mork et al., 2016). High levels of endogenous corticosterone induced by chronic stress also contributed to the increased bile acid secretion in mouse faeces (Silvennoinen et al., 2015). “Intrauterine programming” is defined as a stimulus or insult occurring during critical periods of foetal growth and development that can

an important intermediate for synthesizing steroid hormones (including corticosterone and aldosterone) with the help of a series of adrenal steroid synthetases (Payne and Hales, 2004). We found that the levels of foetal serum cortexolone were significantly decreased by PDE in female and male offspring, which was related to dexamethasone inhibiting adrenal steroidogenesis through negative feedback (Xu et al., 2011). Hyperbileacidemia in utero and adulthood: Bile acids play an important role in lipid metabolism, and their deposition could lead to the occurrence of diseases such as primary biliary cirrhosis and primary sclerosing cholangitis (Pauli-Magnus et al., 2004; Carey et al., 2015; Hirschfield et al., 2013). Clinical and experimental studies found that

Fig. 11. The network of the metabolites and the Kyoto Encyclopedia of Genes and Genomes pathways for male rats in postnatal week 12. 144

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increased protein breakdown and disordered lipid metabolism, and multiple metabolic pathways were changed, displaying certain gender differences. Furthermore, PDE offspring in adulthood showed fewer and inconsistent types of differential metabolites than those in utero, which exhibited significant gender differences. The main changes in differential metabolites induced by PDE included hyperlactacidemia, increased carnitine, decreased cortexolone, hyperbileacidemia, decreased phospholipid, hyperuricemia and changed PAF, which may participate in dexamethasone multi-organ toxicities and multi-disease susceptibility after birth. This study helps to recognize the short and long-term developmental toxicities caused by PDE and elucidate its mechanisms. Moreover, it helps to guide the safe use of drugs during pregnancy, and explore early warning and therapeutic targets for PDE-related foetaloriginated diseases.

permanently alter tissue structure and function (He et al., 2017). In the present study, PDE continuously increased the bile acid levels (mainly for CA and CDCA) in utero and PW12. We also observed that the indexes of acute liver injury (serum ALT, AST and GST) were significantly increased and that of chronic liver injury (serum ALB) in adulthood was decreased by PDE. Taken together, the deposition of bile acid caused by PDE could program from the intrauterine period until after birth, which may lead to the occurrence of liver injury in adulthood. Decreased phospholipid in utero and adulthood: Clinical and animal experiments have shown that overexposure to glucocorticoid during pregnancy will lead to a series of adverse effects to the foetuses, including IUGR, postnatal cognitive impairment, and mental illness (Xu et al., 2011; Zhang et al., 2016; Davis and Sandman, 2010; Isaksson et al., 2015). Phosphatidylcholine is a type of phospholipid molecule that makes up biofilms for neurons and glial cells. Previous studies suggested that the chronic deficiency of vitamin E leads to decreased intracerebral phosphatidylcholine levels and damage to cognitive function (McDougall et al. 2017). Researchers also found that the level of phosphatidylcholine was decreased in the brain tissue and plasma of patients with Alzheimer's disease (Nitsch et al., 1992; Whiley et al., 2014), and phosphatidylcholine supplementation slowed the aging process and improved brain memory function in patients with Alzheimer's disease (Hung et al., 2001). In the present study, the serum phosphatidylcholine levels in both male and female PDE offspring were decreased in utero, but increased to the control level in PDE females but persistently decreased in PDE males at PW12. Our recent study demonstrated that the functions of learning and memory were impaired by PDE in the male offspring rats, accompanying pathologic changes of hippocampal morphology (Dong et al., 2018). Therefore, we speculated that the low-function programming of phosphatidylcholine synthesis by PDE mediated postnatal cognitive dysfunction in the male offspring. Hyperuricemia in utero: We also observed that serum uric acid levels were increased in female and male PDE offspring in utero, and the serum indexes of kidney function (Scr and BUN) were increased in adulthood. Uric acid is a decomposition product of purine nucleotides and is mainly excreted by the kidneys. The serum uric acid is filtered by glomeruli; most is reabsorbed and a small part is excreted (FathallahShaykh and Cramer, 2014). Previous studies reported that the level of uric acid was increased in the cord blood of IUGR foetuses (Mena Nannig et al., 2016). PDE also can lead to foetal kidney damage in goat offspring (Moritz et al., 2011) and changes of kidney gene expression profiles in rats after birth (Sheen et al., 2015). These suggested that the increased level of foetal serum uric acid may be an indicator for kidney developmental toxicity in utero and impaired renal reabsorption in adulthood caused by PDE. Changed PAF in utero and adulthood: PAF is a phospholipid activator involved in the physiological functions of many leukocytes, platelet aggregation and degranulation, inflammation and allergic reactions (Prescott et al., 2000; McIntyre et al., 2009). Studies suggested that the catch-up growth in the IUGR offspring mediated the occurrence of adult allergic diseases (Tedner et al., 2012). Our study found that the foetal serum PAF levels in male and female offspring were reduced by PDE in utero but were increase to the control level in female offspring at PW12. We speculated that the serum PAF level in female offspring after birth was mainly related to catch-up growth. The detailed relationship between PDE-related PAF level and the susceptibility to allergy disease after birth needs further investigation.

Declaration of Competing Interest All of the authors state that they have no conflicts of interest. Acknowledgments This work was supported by grants from the National Natural Science Foundation of China (No.81220108026, 81430089, 81673524,81522047), the National Key Research and Development Program of China (2017YFC1001300), and Hubei Province Health and Family Planning Scientific Research Project (No. WJ2017C0003). Authors’ contributions: Guanghui Chen, Hao Xiao, Jinzhi Zhang, Huizhen Zhang, and Bin Li performed the research; Hui Wang, Huichang Bi, Guanghui Chen, and Hao Xiao designed the research study; Guanghui Chen, Hao Xiao, Tao Jiang, Yajie Wen, Yimin Jiang, Kaili Fu, Dan Xu, Yu Guo, and Ying Ao analysed the data; Guanghui Chen, Hao Xiao, Hui Wang, and Huichang Bi wrote and revised the paper; all authors approved the final manuscript. References Andescavage, N., Limperopoulos, C., Evangelou, I., Murnick, J., du Plessis, A., 2015. Pregnancy outcomes in two growth restricted fetuses with in utero cerebral lactate. J. Neonatal. Med. 8, 269–273. Boehm, G., Senger, H., Braun, W., Beyreiss, K., Raiha, N.C., 1988. Metabolic differences between AGA- and SGA-infants of very low birthweight. I. Relationship to intrauterine growth retardation. Acta Paediatr. Scand. 77, 19–23. Boehm, G., Muller, D.M., Teichmann, B., Krumbiegel, P., 1990. Influence of intrauterine growth retardation on parameters of liver function in low birth weight infants. Eur. J. Pediatr. 149, 396–398. Brindle, J.T., Antti, H., Holmes, E., Tranter, G., Nicholson, J.K., Bethell, H.W., et al., 2002. Rapid and noninvasive diagnosis of the presence and severity of coronary heart disease using 1H-NMR-based metabonomics. Nat. Med. 8, 1439–1444. Cahill, L.S., Rennie, M.Y., 2017. Feto- and utero-placental vascular adaptations to chronic maternal hypoxia in the mouse. J. Physiol. 2017, 1–13. Carey, E.J., Ali, A.H., Lindor, K.D., 2015. Primary biliary cirrhosis. Lancet 386, 1565–1575. Chen, Z., Zhao, X., Li, Y., Zhang, R., Nie, Z., Cheng, X., et al., 2018. Course-, dose-, and stage-dependent toxic effects of prenatal dexamethasone exposure on long bone development in fetal mice. Toxicol. Appl. Pharmacol. 351, 12–20. Crowther, C.A., McKinlay, C.J., Middleton, P., Harding, J.E., 2015. Repeat doses of prenatal corticosteroids for women at risk of preterm birth for improving neonatal health outcomes. Cochrane Database Syst. Rev. 5, Cd003935. Davis, E.P., Sandman, C.A., 2010. The timing of prenatal exposure to maternal cortisol and psychosocial stress is associated with human infant cognitive development. Child Dev. 81, 131–148. Ding, H., Mohan, C., 2016. Connective tissue diseases: promises and challenges of metabolomics in SLE. Nat. Rev. Rheumatol. 12, 627–628. Dong, W., Xu, D., Hu, Z., He, X., Guo, Z., Jiao, Z., et al., 2018. Low-functional programming of the CREB/BDNF/TrkB pathway mediates cognitive impairment in male offspring after prenatal dexamethasone exposure. Toxicol. Lett. 283, 1–12. Elfayomy, A.K., Almasry, S.M., 2014. Effects of a single course versus repeated courses of antenatal corticosteroids on fetal growth, placental morphometry and the differential regulation of vascular endothelial growth factor. J. Obstet. Gynaecol. Res. 40, 2135–2145. Fathallah-Shaykh, S.A., Cramer, M.T., 2014. Uric acid and the kidney. Pediatr. Nephrol. 29, 999–1008. Feng, J.H., Yan, Y.E., Liang, G., Liu, Y.S., Li, X.J., Zhang, B.J., et al., 2014. Maternal and fetal metabonomic alterations in prenatal nicotine exposure-induced rat intrauterine growth retardation. Mol. Cell. Endocrinol. 394, 59–69.

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