LiNA study design and sample collection

The German prospective birth cohort LINA (Lifestyle and environmental factors and their Influence on Newborns Allergy risk) recruited 622 mothers (629 children) at 34 weeks of gestation between May 2006 and December 2008 in Leipzig, Germany. Mothers with severe immune or infectious diseases during pregnancy were excluded from the study. Standardised self-administered questionnaires were collected annually starting in pregnancy, assessing general information about personal lifestyle, housing and environmental conditions and disease state. Study participation was voluntary and written informed consent was obtained by all participants. The study was approved by the Ethics Committee of the University of Leipzig (file ref # 046-2006, 160-2008, 160b/2008, 144-10-31052010, 113-11-18042011, 206-12-02072012, #169/13-ff, #150/14-ff). The LINA study is registered in birthcohorts.net.

Analysis of urinary paraben concentrations in human samples

Urinary concentrations of eight paraben species were determined in 504 maternal urine samples of the 34th week of gestation (reduced sample size due to available urine samples). The samples were prepared and analysed as described and validated by Schlittenbauer et al.39. In brief, urine samples, quality controls, and solvent blanks were thawed at room temperature, vortexed, and centrifuged. Aliquots were spiked with internal standard solution and a deconjugation standard. Hydrolysis was achieved by adding enzyme buffer solution and incubation in an ultrasonic bath. The enzyme was removed by centrifugation/filtration using Amicon® Ultra-0.5 filters. LC-MS analysis was performed on a UPLC™ system (ACQUITY I-Class, Waters Cooperation Milford, MA, USA) coupled to a triple quadrupole mass spectrometer (Xevo TQ-S, Waters Cooperation, Manchester, UK) equipped with an electrospray ionisation (ESI) source. For each analyte (MeP, EtP, iPrP, nPrP, sBuP, iBuP, nBuP, BzP), a quantification (Q) and a confirmation (q) MRM transition was selected. Quality criteria for positive confirmation of peaks were (i) presence of both MRM transitions, (ii) a retention time within 0.03 min, and (iii) a relative ion ratio within 50 to 150% compared to a standard. The limits of quantification (LOQ) were 0.5 µg l−1 for MeP and 0.1 µg l−1 for the others parabens (EtP, iPrP, nPrP, sBuP, iBuP, nBuP, BzP). For 498 participants valid paraben concentrations above the detection limit were obtained (cases used for weight analyses are shown in Supplementary Table 2). For iPrP, sBuP and BzP more than 70% of samples had concentrations below the LOQ and were therefore excluded from further analysis. For paraben analysis of mouse samples 20 µl urine and 180 µl milliQ water was mixed and the same protocol as described above was applied.

Assessment of cosmetic product application

The usage of cosmetic products during pregnancy was assessed within the recruitment period via questionnaires at the 34th week of gestation. Participants could name up to 6 cosmetic products that were used on a daily basis. These cosmetics were categorised in leave-on products (crèmes, body lotion, make-up or leave-on facial cleansings) and rinse-off products (toothpaste, rinse-off facial cleansings, hairstyling, perfume and others). The content of parabens in the named cosmetic products was assessed between April and July 2016 via the TOXFOX app for iOS from the Friends of the earth Germany (Bund für Umwelt und Naturschutz Deutschland), which provides information about the content of endocrine disrupting chemicals like parabens in an online data base of cosmetic products. Insufficiently indicated cosmetic products that could not be assigned specifically to a particular brand/product were excluded from further analysis.

Anthropometric data collection

Children’s body weight and height were assessed annually during clinical visits or were obtained from regular preventive medical check-ups asked for in the LINA questionnaires. At birth and the one-year follow-up children’s length was measured horizontally; afterwards standing height was measured to the nearest 0.1 cm. Body weight was measured to the nearest 0.1 kg. To adjust for child’s age and sex, BMI was classified according to the Extended International (IOTF) Body Mass Index Cut-Offs for Thinness, Overweight and Obesity in Children40 on a detailed monthly basis for children from age 2–8 years. Children with BMI equivalent to an adult BMI ≥ 25 (for example: 36-month-old girl: 17.64 kg m-², 36-month-old boy 17.85 kg m-²) were classified as overweight (OW), children with BMI equivalent to an adult BMI < 25 as non-overweight (NOW). Further, we have classified weight at birth as ≥4000 g (or macrosomia; considering gestational age as a confounding factor in our model analyses) or as birth weight <4000 g (n = 496). Longitudinal BMI information was available for 396 children from year 1–8. Classification in NOW and OW was performed in early to mid-childhood (year 2–8; n = 223). Due to missing reference data the first year could not be included in this analysis. This should not be of relevance for the paraben effect since Körner et al.41 described a longitudinal overweight stabilising effect only after the age of 3 years. We were able to verify the OW classification based on a Bioelectrical Impedance Analysis (BIA) performed at children’s age of eight. Children classified as OW had about 30% higher body fat mass percentage at 8 years of age compared to normal weight children (fat mass overweight = 19.3%, n = 58; fat mass normal weight = 14.9%, n = 138, P < 0.001).

In vitro adipocyte differentiation

Human adipose-derived mesenchymal stem cells (MSC) derived by a female donor (ATCC, LGC Standards (Wesel, Germany), PCS-500-011; LOT #59753760) and appropriate culture media were purchased from ATCC (LGC Standards,Wesel, Germany, PCS-500-030, PCS-500-040 and PCS-500-050). Cells were cultured under normal conditions at 37 °C, 5% CO 2 and 95% humidity according to the manufacturer’s instructions9. In brief, for adipocyte differentiation MSCs passage 3–5 were seeded at 9.000 cells per cm2 in a 96-well plate and grown to 70% confluence. For initiation of differentiation cells were fed with Adipocyte Differentiation Initiation Medium (ADIM; ATCC Adipocyte Differentiation Toolkit PCS-500-050). ADIM was changed to Adipocyte Maintenance Medium (ADMM; ATCC Adipocyte Differentiation Toolkit PCS-500-050) after 4 days and changed regularly. Cells were treated with parabens at different concentrations (working solution in 0.05% ethanol) during the entire differentiation period. Butyl paraben exposure was represented in the experimental setup by nBuP due to its higher human exposure (see Supplementary Table 1) compared to iBuP. Proliferation and differentiation were monitored in real-time by the impedance-based xCELLigence MP System (ACEA Biosciences Inc., San Diego, USA) on a microelectrode 96 well E-View-Plate (ACEA Biosciences Inc.). A cell index was assessed every 10 min by normalising an electrical impedance measurement to a bland value for each well. Measurements were paused for media changes. After a total of 16 days, cells were stained with Oil Red O for triglyceride depots and harvested for qPCR analysis. Oil Red O staining was quantified via absorbance measurement at 510 nm. qPCR was carried out in a Roche Light Cycler 480 system with primer sequences being listed in the Supplementary Table 4. Human adiponectin and leptin protein concentrations were assessed in cell culture supernatants of human MSC derived adipocytes by commercially available ELISA kits (Adiponectin ELISA Kit, Human (KHP0041); Leptin ELISA Kit, Human (KAC2281) from Invitrogen/Thermo Fischer, Ulm, Germany) according to the manufacturer’s instructions. Undiluted samples were measured in triplicates, using a 1× secondary antibody and compared to 8 standards, ranging from 0−1000 pg ml−1 for leptin and 0–23 ng ml−1 for adiponectin. Cytotoxicity of exposed chemicals was assessed prior to and after adipocyte differentiation via a MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyoltetrazoliumbromid) assay.

To follow our translational research design, a validation of the human adipose-derived MSC experiment was performed using adipocyte derived murine MSCs (Cyagen, MUBMD-01001, Strain C57BL/6 Mouse Adipose-Derived Mesenchymal Stem Cells) according to manufacturer’s instructions. In brief, for adipogenic differentiation mMSC were passaged onto 12-well plates at a concentration of 20,000 cells per cm2 and were grown to at least 100% confluence in Basal Media (Cyagen, MUXMX-90011). After that the cells underwent adipogenic differention according to manufacturer’s instructions (using Cyagen GUXMX-90031) following a maximum of 5 cycles of 3 day induction and 1 day maintenance period. With each cycle of induction, cells were exposed to Ethanol control (0.05%), 0.5 µM, 1 µM, or 10 µM nBuP. Rosiglitazone provided by Cyagen was only used as positive control. After the cells formed big and round lipid droplets they were harvested and processed for further analyses (see human MSCs for Oil Red O staining and qPCR analyses).

Reporter gene assay

Butyl paraben was tested in five reporter gene cell lines42 based on the human nuclear receptors peroxisome proliferator activated receptor PPARγ (CellSensor PPARγ-UAS-BLA293-H, Thermo Fisher Scientific), androgen receptor AR (CellSensor AR UAS BLA GRIPTITE), oestrogen receptor ER (CellSensor ER a UAS BLA GRIPTITE), progesterone receptor PR (CellSensor PR-UAS-BLA HEK293T), and glucocorticoid receptor GR (CellSensor GR-UAS-BLA HEK293T). The experiments were performed as described previously43,44 using in parallel reference compounds for quality control and dosing the parabens in methanol assuring that the final methanol was <0.1%. 4000 cells (PPARγ), 3500 cells (ER), or 4500 cells (AR, PR, GR) were seeded per well in 384-well plates with 30 µl of medium and incubated for 24 h at 37 °C and 5% CO 2 . The methanolic stock solutions of the paraben were diluted with assay medium and a 10-step serial dilution-series was prepared in a 96-well dosing plate and 10 µl of each dilution were added to the 384-well cell plates. The dosed cells were incubated another 22 h at 37 °C and 5% CO 2 . Activation of nuclear receptors PPAR, AR, ER, PR, GR was detected after adding 8 µl of FRET detection including ToxBlazer mixture per well followed by 2 h incubation at room temperature. Blue (activated) and green signals (inactive) were detected using an excitation filter of 409 nm and emission filters of 460 nm and 530 nm, respectively. Induction of the transcription factor or, more precisely, expression of the reporter gene β-lactamase was measured by FRET fluorescence. For the cytotoxicity, an excitation filter of 600 nm and an emission filter of 665 nm were used. The activation of ARE was detected by quantification of the luciferase reporter product using luciferin/ATP reagent as described previously43,45. Concentration-response curves were plotted for cytotoxicity and activation of the reporters. The inhibitory concentration for 10% of cytotoxicity IC 10 was deduced from a log-sigmoidal fit of the concentration-cytotoxicity curve43. Only concentrations below IC 10 were further processed for activation. Linear concentration-response curves up to 30% effect level or an induction ratio of 4 for the AREc32 assay were plotted and effect concentration triggering 10% of the maximum effect of a positive control, EC10, or causing an induction ratio IR of 1.5, EC IR1.5 were derived46.

Mice

BALB/cByJ mice (6–8 weeks of age) were obtained from the Elevage Janvier Laboratory (Le Genest St Isle, France). Mice were bred and maintained in the animal facility at the University of Leipzig (Germany) under conventional conditions with 23 °C room temperature, 60% humidity, and 12 h day/night rhythm. Control and nBuP-exposed dams and pups were housed in polyphenylsulfone (PPS) cages and bedded with LIGNOCEL® bedding material. All mice received phytoestrogen-free diet (C1000 from Altromin, Lage, Germany) and water ad libitum. Experiments included groups of 4-6 mice per cage and were performed at least two times according to institutional and state guidelines. Animal protocols used in this study were approved by the Committee on Animal Welfare of Saxony/Leipzig (Permit Number: TVV01/15).

Exposure to nBuP and Aza treatment

To investigate the impact of an intrauterine exposure on weight development in the offspring we exposed pregnant mice to nBuP via subcutaneous (s.c.) injection of 1.75 µg nBuP (Sigma-Aldrich Chemie GmbH, Munich, Germany) in 100 µl corn oil twice per week until weaning when pups were 4 weeks old (perinatal exposure). Control dams received only the vehicle. To investigate a possible involvement of epigenetic alterations offspring were treated i.p. with 160 µg kg−1 body weight Aza (Sigma-Aldrich) solved in PBS 3 times per week starting 1 week after birth until weaning26. Each exposure protocol was performed at least two times from at least 3 dams (each with 2–5 pups with not more than 3 pups per sex).

Assessment of weight and metabolic parameters

Body weight of the pups was measured twice a week and a mean weight per week was calculated for each mouse. At the end of the observation period (12 weeks) whole body composition (fat mass and lean mass) was determined in awake mice based on nuclear magnetic resonance technology using an EchoMRI700™ instrument (Echo Medical Systems, Houston, TX, USA) in the offspring of control and nBuP exposed dams. Food intake was monitored after weaning until week 11. For determination of the adipocyte area visceral adipose tissue was removed and stored into in 4% paraformaldehyde for more than 24 h to complete fixation. Afterwards the tissues were dehydrated, embedded in paraffin and sectioned into 3 μm sections. After a deparaffinization, tissue sections were stained with hematoxylin and eosin (H&E) for the adipocyte area assessment. Insulin tolerance test (ITT) was performed in the offspring 9 weeks after birth. Insulin (0.75 U kg−1 body weight) was injected intraperitoneally. For glucose measurements blood of tail vein was taken at four time points at 0, 15, 30 and 60 min after insulin injection. For the glucose tolerance test (GTT) glucose (2 g kg−1 body weight) was injected i.p. and the measurement was performed equally. Adiponectin, Leptin, Resistin, Insulin, and acetylated Ghrelin serum concentrations were determined by ELISA using mouse standards according to the manufacturer’s guidelines (Mouse Adiponectin, Leptin, Resistin ELISA; R&D Systems/bio-techne, Minneapolis, USA, Mouse/Rat/Human 17-beta Estradiol ELISA; Abcam, Cambrigde, UK, Mouse/Rat Insulin ELISA; bertinpharma, Montigny-le-Bretonneux, France and Mouse/Rat acylated Ghrelin ELISA; BioVendor, Karasek, Czech Republic).

RNA extraction, cDNA synthesis and qPCR

Dissection of the hypothalamus was conducted from the ventral side of the brain. The optic chiasm was removed away from the anterior portion of the hypothalamus. The mammillary nuclei were dissected from the posterior of the hypothalamus. The entire hypothalamus was prepared including the arcuate, ventromedial, dorsomedial, and paraventricular nuclei. Total RNA was extracted from adipocytes of humans, visceral adipose tissue and hypothalamus of mice by using QIAzol Lysis Reagent (QIAGEN, Hilden, Germany) and RNAeasy Plus Mini Kit (QIAGEN) following manufactures instructions. Two hundred nanogram were used for cDNA synthesis by RevertAid™ H Minus Reverse Transcriptase (Thermo Fisher Scientific, MA, USA). qPCR was performed with BIOTAQ™ DNA polymerase (Bioline GmbH, Luckenwalde, Germany) and SYBRgreen I nucleic acid gel stain (Thermo Fisher Scientific) on a LightCycler 480 (Roche Applied Sciences, Penzberg, Deutschland) with the following cycling programme: 10 min at 95 °C, followed by 45 cycles of 95 °C for 10 s, 20 s at 68 °C and 72 °C for 20 s. All reactions were performed in duplicates. Primers were designed exon spanning. Primer sequences of target genes are listed in Supplementary Table 6. Expression values of target genes were evaluated using 2-ΔΔCT method with U6 small nucleolar RNA (u6), beta-actin (actb), the beta-glucuronidase (gusb, GUSB) and phosphoglycerate kinase 1 (PGK1) as reference genes and normalised to the lowest measured value.

Pyrosequencing

For DNA methylation analysis of POMC promoter and enhancers genomic DNA (gDNA) of the hypothalamus was isolated using the DNeasy Blood and Tissue Kit (QIAGEN). Two hundred nanogram gDNA was bisulfite converted using the EZ DNA Methylation™ Kit (ZymoResearch, CA, USA). The pyrosequencing assay for DNA-methylation analysis of enhancers was designed on the forward strand and on the reverse complement-strand for POMC promoter using the PyroMark Assay Design version 2.0.1.15 software (Qiagen, POMC enhancer nPE1: forward primer 5’-GTGGGTAAGTTTGAGTTTTGAATG-3’, reverse primer 5´-biotin-ACCCTTCCTCAAAAATACAAAATTC-3’, sequencing primer 5’-AGTTTGAGTTTTGAATGT-3’, amplicon coordinates: chr12: 3942231-3942372; POMC enhancer nPE2: forward primer 5’-TTTTTTTGTTTTGTGGGGTATGTAGT-3’, reverse primer 5´-biotin-AAAAACCCTAATAAAAAACCCCTTAA-3’, sequencing primer: 5’-AATATATGTATTAGTGGATGAAA-3’, amplicon coordinates: chr12: 3944766-3944876; POMC promoter: forward primer 5’-GTTGGGTGGGTGAGTTTT-3’, reverse primer 5´-biotin-CACCATTCTTAATTAAATTCTTCCTAACC-3’, sequencing primer: 5’-GTGGGTGAGTTTTGGA-3’, amplicon coordinates: chr12:3954708-3954911). The genomic position of the amplicons for DNA-methylation of the POMC promoter and enhancers nPE1/nPE2 were determined based on Rubinstein et al.33, Drouin et al.47, and Langlais et al.48. (see Supplementary Fig. 7).

Bisulfite treated gDNA was amplified with the HotStar Taq DNA Polymerase Kit (QIAGEN) with the following cycling programme: 15 min at 95 °C, followed by 45 cycles of 94 °C for 30 s, 30 s at 56 °C and 72 °C for 50 s, and a final elongation for 10 min at 72 °C. DNA methylation was assessed by pyrosequencing on a PyroMark Q48 (QIAGEN) following manufacture’s instruction.

Statistical analysis

LINA study data were evaluated by STATISTICA for Windows, Version 12 (Statsoft Inc., USA) and STATA version 15.1 (StataCorp LLC, USA). A GEE model with exchangeable correlation matrix and cubic splines with knot points at 12, 25, 38.2, 61 and 98,2 months was applied to test for longitudinal differences in BMI between low (1st tertile, reference), medium (2nd tertile) and high (3rd tertile) paraben exposure groups. Logistic regression models were performed to analyse the impact of prenatal paraben exposure on infants’ risk for macrosomia at birth and overweight development afterwards (year 2–8). Both models were adjusted for the sex of the child, smoking during pregnancy, parental school education, gestational week at delivery, existence of siblings, breast feeding duration (not for models with only birth weight as outcome) and age of the mother at birth. All confounders were chosen according to their potential weight association after a literature review. To test the equal distribution of parameters in the analysed sub-cohort and the entire LINA cohort the chi squared test was performed. Mann–Whitney-U-test was used for the comparison of median paraben levels of mothers that used paraben containing vs. paraben free cosmetic products. Experimental data sets from in vivo and in vitro studies were processed and analysed in GraphPad PRISM 7.02 for windows (GraphPad Software, Inc.) and R version 3.5.1. All p-values are derived by GEE models with exchangeable correlation matrix for longitudinal mouse data, ANOVA or unpaired t-test.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.