SELMA is a pregnancy cohort following +2,000 mother child pairs from early pregnancy over birth and up in school age with focus on prenatal exposure

Transkript

1 SELMAisapregnancycohortfollowing+2,000mother childpairsfromearlypregnancyoverbirthandupin schoolagewithfocusonprenatalexposureforchemicals withendocrinepropertiesandtheimportanceforhealth anddevelopmentinchildren CarlGustafBornehag,MariaUnengeHallerbäck,SverreWikström,LauravonKobyletzki, EewaNånberg,BirgittaSundström,MariaMarinpoulou,MalinKnutz 4/20/2015

2 1 SCIENTIFICQUESTION...4 ENDOCRINEDISRUPTINGCHEMICALSAREAMAJORGLOBALCONCERN...4 SELMAANDEDCMIXRISK...5 GENERALQUESTIONSRELATEDTOENDOCRINEDISRUPTERS...6 EDCSINFOCUSINTHESELMASTUDY...8 Eightchemicalsclasses(51EDCs)underinvestigation...9 HEALTHANDDEVELOPMENTOUTCOMESINSELMA...12 Sexualdevelopment...12 Neurodevelopment...14 Metabolismandgrowth...16 Asthmaandallergy...17 Serumvaccineantibodyconcentration...18 MODEOFACTION...18 Inflammation...19 Epigeneticandgeneticmarkers...20 DESCRIPTIONOFTHESELMASTUDY...23 SELMAI...23 AimwithSELMAI...23 Establishmentofthecohort...24 Datacollectionandsamplingprocedures...24 Biologicalsampling...24 Interviewsofthepregnantwomen...25 Environmentalsampling...25 Selfadministeredquestionnaires...26 Routinelyperformedexaminationsofthechild...27 AvailabledatafromSELMAI...27 PrenatalEDCexposure...27 Thepregnantwomen shealth...29 Storedairanddustsamples...29 Lifestyleandhealthforthefamily...29 Datafromroutinelymadeexaminationofthechild...30 DatabaseforSELMAI...30 SELMAII...30 AimswithSELMAII...30 InvitationofthechildandthefamilytoSELMAII...34 DatacollectioninSELMAII...35 Biosamplestobecollectedfromthechild...35 Examinationofhealthanddevelopmentofthechild...36 Semistructuredinterviewwiththecustodians...40 Questionnairetothefamily...40 Collectionofdatafromregistersandmedicalrecords...41 Chemicalanalysesofbiomarkersinbiosamples...41 BIOSTATISTICALMODELLINGOFDATA...42 Constructionofcompositescoresforeachhealthdomain...42 Statisticalpower...43 Statisticalanalysesofcodeddata...44 ETHICALAPPROVALSFORTHESELMASTUDY...44 PUBLISHEDANDPRELIMINARYRESULTSINSELMA...45 SCIENTIFICVALUEOFTHESELMASTUDY...46

3 ETHICALCONSIDERATIONS...49 RECRUITMENTPROCEDURESINSELMAII...49 COLLECTION,STORAGEANDPROTECTIONOFSENSITIVEDATA...50 HANDLINGOFGENETICINFORMATION...52 PERFORMANCEOFBIOSAMPLING...52 BIOBANKINGOFSAMPLES...53 RISKSFORPARTICIPATINGCHILDRENANDFAMILIES...53 HANDLINGOFIDENTIFIEDDISEASES/DIAGNOSES...54 THEFAMILIESRIGHTTOOBTAINDATAFROMTHESTUDY...55 TRANSFEROFBIOSAMPLESANDDATAINEDCMIXRISKCONSORTIUM...55 PUBLICATIONSANDREPORTS...56 REFERENCES...57 APPENDIXA...72 Studyprotocolforhealthexaminationofchildren...72 APPENDIXB...86 Invitationletter...86 APPENDIXC...92 Consentletters...92 APPENDIXD Samplingprocedures APPENDIXE Biomarkerstobemeasured APPENDIXF Examinationstobeconductedregardingsexualdevelopment APPENDIXG Examinationstobeconductedregardingneurodevelopment APPENDIXH Examinationstobeconductedregardingmetabolismandgrowth APPENDIXI Examinationstobeconductedregardingasthmaandallergy APPENDIXJ Semistructuredinterviewofparents APPENDIXK Postalquestionnairestothefamily APPENDIXL Modeofactionmeasurements APPENDIXM Codingofcollectedsamplesandinformation APPENDIXN Handlingofadversehealthoutcomes

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5 SCIENTIFICQUESTION Human and wildlife health depends on the ability to reproduce and develop normally. This is not possible without a healthy endocrine system. This is the first of 14 key concerns expressed by UNEP/WHO (Bergman et al., 2013). Today we know that the endocrine system is of greatest importance for a healthy development - from the time of conception until death - for both animals and humans. It is therefore of global concern that the entire human population, foetuses, infants, children and adults, are constantly exposed to low levels of anthropogenic chemicals, some of which are endocrine disrupting chemicals (EDCs) that may interact with our natural endocrine functions with adverse health effects as result. Endocrinedisruptingchemicalsareamajorglobalconcern Based on present knowledge, it is evident that there are an uncountable number of common consumer products and materials that contain actual or potential EDCs among which many migrate out of the materials to the surrounding environment, over its entire lifespan. This chemical leakage is leading to environmental discharges, contamination and human uptake and exposure. In fact, everyone is exposed, as supported by global bio-monitoring data showing that EDCs and/or their metabolites are routinely detected in human fluids such as urine, blood, breast milk, and recently in amniotic fluid showing that they pass placenta and exposing the fetus at vulnerable developmental stages. Exposure to EDCs during windows of susceptibility during fetal development, even at low doses and in complex mixtures, is of particular concern for developmental programming and trans-generational effects on the proteome, transcriptome and epigenome. These changes underlie disorders that may manifest in adult life and contribute to a multitude of chronic diseases. A large body of epidemiological studies have also reported that early life exposure to EDCs is associated with health effects later on in life such as sexual development and reproduction problems, neurodevelopmental disorders and behaviour, metabolic malfunctions incl. overweight/obesity, as well as asthma and allergy, as described in a recent overview from WHO/UNEP (Bergman et al., 2013). However, even if the scientific knowledge regarding health risks related to EDC exposure have been growing during the last decade there are a number of critical questions that have to be answered. New information here is of the greatest importance not at least for better risk assessment of EDCs and protection of vulnerable parts of the population, and there is an intensive work within the EU how to regulate EDCs with requests of more research and a number of recent call for scientific research proposals are focusing on this questions. 4

6 SELMAandEDCMixRisk With the overall aim to investigate the importance of prenatal exposure for chemicals with endocrine disrupting properties for children s health, the SELMA study started in 2007 in Sweden. The study is investigating prenatal and postnatal exposure for EDCs both as single compounds and as mixtures for health outcomes and development in four different domains in children including sexual development, neurodevelopment and behavior, metabolism and growth, and asthma and allergy, and to examining mode-ofactions including inflammation and epigenetic mechanisms. SELMA is currently following +2,000 mother child pairs from 1 st trimester over birth and up in school age. EDC-MixRisk is a study recently awarded by Horizon 2020 with a 6.2 million Euro grant where SELMA is included. The long reaching goal of EDC-MixRisk is to move forward and meet the societal need of improved decision making regarding human exposure risks to mixtures of anthropogenic chemicals over the whole life span. Hence the project will determine and assess the risk for multiple adverse health outcomes based on molecular mechanisms involved, after early life exposure to complex mixtures of endocrine disrupting chemicals (EDCs 1 ). To enable us to address such a complex research task, the project relies on interaction between advanced expertise in epidemiology, toxicology and risk assessment, i.e., toxicological sciences. The value of this combined research effort is: Identification of mixtures of EDCs that are associated with multiple adverse health outcomes in three health domains (sexual development, metabolism and growth, and neurodevelopment) in two epidemiological pregnancy cohort studies (SELMA and Life Child study) by use of novel and advanced bio-statistical methods. Identification of molecular mechanisms and pathways underlying the associations between exposure to EDC mixtures and adverse health outcomes by the use of experimental animal and cell models. Development of a transparent, consistent and systematic (TCS) framework in risk assessment - sometimes referred to as a weight of evidence approach 2 - for integrating epidemiological and experimental research to facilitate risk assessment for EDCs and mixtures. 1 An endocrine disruptor is an exogenous substance or mixture that alters function(s) of the endocrine system and consequently causes adverse health effects in an intact organism, or its progeny or (sub) populations (IPCS 2002). 2 Weight of Evidence (WoW) is commonly used term which lately has been questioned due to ambiguity, e.g., by National Academy of Sciences (USA) and commonly among scientists. The term is therefore not used herein and TCS is applied instead. 5

7 Generalquestionsrelatedtoendocrinedisrupters The global concern about exposure for EDCs during critical developmental periods of life (Bergman et al., 2013) includes a number of issues. Low dose EDC exposure during fetal development and related health effects Multiple EDC exposure in mixtures and related health effects Co-morbidity between different health domains related to EDC exposure Different health effect in males and females (i.e., sexual dimorphism) Epigenetic programming during development as a possible biological mechanism Low dose EDC exposure during fetal development and related health effects can be effectively investigated in epidemiological studies. However, there is a need of studies including EDC exposure from the sensible early pregnancy period during critical biological development (e.g., regarding brain differentiation and sexual development) and follow up data of the children later on. Such data are rare, and when available there are often problems with (1) small sample sizes; (2) differences in assessment instruments; (3) variations in child age and sex at testing; (4) large variation in socio-demographics. In SELMA we can meet these shortcomings. The study is further totally integrated within the Swedish health care system which is unique in the world with great possibilities to follow the families during the pre- and postnatal period with access to good health record systems. Multiple EDC exposure in mixtures and related health effects has recently been in focus in several calls for proposals in EU (Horizon 2020) and US NIH, etc. Humans are exposed to environmental chemical mixtures which is also evident from SELMA data where all 2,356 pregnant women had all 20 investigated EDCs in their urine or serum above levels of detection. From preliminary analyses we also know that several of these compounds are strongly inter correlated which is complicating the traditional biostatistical analyses. We have started collaboration with Icahn School of Medicine at Mount Sinai (MSSM), NY, USA (prof. Chris Gennings). MSSM have a world leading team developing novel biostatistical methods for handling this urgent problem, a collaboration that is also a part in the EDC-MixRisk project. The essential question of mixtures is in focus in the SELMA study which with its design, size and number of EDC investigated is a very important resource with great possibility for providing new valid information. Co-morbidity between different health outcomes that are related to EDC exposure is an important issue very seldom covered in epidemiological studies. Chemicals with endocrine properties interacting with e.g., estrogen and androgen receptors as in the case of phthalates, PFASs and BPA are of importance for sexual development but also for fetal brain development. It is therefore of interest to study these two health outcomes and others jointly since there might be a common pathway. Several other potential comorbidity have 6

8 been expressed in the literature (Bergman et al., 2013). In SELMA we are studying four different health domains (see below) and the relationship to prenatal EDC exposure which is unique. Preliminary data described below are also indicating that prenatal exposure for e.g., phthalates may be related to sexual development (a shorter anogenital distance in baby boys) as well as neurodevelopmental symptoms and respiratory diseases in children. This is an indication that there might be a common biological mechanism behind, e.g., inflammation. Different effect in males and females (sexual dimorphism) is one of the hottest topics discussed at recent scientific meetings. For example, androgen action during the fetal masculinization-programming window determines the maximum potential for growth of androgen-dependent organs. Such androgen actions are potentially related to sexual development (and fertility later on in life) but as described above it is also known that the fetal brain development is tightly regulated by the maternal endocrine system under influence of both estrogen and testosterone. If there are effects related to the steroid hormone system (e.g., down regulation of testosterone) one may expect different effects in males and females, i.e., sexual dimorphic effects. Supporting such a hypothesis is the observations in numerous epidemiological studies that several adverse health effects show different prevalence s in boys and girls. Studies have shown that effects in the reproduction system are mainly found in baby boys, such as malformation of genitals and shorter anogenital distance (Bornehag et al., 2015, Swan et al., 2005, Swan et al., 2015); neurodevelopmental disorders such as autism and ADHD are more than four times more commonly diagnosed in boys than in girls (Miodovnik, 2011); respiratory diseases including asthma are more common in boys at young ages while a shift have been reported in puberty when the incidence is increasing in girls (Bornehag and Nanberg, 2010); while regarding metabolic disorders including overweight and obesity the picture is more unclear. In SELMA we have a unique possibility to analyse the sexual dimorphic hypothesis in all four health outcome domains and we are collaborating with MSSM, NY, and prof. Shanna Swan which is world leading expert in this area. Our preliminary data below also support this hypothesis of sexual dimorphic outcomes. Epigenetic programming during development as a possible biological mechanism is one promising mechanisms for explaining how EDC exposure during critical developmental periods may result in diseases and disorders later on. Emerging evidence suggests that exposure to environmental factors during development can induce changes that - like genetic mutations - are persistent during a lifetime and can even be transferred to subsequent generations (so-called epigenetic changes) without sequence changes of the genome. The exposure for EDCs started as early as in the thirties but the major increase happened in the fifties and onward. Thus, children born in the eighties and later belong to the first generation born to parents that were exposed during embryonic development. The germ cells that give rise to the next generation are formed during embryonic 7

9 development. Therefore, on top of adverse effects during their own development, children born in the eighties might have inherited unfavorable changes from their parents germ cells. We are hereby collaborating with Swetox and groups at Karolinska Institutet in order to integrate epidemiological studies with experimental investigations in animal and cell models, to obtain founded knowledge about the impact of exposure to endocrine disrupting chemicals (EDCs) on neurodevelopmental disorders in children. The idea is to in a first step, by the use of epidemiological data in SELMA, identify EDCs that are associated with impaired cognitive, motor, behavioural and language development in children. In a second step, the effects of these compounds will be tested on cell differentiation and epigenetic patterning in vitro and on neurodevelopment and behaviour in mice in order to verify epidemiological findings, and to identify epigenetic markers correlating with exposure. Finally, in a third step, these epigenetic markers will be measured in children with high and low prenatal phthalate exposure levels in SELMA. EDCsinfocusintheSELMAstudy Within SELMA we are examining both non-persistent organic compounds and persistent organic pollutants (POPs), all with documented EDC properties, known as additives in materials of common consumer products, known to occur in the general environment and detected in humans globally. All these compounds or metabolites of them can be analysed in human fluids, and in this case we have used urine and serum from the pregnant women and the child. Non-persistent organic compounds The selected non-persistent organic compounds represent pollutants that are stable enough to migrate out of materials unchanged and distribute in the environment but as soon as taken up by an organism their inherent short biological half-life confirm their non-persistent characteristics. These are chemicals that are taken up by ingestion of contaminated food/water and dust, via inhalation of dust and dermal uptake. All these chemicals and/or their metabolites are routinely found in human fluids such as urine, serum, breast milk and amniotic fluids (pregnant women) confirming general exposure globally. Several of these compounds are sometimes referred to as pseudo-persistent chemicals due to the fact that humans may establish steady-state concentrations internally due continuous exposure. Persistent organic pollutants The persistent organic pollutants (POPs) are defined in the Stockholm convention, i.e. are only slowly transformed abiotically, widely distributed in the environment, lipophilic (e.g. PCBs, DDTs) or proteinophilic (e.g. PFOS) and toxic POPs are biomagnified in the food webs. They are in general accurately measured even in very small quantities of maternal serum, mothers milk and/or other fat rich matrices when lipophilic. The POPs we are focusing on are known as EDCs (Bergman et al., 2013). 8

10 Eightchemicalsclasses(51EDCs)underinvestigation The overreaching goal of this project is to estimate associations between prenatal and postnatal exposure to 51 EDCs and health outcomes in children. Our target EDCs are 21 non-persistent compounds representing four classes of pollutants: Phtalic acid esters ( phthalates ), alkyl phenols, polycyclic aromatic hydrocarbons (PAHs), and pesticides, and 30 persistent organic compounds (POPs) of four classes: proteinophilic per- and polyfluorinated alkyl substances (PFAS), lipophilic, polychlorinated biphenyls (PCBs), polybrominated diphenyl ethers (PBDEs), and organochlorine pesticides. Several of these compounds have already been analyzed in prenatal urine or serum in SELMA phase I (Table 3) and some of them will be measured by funding from EDC- MixRisk (Table 2). The relevance of the selected classes of pollutants is addressed below. Phthalic acid esters (phthalates) have for long been used as plasticizers in e.g. polyvinyl chloride (PVC). Phthalates are found everywhere and often in high concentrations. Since phthalates are used as additives they are commonly found in consumer products including food, building materials, plastics, cosmetics, cleaning products, packages, toys, etc. (Bornehag et al., 2005a, Dodson et al., 2012, Rudel et al., 2010). As a result they are routinely found in indoor air and dust (Bergh et al., 2011, Rudel and Perovich, 2009, Bornehag et al., 2005a) as well as in food and water. Phthalates are ubiquitous in the environment and humans are exposed via ingestion, dermal uptake and inhalation. It has recently been shown that e.g. exposure to di-2-ethylhexyl phthalate (DEHP) primarily occur via food and for children also via mouthing of plastics, while exposure to diethyl phthalate (DEP) and benzyl butyl phthalate (BBzP) primarily occur via dermal and inhalation pathways (Carlstedt et al., 2013, Wittassek et al., 2011, Koch et al., 2013). Robust biomonitoring data show phthalate metabolites in human fluids such as urine (both in children and adults), blood and breast milk (Wittassek et al., 2011, Langer et al., 2014, Wan et al., 2013, Frederiksen et al., 2010, Fromme et al., 2011). These compounds have also been found in amniotic fluid, suggesting that they cross the placental barrier and convey foetal exposure (Jensen et al., 2012). Thirteen phthalates are today on the ECHA candidate list ( In the SELMA study we could identify phthalate metabolites in the urine of all investigated 2,356 pregnant women (Table 3). Many of the phthalates are individually known as EDCs. Alkyl phenols and non-halogenated phenols are used in plastics as additives (alkyl phenols such as nonyl and octyl phenols) or precursors (e.g. bisphenols). Bisphenol A (BPA) is among the most thoroughly researched EDCs. It is an estrogenic compound while far less is known about closely related bisphenols (e.g. bisphenol S and F). Epoxy resins containing BPA are used as coatings on the inside of food cans and for relineing of water pipes in buildings/apartment houses. BPA has recently been replaced with e.g., bisphenol F in many consumer goods. Some low chlorinated phenols are used as 9

11 bactericides in e.g. food contact materials. The most well-known compound is triclosan which is an antibacterial agent found in consumer products, including soaps, toothpaste detergents, toys and surgical cleaning treatments. Its efficacy as an antimicrobial agent and the risk of inducing bacterial resistance remains controversial. Additional research seeks to understand its potential effects on organisms and environmental health. BPA and triclosan are routinely found in human urine both for infants, children and adults and it has been identified in amniotic fluid (Rochester, 2013). In SELMA (Table 3) we could identify BPA and triclosan in urine above LOD for (100%) all 2,356 pregnant women (Shu et al., submitted). Polycyclic aromatic hydrocarbons (PAHs) are the oldest known environmental contaminants researched with a history back to the 18 th century. PAHs are part of crude oil, certain petrochemical refined products, formed at insufficient efficient combustions from waste incineration, gasoline and diesel engines, tobacco smoking and cooking. A very large number of PAHs exists among which a number are also rated as carcinogens (IARC 2013; 2010). PAHs are non-persistent compounds with short half-lives in humans and wildlife. Due to their ubiquitous formation they are also distributed randomly. PAHs are sometimes referred to as pseudopersistent chemicals. The exposure to a set of PAHs, as determined via PAH metabolite assessment determine significant PAH human exposure in a NHANES related study (Clark et al., 2012). Benzo(a)pyrene, representing the PAH group of chemicals is associated with endocrine related diseases and disorders such as endocrine related cancer, adrenal disorders and immune functions (UNEP/WHO, 2013). Pesticides are used in agricultural activities but also in households meaning that the general population may be exposed. Biomonitoring data is also showing that such compounds can be found in human fluids (such as urine). Several of these compounds are known to have endocrine disrupting properties from both animal and human models. Per- and polyfluorinated alkyl substances (PFAS) are man-made, extremely stable as such or as their primary transformation products. PFASs consist of a very large number of structurally related chemicals, the sulfonic acids/salts and carboxylic acids being most well researched and both types of chemicals being identified as EDCs (Bergman et al., 2013). PFASs are widely spread throughout the environment due to their use in e.g. nonstick pans, carpets, furniture, household cleaners, shampoos, shoes, clothing, and convenience food contact materials. The use of PFASs in fire-fighting foams have severely contaminated drinking water wells in many areas and is the cause of heavy exposure to perfluorooctane sulfonate (PFOS), in particular. PFASs are routinely found in human serum globally. In Sweden, 6 PFASs (out of 8) were found above the limit of detection in all (100%) 2,356 women in the SELMA study as described in (Table 3) (Boman Lindström et al., in manuscript). PFASs have been shown to cross the placenta, 10

12 and the fetus s blood-brain-barrier meaning that the developing fetus is exposed in utero (Lau et al., 2006). Polychlorinated biphenyls (PCBs) are well-known POPs with a long history but banned and removed from the market already in the first part of the 1970 s, in many countries. They are still a major group of pollutants globally and a source for mixture exposure. PCBs form two types of highly relevant metabolite classes. Hydroxylated PCBs being related to thyroidogenicity and PCB methyl sulfones being related to glucocorticioid activities in the adrenals (Bergman et al., 2013). The different PCB congeners have a distinct toxicological activity profile. Still today, regardless of the production and application bans set in the 1970 s, important sources of human exposure are fish and breast milk. Polybrominated diphenyl ethers (PBDEs) are banned flame-retardants that still exists in e.g. not yet recycled furniture and wall-to-wall carpets. The European Food Safety Authority (EFSA) has recently reviewed PBDEs in food (EFSA, 2011). PBDEs and their metabolites associate with endocrine related effects in the thyroid system in particular. Hydroxylated PBDEs have a significant structural similarity to thyroxin (both T4 and T3). In a recent study of Mexican-American children living in an agricultural community (CHAMACOS), the most frequently detected PBDE congeners in children s serum (PBDE-47, -99, -100, and -153) were measured in >99% of the children (Eskenazi et al., 2011). Braun et al (2014) found several PBDEs in % of 175 pregnant US women in the Health Outcomes and Measures of the Environment study, in which samples were collected during the period (Braun et al., 2014). PBDEs can cross the placenta, and animal models of their exposures have shown long-lasting changes in spontaneous motor activity as well as learning and memory problems. Organochloride pesticides (OCs) are the historically important and well-studied synthetic pesticides including the infamous DDT, its metabolites and hexachlorobenzene (HCB), but also the less well-known POPs, trans-nonachlor and pentachlorobenzene (PeCB). In the past, PeCB was a component of the chlorobenzene mixture that was used to reduce the viscosity of PCB products. HCB is an organochlorine chemical that has been used in agriculture and industry but is today mainly formed as a byproduct of numerous industrial processes. Trans-nonachlor is an OC that was used in the U.S. until 1983 as an insecticide for crops like corn and citrus, and on lawns and domestic gardens. Although they have been banned across much of the world and concentrations have been decreasing over the past 30 years, these compounds are still detectable in the blood of current generations. For example, Braun et al., found HCB in serum in 94% of 175 pregnant US women in samples collected between 2003 and 2006 in USA (Braun et al., 2014). 11

13 HealthanddevelopmentoutcomesinSELMA Most of our target chemicals and their by-products (metabolites) are of health concern because of their potential toxicity to humans and wildlife as well as their potential impact on and interaction with the human endocrine system. In the following and in Table 1 we briefly summarize the epidemiological literature on sexual development, neurodevelopment, growth and metabolism, and asthma and allergy related to these exposures. Sexualdevelopment Concern of prenatal EDC exposure on reproductive health arose from the early dramatic observation of vaginal carcinoma in the adult female offspring of mothers exposed to diethylstilbestrol (DES) during pregnancy (Herbst et al., 1971). Since then a large body of accumulating data indicate that foetal and early life exposure to EDCs may contribute to adverse reproductive health outcomes in humans. Among males those include cryptorchidism, hypospadias, testicular cancer, and especially the reports on decreasing sperm counts have intensified the debate about the potential effects of EDCs on reproductive performance in humans (Sharpe and Skakkebaek, 1993, Jensen et al., 1995). Recent reviews have also concluded that female reproductive performance may be on decline, and threatened by EDCs (Crain et al., 2008, Diamanti-Kandarakis et al., 2009, Bergman et al., 2013). Female conditions that have been linked to ECD exposure include premature ovarian failure, infertility, intrauterine growth restriction, and small birth weight. Androgens and the androgen receptor play a pivotal role in the development of male external genitalia. Phenotypic features of an abnormal development of reproductive organs, which are associated with insufficient androgen action during the sensitive masculinization window in male foetuses, are hypospadias, micropenis and shortened anogenital distance (AGD). Multiple evidence points out those environmental factors can 12

14 contribute significantly to the occurrence of under-masculinization. Exposure of male foetuses in utero to different environmental chemicals such as vinclozolin, PCBs, phthalates and dioxins has been shown to induce hypospadias in boys of the resident families (Dolk, 1998). Thus, EDCs can contribute to the pathogenesis of hypospadias by disturbing androgen-dependent signalling pathways associated with proliferation and differentiation of human foetal penile cells (hfpcs) and/or by suppressing androgen production by human foetal Leydig cells (hflcs) causing under-masculinization of the male external genitalia. Animal studies have confirmed a critical time period during early gestation termed the male programming window (MPW) where androgen deficiency results in outcomes described above. AGD is reduced in infants with cryptorchidism (Jain and Singal, 2013, Thankamony et al., 2014) and especially in those with hypospadias (Thankamony et al., 2014) and it appears to provide a reliable guide to foetal androgen exposure (Dean and Sharpe, 2013). Reports of shorter AGD in male infants related to prenatal exposure to EDCs such as phthalates (Suzuki et al., 2012, Swan et al., 2005), BPA (Miao et al., 2011), and dioxins (Vafeiadi et al., 2013) support the utility of AGD measurement as a biomarker of endocrine disruption in humans but larger studies are needed. In the SELMA study we have found a significant association between prenatal exposure for DiNP-metabolites (and a non-significant association for DEHP-metabolites) and a shorter AGD in 196 baby boys at the age of 20 months (Bornehag et al., 2015). Finger ratio or digit length ratio has also been explored as an indicator of foetal androgen exposure (Dean and Sharpe, 2013). Among females early menarche has been associated with a greater risk of several major chronic diseases (Dossus et al., 2012) and may have a connection with early growth as in girls most studies document a relationship between intra-uterine growth retardation and earlier pubertal development or normal timing but with rapid progression (Hernandez and Mericq, 2008). In particular with respect to puberty onset, exposure to EDCs showing pro-oestrogenic, anti-androgenic activity or interaction with hypothalamic-pituitarygonadal (HPG) axis is considered biologically plausible and deserving attention (Maranghi and Mantovani, 2012). In girls PCBs, DDE, DDT, pesticides and phthalates have been associated with earlier menarche, pubarche, thelarche and central precocious puberty, and PCDD/Fs are associated with delayed breast development (Toppari and Juul, 2010). A Danish study showed a 1.5-year advancement in age at breast development in the female offspring of women who had been occupationally exposed to approximately 200 pesticides before and at the beginning of pregnancy (Wohlfahrt-Veje et al., 2012). It is likely that EDCs can also directly affect the reproductive capacity by regulating follicle health and maturation, since the ovary expresses both ER, a target of many phytoestrogens, and AHR, a target of many PAHs. Maternal smoking (source of many AHR ligands) during pregnancy is associated with a reduced adult fecundity in daughters. It was shown that smoking increases the exposure of the female foetus to PAHs, increases foetal circulating oestrogens and dysregulates ovarian development and 13

15 paracrine/endocrine signalling (Fowler et al., 2014). Activation of the AHR by a component of cigarette smoke was shown to reduce germ cell proliferation in the human foetal ovary (Anderson et al., 2014). Evidence suggests that these effects may be mediated through induction of AHR pathways and AHR-ER crosstalk (Fowler et al., 2014). In rodents, activation of AHR by benzo(a)pyrene directly triggers apoptosis in ovarian follicles, and simultaneous treatment with AHR antagonists protects the follicles from such damage (Matikainen et al., 2001, Neal et al., 2010, Mattison and Thorgeirsson, 1979). Neurodevelopment Prenatal exposure to several of our target compounds or classes of chemicals, including pharmaceuticals, has been examined in relation to neurotoxicity and neurodevelopmental outcomes. Epidemiologic evidence for a relationship between exposure to PFASs and neurotoxic outcomes is limited and contradictory. A recent review by Roth et al (2014) identified 12 relevant epidemiologic studies published after Different neurodevelopmental and neurobehavioural outcomes were identified, with mixed results (Roth and Wilks, 2014). However, those studies were limited by the potential for uncontrolled confounding and small sample size. To date, no study has focused on the contribution of PFASs to autism. Several epidemiologic studies have reported associations between prenatal exposure to PCBs and poorer cognitive functioning during infancy and childhood, including deficits in IQ, motor activity, verbal abilities, attention, and memory (Jacobson et al., 1985, Jacobson and Jacobson, 1997). However, negative results have also been reported in multiple cohorts (Schantz et al., 2003, Orenstein et al., 2014). A recent review of environmental pollutants and risk of autism spectrum disorders (ASD) found that to date only a few case series and small case-control studies have examined biomarkers of PCBs in relation to ASD risk (Rossignol et al., 2014). In those studies, exposure was determined postnatally or after diagnosis. PBDEs can cross the placenta, and animal models of their exposures have shown longlasting changes in spontaneous motor activity as well as learning and memory problems. Direct effects on neurotransmitters relevant for brain development have also been reported (Costa and Giordano, 2007). In humans, maternal prenatal and childhood PBDE exposures have been associated with poorer cognition, including adverse impact on IQ, verbal abilities, attention, and motor coordination. Adverse effects were evident even after low-level exposure (Herbstman et al., 2010, Hoffman et al., 2012). Only one study examined PBDEs in serum from cases of autism/asd and controls (total N=100) (Hertz- Picciotto et al., 2011). They did not find a statistically significant association; however, possibly explained by serum levels at the time of diagnosis do not reflect exposure during the critical developmental window. 14

16 Behavioural impairments after OC exposure, as well as interference with GABAergic neurotransmission have been described in animal models, but little information is available for humans. Data from two Spanish birth cohorts (recruited in 1997 and 1999) showed that an elevated HCB level (>1.5 ng/ml) in cord blood was significantly associated with a 4-fold increase of having a poor social competence score and a 2.7-fold increase of having ADHD symptoms in 4-year-old children (n=475). No association was found with cognitive or psychomotor performance (Ribas-Fito et al., 2007). In another Spanish pregnancy cohort of 1,391 mother-child pairs, prenatal exposure to HCB was not associated with cognitive impairment (Forns et al., 2012). Braun et al (2014) measured 25 PCBs, 6 OCs, 8 PBDEs, and 4 PFASs in blood or urine samples from 175 pregnant women in the Health Outcomes and Measures of the Environment Study (Cincinnati, OH) (Braun et al., 2014). When children were 4-5 years old, mothers completed the Social Responsiveness Scale, a measure of autistic behaviours. As serum concentrations of PBDE-28 and trans-nonachlor increased, so did the level of autistic behaviour. In contrast, less autistic behaviour were observed among children born to women with a nondetectable level of PCB-178 or PBDE-85 and with higher PFAS (PFOA) concentration. A number of epidemiologic studies have evaluated associations between prenatal phthalate exposures and child mental, motor and behavioural development (Kim et al., 2011, Kobrosly et al., 2014, Swan et al., 2010, Whyatt et al., 2012). Several crosssectional studies and experimental animal studies provide limited support for the longitudinal birth cohort study results. Sex-specific effects are often seen, with males more affected than females, but masculinization of female behaviour has also been reported. Such sexually dimorphic effects are likely given the potential mechanisms of action. Taken together, these results suggest that prenatal/early postnatal exposure to phthalates may have substantial adverse effects on child neurodevelopment. Preliminary results in the current SELMA study show that prenatal phthalate exposure was associated to language delay at 2.5 years of age (Unenge-Hallerbäck et al., in manuscript). Prenatal BPA exposures and child behaviour have been examined in three longitudinal birth cohort studies. In one study (n=244), prenatal urinary BPA concentrations were associated with externalizing behaviours among girls (hyperactivity and aggression) at age 2 years (Braun et al., 2009), and anxiety, hyperactivity, depression, emotional control and inhibition at age 3 years (Braun et al., 2011). In the second study (n=198), prenatal urinary BPA concentrations were significantly associated with increases in emotional reactivity and aggressive behaviours in boys and less anxious/depressed and aggressive behaviours in girls (Perera et al., 2012). In a more recent study (n=292), prenatal BPA was associated with internalizing behaviours (including anxiety and depression) among boys based on maternal report, and with aggression based on teacher report (Harley et al., 2013). In addition, a recent study that compared effects of dental amalgams in 534 children ages 6-10 at baseline found that the children with higher cumulative exposure to 15

17 BPA-type amalgams evidenced worse self-reported psychosocial functioning at the 5 year follow-up. Numerous experimental bioassays have evaluated the impact of prenatal BPA exposure on postnatal behaviour with significant effects seen on social behaviour, learning and anxiety, albeit with some inconsistencies (reviewed in (Wolstenholme et al., 2011)). Metabolismandgrowth The widely accepted Barker s hypothesis states that low birth weight across the whole range of birth weights in relation to the length of gestation is later in life associated with increased rates of coronary heart disease and the related disorders, such as insulin resistance, type 2 diabetes mellitus, arterial hypertension, dyslipidemia, overweight, obesity, metabolic syndrome and stroke. Slow growth during infancy and rapid weight gain after the age of two years (or in early life/thereafter) exacerbate the effect of slow fetal growth (Barker, 2006, Saggese et al., 2013). Low-level exposure to PCBs or correlated exposures (such as dioxins) impairs fetal growth (Govarts et al., 2012, Lopez-Espinosa et al., 2011). It has been proposed that these findings may be attributable to confounding by maternal weight gain during pregnancy (Verner et al., 2013), but a recent study adjusting for maternal weight gain still found this association (Vafeiadi et al., 2014). There is also some limited evidence for a relationship between prenatal exposure for PFASs and low birth weight, and preliminary data from the current SELMA study is showing significant negative associations between prenatal exposure for 5 PFASs and low birth weight when adjusted for maternal weight, socioeconomic status and smoking (Wikström et al. manuscript) and that these effects seems to be much stronger for girls which may indicate sexual dimorphism. Low birth weight may be a consequence of an undernourished intra-uterine environment, eg. through disturbances in lipid metabolism. It has been proposed that prenatal exposure to certain environmental chemicals ( obesogens ) during this sensitive developmental window may permanently alter the metabolic set point to promote an energy sparing state including altered adipocyte differentiation and lipid storage (Grün et al 2009, Janesick et al. 2011). Those exposed are then put at a higher risk of overweight and obesity later in life, in line with the Barker hypothesis. For humans tobacco smoke is an established prenatal obesogen (Cupul-Uicab et al., 2012, Ino, 2010, Oken et al., 2008). Animal experiments and epidemiological studies have listed other suspected obesogens such as BPA, OCs, PCBs, phthalates and PFASs (Holtcamp, 2012, Legler et al., 2011, Newbold, 2010). For example, a number of studies have found an association between prenatal organohalogen exposure and increased BMI in infancy (Mendez et al., 2011), childhood (Smink et al., 2008, Verhulst et al., 2009) and adulthood (Halldorsson et al., 2012, Karmaus et al., 2009). In line with the risk profile of Barker s hypothesis, prenatal exposure to a mixture of modern pesticides have been 16

18 associated with smaller birth weight and weight for gestational age, but larger increase in BMI Z-score from birth to school age (Yanagisawa et al., 2008, Wohlfahrt-Veje et al., 2011). In general however, previous findings of epidemiological as well as animal studies on single chemicals are inconsistent although indicating sexual dimorphism and nonlinear dose-response relationships (Hatch et al. 2010, Merril et al. 2011). Prenatal exposure to several EDCs has also been associated with biochemical metabolic markers in offspring and in cross sectional studies childhood levels of exposure have been correlated with various biochemical endpoints. These include insulin resistance measures (eg., PFAS, BPA, phtahlates; Rochester 2013, Smerieri et al., 2015, Timmermann et al., 2014), thyroid hormones (eg. BPA, DDE, PFAS; de Cock et al. 2014, Romano et al., 2015, Geens et al, 2015), triglycerides, leptin and adiponectin (eg., PFAS, BPA, phthalates; Halldorsson et al. 2012, Timmermann et al. 2014). Animal investigations of metabolic biomarkers in relation to the same EDCs also indicate alterations of glucose tolerance, fatty acid metabolism, cholesterol synthesis, inflammation and adipokine levels (Lau et al. 2007). Asthmaandallergy Asthma and atopic dermatitis (AD) are the most common chronic disease among children; their cumulative prevalence reaches up to 40% (WHO, 2013) with a heavy burden on the health care system. More than half of asthma and AD are atopic diseases and IgE mediated. Allergic diseases are associated with environmental factors like smoking and ambient air pollutions related to traffic. However, recent studies have identified chemicals related to building materials and many consumer products as risk factors for asthma and AD, chemicals with suspected endocrine disrupting properties. We have earlier reported that PVC flooring material in the home release phthalates (DEHP and BBzP) to indoor dust particles (Bornehag et al., 2005b, Kolarik et al., 2008), and that infants ingest these phthalates (Carlstedt et al., 2013). PVC flooring material has been related to airways symptoms in children in 7 cross sectional studies described in a recent review (Bornehag and Nanberg, 2010) and in two prospective studies (Larsson et al., 2009, Shu et al., 2013). BBzP and DEHP in dust from the bed room has been associated with airway symptoms in cross sectional data (Bornehag et al., 2004c, Callesen et al., 2011, Hsu et al., 2012, Kolarik et al., 2008, Bornehag et al., 2004b). Cross sectional studies have also reported associations between urinary levels of phthalate metabolites and airways symptoms (Hoppin et al., 2013, Callesen et al., 2011, Bekö et al., 2013). Of specific interest is a cross sectional Norwegian study reporting an association between DiNP-metabolites and asthma (Bertelsen et al., 2013). The most interesting study is a longitudinal cohort conducted by our partner at Columbia University which reports an association between prenatal BBzP exposure and infant eczema (Just et al., 17

19 2012) and a follow up at childrens` age 5-11 years showed an association between prenatal exposure to BBzP and DnBP with asthma (Whyatt et al., 2014b). In summary, epidemiological studies and findings in animal and cell models indicate that exposure to phthalates early in life increase asthma and allergy (Bornehag and Nanberg, 2010). But the epidemiological studies suffer from weaknesses regarding study design (most cross-sectional), most have not measured prenatal phthalate exposure, low representativeness and small numbers which limits possibilities to model data. Allergic or non-allergic asthma Limited evidence is available for the prenatal exposure to phthalates and IgEsensitization relationship. Two studies, the Manhattan cohort mentioned above and data from Taiwan (Wang et al., 2013) as well as Swedish data (Bornehag et al., 2004b) couldn t find any relationship to sensitization. But the study sizes were quite small with limited potential regarding stratification which doesn t allow drawing final conclusions. Preliminary data in SELMA is showing an association between prenatal exposure for phthalates mainly BBzP- and DEHP-metabolites and airway symptoms in infant children (Shu et al., in manuscript) which is in line with recent results from the Manhattan cohort (Whyatt et al., 2014c). Epidemiological data and preliminary data from the SELMA study suggest that exposure to phthalates early in life can be related to airways diseases in children. Serumvaccineantibodyconcentration Perfluorinated compounds have emerged as important food contaminants. They cause immune suppression in a rodent model at serum concentrations similar to those occurring in the US population, but adverse health effects of PFAS exposure are poorly understood. Recent data from Faroe Islands reported a significant association between both prenatal and postnatal exposure for PFASs and down regulation of antibody levels for tetanus and diphtheria in 587 children aged 5-7 years (Grandjean et al., 2012) where part of the children showed antibody levels below recommended values. A small cross sectional study from Norway found that PFAS exposure was associated to immunological responses in children. Modeofaction Defined by their capacity to interact with and disrupt normal endogenous hormonal signaling, EDCs have been shown to interact with a large number of nuclear hormone receptors and thereby having effects on a large variety of cellular functions both at the level of gene regulation and cell signaling mechanisms. For example, selected phthalates promotes signaling via nuclear PPAR-, the AhR, have pro-estrogenic and antiandrogenic affects and interacts with ion-channels and other cell signaling molecules, making the entangling of EDC target mechanisms linked to specific outcomes in humans 18

20 a big challenge. Within the SELMA study and EDC-MixRisk, several research groups are involved in projects using cell and animal model systems to identify mode-of-action mechanisms that may explain how early prenatal EDC exposures (individual and mixtures) can contribute to health outcomes in children. Within the SELMA II study we focus on two basic mode-of-action mechanisms, inflammatory responses and epigenetic regulation. Inflammation Several of the described domains and conditions for which epidemiological studies have identified a positive association between prenatal and early life exposure to EDCs, see above, have a common component of inflammation. However, the important question if early life EDC exposure attenuates normal functionality of the immune system including inflammatory mechanisms directly or if altered inflammatory responses are secondary to other initial pathophysiological mechanisms leading to cell and tissue damage is largely unknown. One hypothesis is that prenatal exposure to certain EDCs, such as phthalates, primes the innate immune system towards higher inflammatory reactivity visible later on in life. This involves a higher sensitivity and a dysregulated response to normal proinflammatory stimuli such as microbes activating Toll-like receptors (TLRs) and oxidative stress-related cell damage. The dysregulated response makes the individual more susceptible to natural environmental factors with increased risk for development of inflammatory disease as a health outcome. Dysregulated inflammatory responses and defense mechanisms counteracting oxidative stress-induced damage will increase the risk of inflammation-dependent pathological conditions but also the risk to develop chronic sub-clinical inflammation and thereby an increased susceptibility to other risk factors like genotype, diet and environmental risk factors. In asthma, oxidative stress induced by e.g. neutrophil activation (a defense mechanism also hitting normal cells and tissues) plays a strong role driving inflammation of the airways and development of hyper-reactivity (Galli et al., 2008, Lee and Yang, 2013, Esposito et al., 2014). A factor of oxidative stress and inflammation has also been suggested in several neurodevelopmental conditions (Mitchell and Goldstein, 2014), neuropsychiatric disorders (Assies et al., 2014) neurodegenerative disorders (Urrutia et al., 2014) metabolic diseases (Du et al., 2013, Montane et al., 2014) and references therein, and male fertility (Walczak-Jedrzejowska et al., 2013). The degree of co-morbidity of inflammatory diseases, e.g., asthma and ADHD (Mogensen et al., 2011), depression and cardiovascular disease (Assies et al., 2014) (and references therein) speaks in favor of common risk factors related to the innate immune system and inflammatory status. 19