Application of AOPs to assist regulatory assessment of chemical risks - Case studies, needs and recommendations.

While human regulatory risk assessment (RA) still largely relies on animal studies, new approach methodologies (NAMs) based on in vitro, in silico or non-mammalian alternative models are increasingly used to evaluate chemical hazards. Moreover, human epidemiological studies with biomarkers of effect (BoE) also play an invaluable role in identifying health effects associated with chemical exposures. To move towards the next generation risk assessment (NGRA), it is therefore crucial to establish bridges between NAMs and standard approaches, and to establish processes for increasing mechanistically-based biological plausibility in human studies. The Adverse Outcome Pathway (AOP) framework constitutes an important tool to address these needs but, despite a significant increase in knowledge and awareness, the use of AOPs in chemical RA remains limited. The objective of this paper is to address issues related to using AOPs in a regulatory context from various perspectives as it was discussed in a workshop organized within the European Union partnerships HBM4EU and PARC in spring 2022. The paper presents examples where the AOP framework has been proven useful for the human RA process, particularly in hazard prioritization and characterization, in integrated approaches to testing and assessment (IATA), and in the identification and validation of BoE in epidemiological studies. Nevertheless, several limitations were identified that hinder the optimal usability and acceptance of AOPs by the regulatory community including the lack of quantitative information on response-response relationships and of efficient ways to map chemical data (exposure and toxicity) onto AOPs. The paper summarizes suggestions, ongoing initiatives and third-party tools that may help to overcome these obstacles and thus assure better implementation of AOPs in the NGRA.

Dietary habits among men and women in West Greenland: follow-up on the ACCEPT birth cohort.

BACKGROUND: In the past decades, the diet in Greenland has been in transition resulting in a lower intake of traditional food and a higher intake of imported western food. This diet transition can affect public health negatively, and thus, continued monitoring of dietary habits is important. The present study aimed to follow up on the dietary habits of pregnant women included in the Greenlandic ACCEPT birth cohort (2013-2015) and the children's father. METHODS: The follow-up food intake was assessed in 2019-2020 using food frequency questionnaires for 101 mothers and 76 fathers aged 24-55 years living in Nuuk, Sisimiut, and Ilulissat. Non-parametric statistical methods were used (Mann-Whitney U test/Spearman correlation) to assess the dietary pattern and influencing factors. RESULTS: The proportion of traditional and imported food was 14 and 86%, respectively. Intake frequency differed by gender (vegetables, fruits, fast food), the living town (terrestrial animals, vegetables, fruits), and age (fish, meat products, fruits, fast food). Socioeconomic and lifestyle factors significantly correlated with the intake frequency of several traditional and imported foods. Few changes in the mother's dietary habits from inclusion (during pregnancy) to follow-up (3-5 years later) were found, showing less frequent intake of seabirds and fruits and more frequent meat intake. CONCLUSION: We identified several factors that could affect dietary habits, and the results may be used to target future food recommendation for relevant population groups.

Exposure to Perflouroalkyl acids and foetal and maternal thyroid status: a review.

BACKGROUND: Exposure to perfluorinated-alkyl-acids (PFAAs) is ubiquitous. PFAAs are hormone-disrupting compounds that are strongly suspected to affect mother-child-health such as fetal growth. Thyroid disruption is a plausible mechanism of action. We aim to summarize the epidemiological evidence for the relation between prenatal and postnatal exposure to PFAAs and disruption of thyroid homeostasis in mothers and/or infants. METHOD: Fifteen original publications on PFAAs concentrations and thyroid hormones (TH) in pregnant women and/or infants were found upon a literature search in the PubMed database. Information on exposure to seven PFAAs congeners [Perfluorooctane sulfonate (PFOS), Perfluorooctanoate (PFOA), Perfluorohexane sulfonate (PFHxS), Perfluorononanoic acid (PFNA), Perfluorodecanoic acid (PFDA), Perfluoroundecanoic acid (PFUnA), and Perfluorododecanoic acid (PFDoA)] and thyroid stimulating hormone (TSH), free and total thyroxine (FT4 and TT4), free and total triiodothyronine (FT3 and TT3), T3RU (Free triiodothyronine resin uptake) and FT4-index (FT4I) levels were recorded. We evaluated sampling of maternal TH by trimester, and infant TH by sex stratification. Reported associations between mother or infant PFAAs and TH were not uniformly assessed in the selected studies. RESULTS: Ten out of the fifteen studies examined maternal PFAAs concentration and TSH level. Seven studies showed significant associations between TSH and exposure to six PFAAs congeners, most of them were positive. Maternal T4 and T3 were investigated in nine studies and five studies found inverse associations between exposure to six PFAAs congeners and TH (TT3, TT4, FT3, FT4 and FT4I) levels. Eight of the fifteen studies investigated PFAAs concentrations and infant TSH. Infant TSH level was significantly affected in four studies, positively in three studies. Nine studies investigated infant T4 and T3 and seven studies found significant associations with PFAAs exposure. However, both inverse and positive significant associations with infant TH were found eliciting no clear direction. CONCLUSION: Results indicate a mainly positive relationship between maternal PFAAs concentrations and TSH levels, and suggestion of an inverse association with T4 and/or T3 levels. Associations of infant TH with PFAAs concentration were less consistent.

Serum levels of environmental pollutants is a risk factor for breast cancer in Inuit: a case control study.

BACKGROUND: Environmental Persistent Organic Pollutants (POPs) can alter the hormone homeostasis by mimicking, interfering or blocking the function of hormones; moreover POPs are hypothesized to modify the risk of breast cancer. The association between POPs and breast cancer has been widely studied but the conclusions are inconsistent. The present study examined the associations between serum levels of POPs and breast cancer with focus on the highly exposed Greenlandic Inuit population. METHODS: The study design was a case-control study of Inuit women from Greenland. The participants were asked to complete a questionnaire with information on reproductive history and lifestyle and to provide a blood sample. The sampling was carried out in two time periods (2000-2003 and 2011-2014). The serum levels were determined of 14 polychlorinated biphenyls (PCBs), 11 organochlorine pesticides (OCPs), 16 perfluoroalkyl acids (PFAAs), 1 polybrominated biphenyl (PBB), and 9 polybrominated diphenyl ethers (PBDEs). Independent samples t-test was used to compare differences between cases and controls and odds ratios (OR) adjusted for identified confounders were obtained using logistic regression. RESULTS: The study population included 77 breast cancer cases and 84 controls. The majority of the measured compounds declined significantly from 2000 - 2003 to 2011-2014. However, for the perfluorinated carboxylic acids (PFCAs) an increase was observed. The serum levels were significantly higher in cases compared to controls for the majority of the compounds, and after adjusting for age the difference was maintained for ∑OCP, dichlorodiphenyldichloroethylene (p,p'-DDE), ∑PFAA, ∑perfluorinated sulfonic acids (PFSA), perfluorohexane sulfonate (PFHxS), and perfluorooctane sulfonate (PFOS). For the lipophilic POPs, high serum levels (middel/highest vs. lowest tertile) of ∑PCB, ∑estrgoenicPCB, PCB99, PCB138, PCB153, PCB170, PCB170, and PCB183 was associated with breast cancer risk; for the amphiphilic PFAAs, high serum levels of ∑PFAA, ∑PFCA, ∑PFSA, perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), perfluorodecanoic acid (PFDA), PFHxS, and PFOS were associated with breast cancer risk. CONCLUSION: Significant, positive associations between breast cancer risk and PCBs and PFAAs were observed. The associations indicate that environmental exposure to POPs can be a factor increasing the risk for breast cancer in Inuit women.

Metal exposure in the Greenlandic ACCEPT cohort: follow-up and comparison with other Arctic populations.

Humans are exposed to metals through diet and lifestyle e.g. smoking. Some metals are essential for physiologically body functions, while others are non-essential and can be toxic to humans. This study follows up on metal concentrations in the Greenlandic ACCEPT birth-cohort (mothers and fathers) and compares with other Arctic populations. The data from 2019 to 2020 include blood metal concentrations, lifestyle and food frequency questionnaires from 101 mothers and 76 fathers, 24-55 years, living in Nuuk, Sisimiut, and Ilulissat. A high percentage (25-45%) exceeded international guidance values for Hg. For the mothers, the metal concentrations changed significantly from inclusion at pregnancy to this follow-up 3-5 years after birth; some increased and others decreased. Most metals differed significantly between mothers and fathers, while few also differed between residential towns. Several metals correlated significantly with marine food intake and socio-economic factors, but the direction of the correlations varied. Traditional marine food intake was associated positively with Se, As and Hg. To the best of our knowledge, this study provides the most recent data on metal exposure of both men and women in Greenland, elucidating metal exposure sources among Arctic populations, and documents the need for continuing biomonitoring to follow the exceeding of guidance values for Hg. [Figure: see text].

Maternal serum dioxin-like activity and gestational age at birth and indices of foetal growth: The Aarhus birth cohort.

Human exposure to lipophilic persistent organic pollutants (lipPOP) is ubiquitous and life-long, beginning during foetal development. Exposure to lipPOP elicits a number of species and tissue specific responses including dioxin-like activity which involve the activation of aryl hydrocarbon receptor (AhR). This study aims i) to describe the combined dioxin-like activity in serum from Danish pregnant women collected during 2011-2013; ii) to assess the association between maternal serum dioxin-like activity, gestational age at birth and foetal growth indices. The serum lipPOP fraction was extracted using Solid Phase Extraction and cleaned-up on Supelco multi-layer silica and florisil columns. The combined dioxin-like activity of the extract was determined using the AhR reporter gene bioassay, expressed as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxic equivalent (TEQ) [AhR-TEQ (pg/g lipid)]. The associations of AhR-TEQ and foetal growth indices (birth weight, birth length and head circumference) and gestational age were assessed by linear regression models. We detected AhR-TEQ in 93.9 % of maternal first trimester serum samples, with a median level of 185 pg/g lipid. Each ln-unit increase in AhR-TEQ was associated with an increase in birth weight of 36 g (95 % CI: 5; 68), birth length of 0.2 cm (95 % CI: 0.01; 0.3) and pregnancy duration of 1 day (95 % CI: 0; 1.5). In women who never smoked, higher AhR-TEQ values were associated with higher birth weight and longer duration of gestation, while in smokers the association was the opposite. Mediation analyses suggested that gestational age may mediate the association of AhR-TEQ with foetal growth indices. We conclude that AhR activating substances are present in the bloodstream of almost all pregnant women in Denmark and the AhR-TEQ level was around four times higher than previously reported. The AhR-TEQ was associated with slightly longer gestational duration and thereby higher birth weight and birth length.

Exposure to persistent organic pollutants in Danish pregnant women: Hormone levels and fetal growth indices.

This study examines possible associations of maternal Persistent Organic Pollutants (POP) exposure during pregnancy, maternal hormone levels and fetal growth indices (FGI). During 1st trimester, we measured maternal thyroids, androgens and estrogens, lipophilic POP and perfluorinated-alkyl-acid (PFAA) levels in serum from nulliparous women. Adjusted multivariate-linear regression models assessed associations between exposure and outcomes. Maternal characteristics and POP exposures associated with maternal hormone levels. Lipophilic POP elicited inverse association with androgen and estrogen levels but no strong association with thyroids. Higher level of PFAA was associated with higher thyroid and androgen levels. The PFAA did not associate with estrogens. Higher thyroid-peroxidase-antibody (TPO-Ab) and estradiol level associated with higher birth weight and length in sons. For daughters, the TPO-Ab associations were the opposite being inversely associated with birth weight and length, and higher TPO-Ab and estradiol associated with lower gestational age. Mediation analyses suggested that TPO-Ab mediates the association of PFAA with FGI.

Xeno-estrogenic activity of real-life mixtures of perfluoroalkylated substances in human placenta homogenates.

Humans are simultaneously exposed to complex chemical mixtures, and its combined effect can affect human health. As part of the HBM4EU project, the actual mixture of perfluoroalkylated substances (PFAS) in 25 human placenta samples was extracted by chromatographic methods and assessed for xeno-estrogenic activity using two in-vitro bioassays: the estrogen receptor transactivity and the E-Screen assay. Most of the PFAS extracts displayed xeno-estrogenic activity, in one or both assays. The xeno-estrogenic activities in the two bioassays were not correlated, but both assays showed an overall negative correlation with placenta concentrations of single PFAS. Xeno-estrogenic activities were significantly related to maternal characteristics; being higher in young, smokers and primiparous women, but not with fetal growth (birth weight, birth length, head circumference, gestational age, placenta weight). The presented extraction method can be used to study the combined effect of real-life mixtures of PFAS in relation to health outcomes in large-scale human biomonitoring studies.

Implementation of effect biomarkers in human biomonitoring studies: A systematic approach synergizing toxicological and epidemiological knowledge.

Human biomonitoring (HBM) studies have highlighted widespread daily exposure to environmental chemicals. Some of these are suspected to contribute to adverse health outcomes such as reproductive, neurological, and metabolic disorders, among other developmental and chronic impairments. One of the objectives of the H2020 European Human Biomonitoring Initiative (HBM4EU) was the development of informative effect biomarkers for application in a more systematic and harmonized way in large-scale European HBM studies. The inclusion of effect biomarkers would complement exposure data with mechanistically-based information on early and late adverse effects. For this purpose, a stepwise strategy was developed to identify and implement a panel of validated effect biomarkers in European HBM studies. This work offers an overview of the complete procedure followed, from comprehensive literature search strategies, selection of criteria for effect biomarkers and their classification and prioritization, based on toxicological data and adverse outcomes, to pilot studies for their analytical, physiological, and epidemiological validation. We present the example of one study that demonstrated the mediating role of the effect biomarker status of brain-derived neurotrophic factor BDNF in the longitudinal association between infant bisphenol A (BPA) exposure and behavioral function in adolescence. A panel of effect biomarkers has been implemented in the HBM4EU Aligned Studies as main outcomes, including traditional oxidative stress, reproductive, and thyroid hormone biomarkers. Novel biomarkers of effect, such as DNA methylation status of BDNF and kisspeptin (KISS) genes were also evaluated as molecular markers of neurological and reproductive health, respectively. A panel of effect biomarkers has also been applied in HBM4EU occupational studies, such as micronucleus analysis in lymphocytes and reticulocytes, whole blood comet assay, and malondialdehyde, 8-oxo-2'-deoxyguanosine and untargeted metabolomic profile in urine, to investigate, for example, biological changes in response to hexavalent chromium Cr(VI) exposure. The use of effect biomarkers in HBM4EU has demonstrated their ability to detect early biological effects of chemical exposure and to identify subgroups that are at higher risk. The roadmap developed in HBM4EU confirms the utility of effect biomarkers, and support one of the main objectives of HBM research, which is to link exposure biomarkers to mechanistically validated effect and susceptibility biomarkers in order to better understand the public health implications of human exposure to environmental chemicals.

Dioxin-like Activity in Pregnant Women and Indices of Fetal Growth: The ACCEPT Birth Cohort.

Exposure to lipophilic persistent organic pollutants (lipPOPs) elicits a number of species- and tissue-specific toxic responses, many of which involve the aryl hydrocarbon receptor (AhR). This study aims to measure the combined serum dioxin-like activity of lipPOPs in Greenlandic Inuit pregnant women and the associations with fetal growth indices. The combined dioxin-like activity of serum lipPOPs extracts was determined using the AhR reporter gene bioassay and expressed as pico-gram (pg) TCDD equivalent (TEQ) per gram serum lipid [AhR-TEQ (pg/g lipid)]. Significant AhR-TEQ was found in >87% of serum samples with the median level of 86.2 pg TEQ/g lipid. The AhR-TEQ level positively correlated with the marine food intake biomarker n-3/n-6 polyunsaturated fatty acids ratio, while negatively correlated with body mass index and parity. Women giving birth to infants with low birth weight (<2500 g) and length (<50 cm) had higher AhR-TEQ level compared to those with normal weight and length infants. For previous smokers, we found significant inverse associations between maternal AhR-TEQ level and fetal growth indices. In conclusion, exposure of Greenlandic Inuit pregnant women to dioxin-like compounds through traditional marine food can adversely influence the fetal growth via induced AhR activity. Smoking might have modifying effects.

Persistent organic pollutant exposures among Greenlandic adults in relation to lifestyle and diet: New data from the ACCEPT cohort.

High concentrations of persistent organic pollutants (POPs) in blood of the Greenlandic population are well known. The exposure is mainly through traditional food intake, including marine mammals and seabirds. The present study aimed to follow up on POP concentrations (organochlorine pesticides, polychlorinated biphenyls, per- and polyfluoroalkyl substances, and halogenated flame retardants (HFRs)) and relations to lifestyle and diet of the mothers included in the Greenlandic ACCEPT cohort (3-5 years after inclusion in 2013-15) and to include the children's fathers. This new data collection in 2019-20 included blood samples for measurement of POP concentrations and lifestyle and food frequency questionnaires from 101 mothers and 76 fathers aged 24-55 years living in Nuuk, Sisimiut, and Ilulissat, Greenland. The mothers' intra-individual median percentage decrease in POP concentrations from inclusion to this follow-up (3-5 years later) was 16-58%, except for mirex (0% change). Median concentrations of POPs were 1.4-4.6 times higher in fathers than in mothers. The POPs differed by residential town with generally higher concentrations in Ilulissat compared to Sisimiut and Nuuk. We report, for the first time, novel HFRs in human samples from Greenland. However, concentrations were low and only dechlorane plus (with its anti-isomer) was detected in >50% of the samples. Most POPs correlated positively with age and n-3/n-6 fatty acid ratio. The lipophilic POPs correlated positively with the percentage of life lived in Greenland, whereas few POPs correlated positively with BMI, income (personal and household), education, and alcohol intake. The POPs generally associated positively with the intake of marine mammals, seabirds, and dried fish, while few POPs associated positively with Greenlandic fish intake. In contrast, POPs generally associated negatively with imported meat products intake. The study findings may be of interest for future dietary recommendations in Greenland. We discuss the potential explanations for the findings and suggestions for future research.

Reduced Birth Weight and Exposure to Per- and Polyfluoroalkyl Substances: A Review of Possible Underlying Mechanisms Using the AOP-HelpFinder.

Prenatal exposure to per- and polyfluorinated substances (PFAS) may impair fetal growth. Our knowledge of the underlying mechanisms is incomplete. We used the Adverse Outcome Pathway (AOP)-helpFinder tool to search PubMed for studies published until March 2021 that examined PFAS exposure in relation to birth weight, oxidative stress, hormones/hormone receptors, or growth signaling pathways. Of these 1880 articles, 106 experimental studies remained after abstract screening. One clear finding is that PFAS are associated with oxidative stress in in vivo animal studies and in vitro studies. It appears that PFAS-induced reactive-oxygen species (ROS) generation triggers increased peroxisome proliferator-activated receptor (PPAR)γ expression and activation of growth signaling pathways, leading to hyperdifferentiation of pre-adipocytes. Fewer proliferating pre-adipocytes result in lower adipose tissue weight and in this way may reduce birth weight. PFAS may also impair fetal growth through endocrine effects. Estrogenic effects have been noted in in vivo and in vitro studies. Overall, data suggest thyroid-damaging effects of PFAS affecting thyroid hormones, thyroid hormone gene expression, and histology that are associated in animal studies with decreased body and organ weight. The effects of PFAS on the complex relationships between oxidative stress, endocrine system function, adipogenesis, and fetal growth should be further explored.

Time Trend of Persistent Organic Pollutants and Metals in Greenlandic Inuit during 1994-2015.

Persistent organic pollutants (POPs), including polychlorinated biphenyls (PCBs), organchlorine pesticides and perfluoroalkylated substances (PFASs) and heavy metals bioaccumulate in the marine food chain in the Arctic regions, and thus, the Greenlandic population has a higher body burden due to relatively high intake of marine mammals. We assessed the temporal trend for POPs, including PCB 153; 1,1-dichloro-2,2-bis (p-chlorophenyl)-ethylene (p,p'-DDE); oxychlordane; six PFASs; mercury; lead and selenium in Inuit from Ilulissat, Nuuk, and across Greenland (including thirteen towns/districts), from 1994 to 2015. Data showed a significant annual decrease of 6.85-8.61% for PCB153, 6.67-8.61% for p,p'-DDE, 6.11-9.52% for oxychlordane, 5.92-6.76% for mercury and 6.48-9.43% for lead in Inuit women from Nuuk, Ilulissat, and across thirteen Greenlandic districts. The blood selenium level of all Greenlandic women increased 1.01% annually, while the trend direction was negative for Nuuk women. A similar pattern was seen for men across Greenland, with a yearly decrease of 11.3% for PCB 153, 8.61% for p,p'-DDE, 15.6% for oxychlordane, 13.1% for mercury and 12.2% for lead. Perfluorooctane sulfonate, perfluorohexane sulfonate and perfluorooctanoic acid significantly decreased 5.82-11.7% annually for both women and men across Greenland. For perfluorononanoic acid, perfluorodecanoic acid and perfluoroundecanoic acid, we observed an increasing trend for women across Greenland. In conclusion, there was a decreasing trend of the regulated POPs and metals but a potential increasing trend of the nonregulated PFASs in the Greenlandic population between 1994 and 2015. The continuing biomonitoring of contaminants of concern is important to protect the Arctic population heath.

Prenatal exposure to persistent organic pollutants and metals and problematic child behavior at 3-5 years of age: a Greenlandic cohort study.

High levels of persistent organic pollutants (POPs) and heavy metals are found in Arctic populations. POP and heavy metals are linked to impaired cognitive development. This study examined associations between prenatal POP and metals exposure and problematic child behavior using the Strength and Difficulties Questionnaire (SDQ). POPs and metals were measured in 102 pregnant Greenlandic women. During follow-up at 3-5 years, parents answered an assisted questionnaire including children's SDQ scores. Associations were analyzed using linear and logistic regression analyses and adjusted for maternal plasma cotinine, educational level and age at delivery. In the adjusted analyses, the medium tertile of hexachlorobenzene (ß = 3.06, p = 0.010), ß-hexachlorocyclohexane (ß = 3.58, p = 0.004) and trans-nonachlor (ß = 2.06, p = 0.082) were positively associated with SDQ scores. The continuous cis-nonachlor (OR = 1.09, p = 0.079), dichloro-diphenyl-dichloroethylene (OR = 1.01, p = 0.077), trans-nonachlor (OR = 1.01, p = 0.091), and sum Organochlorine-Pesticides (OR = 1.00, p = 0.094) were positively associated with abnormal SDQ score and the continuous mirex (OR = 1.28, p = 0.096), oxychlordane (OR = 1.04, p = 0.066), and trans-nonachlor (OR = 1.02, p = 0.071) with abnormal hyperactivity score. We found no consistent evidence of associations between polychlorinated biphenyls, perfluoroalkylated substances and heavy metals and problematic behavior. Prenatal organochlorine pesticide exposure associated significantly with problematic behavior in 3-5 year old children.

Anthropometric measures and blood pressure of Greenlandic preschool children.

The study aims to describe anthropometric data of Greenlandic preschool children, blood pressure (BP) measures and effects of maternal smoking during pregnancy in a follow-up of the ACCEPT birth cohort. The study included 102 children (55 boys and 47 girls) aged 3.5-5.5 years. Anthropometric measures included weight, height, head circumference (HC), body mass index (BMI), waist and hip circumference, ratio for height/weight, waist/height, waist/hip and BP measurements. Overweight and obesity-prevalence was determined using the international obesity task force BMI references for children. Significant increases in anthropometrics from 3.5 to 5.5 years included weight, height, HC and hip circumference, whereas ratio for height/weight, waist/hip and waist/height decreased. Boys had significantly larger HC than girls. The prevalence of overweight and obesity was similar (35.1%) between genders. Mean systolic and diastolic BP were 104 and 68 mmHg, respectively. Children of mothers smoking during pregnancy had higher hip circumference, lower waist/hip ratio and higher diastolic BP. The study present updated anthropometric data and BP of Greenlandic preschool children and effects of maternal smoking during pregnancy. The prevalence of overweight and obesity was higher than previously reported and maternal smoking during pregnancy affected the child data.Abbreviations ACCEPT: Adaptation to Climate Change, Environmental Pollution and dietary Transition; BMI: Body Mass Index; BP: Blood Pressure; Circ: Circumference; DBP: Diastolic Blood Pressure; FFH: Fisher-Freeman-Halton exact test; HC: Head Circumference; H: Height; HDL: high-density lipoprotein; HiC: Hip Circumference; IOTF International Obesity Task Force, Max: maximum, Min: minimum, P25: 25th percentile, P75: 75th percentile, SBP: Systolic Blood Pressure, SD: Standard Deviation; VDLD: very-low-density lipoprotein; WaC: Waist Circumference, W: Weight, WHO: World Health Organization.

Receptor-based in vitro activities to assess human exposure to chemical mixtures and related health impacts.

Humans are exposed to a large number of chemicals from sources such as the environment, food, and consumer products. There is growing concern that human exposure to chemical mixtures, especially during critical periods of development, increases the risk of adverse health effects in newborns or later in life. Historically, the one-chemical-at-a-time approach has been applied both for exposure assessment and hazard characterisation, leading to insufficient knowledge about human health effects caused by exposure to mixtures of chemicals that have the same target. To circumvent this challenge researchers can apply in vitro assays to analyse both exposure to and human health effects of chemical mixtures in biological samples. The advantages of using in vitro assays are: (i) that an integrated effect is measured, taking combined mixture effects into account and (ii) that in vitro assays can reduce complexity in identification of Chemicals of Emerging Concern (CECs) in human tissues. We have reviewed the state-of-the-art on the use of receptor-based in vitro assays to assess human exposure to chemical mixtures and related health impacts. A total of 43 studies were identified, in which endpoints for the arylhydrocarbon receptor (AhR), the estrogen receptor (ER), and the androgen receptor (AR) were used. The majority of studies reported biological activities that could be associated with breast cancer incidence, male reproductive health effects, developmental toxicities, human demographic characteristics or lifestyle factors such as dietary patterns. A few studies used the bioactivities to check the coverage of the chemical analyses of the human samples, whereas in vitro assays have so far not regularly been used for identifying CECs in human samples, but rather in environmental matrices or food packaging materials. A huge field of novel applications using receptor-based in vitro assays for mixture toxicity assessment on human samples and effect-directed analysis (EDA) using high resolution mass spectrometry (HRMS) for identification of toxic compounds waits for exploration. In the future this could lead to a paradigm shift in the way we unravel adverse human health effects caused by chemical mixtures.

Assessment of chemical mixtures using biomarkers of combined biological activity: A screening study in human placentas.

Humans are simultaneously exposed to complex mixtures of chemicals with limited knowledge on potential health effects, therefore improved tools for assessing these mixtures are needed. As part of the Human Biomonitoring for Europe (HBM4EU) Project, we aimed to examine the combined biological activity of chemical mixtures extracted from human placentas using one in vivo and four in vitro bioassays, also known as biomarkers of combined effect. Relevant endocrine activities (proliferative and/or reporter gene assays) and four endpoints were tested: the estrogen receptor (ER), androgen receptor (AR), and aryl hydrocarbon receptor (AhR) activities, as well as thyroid hormone (TH) signaling. Correlations among bioassays and their functional shapes were evaluated. Results showed that all placental extracts agonized or antagonized at least three of the abovementioned endpoints. Most placentas induced ER-mediated transactivation and ER-dependent cell proliferation, together with a strong inhibition of TH signaling and the AR transactivity; while the induction of the AhR was found in only one placental extract. The effects in the two estrogenic bioassays were positively and significantly correlated and the AR-antagonism activity showed a positive borderline-significant correlation with both estrogenic bioassay activities. However, the in vivo anti-thyroid activities of placental extracts were not correlated with any of the tested in vitro assays. Findings highlight the importance of comprehensively mapping the biological effects of "real-world" chemical mixtures present in human samples, through a battery of in vitro and in vivo bioassays. This approach should be a complementary tool for epidemiological studies to further elucidate the combined biological fingerprint triggered by chemical mixtures.

Persistent organic pollutants in Greenlandic pregnant women and indices of foetal growth: The ACCEPT study.

The Greenlandic population has some of the highest levels of environmental persistent organic pollutants (POPs) globally. Studies have previously found POPs to be linked with disturbance of child development, immune function and reproductive abilities. We investigated the associations between serum POP levels of pregnant women in Greenland and their infant's birth weight, length, head circumference and gestational age (GA) at birth. Pregnant Greenlandic women (n = 504) were enrolled during pregnancy and serum levels of the lipophilic POPs (Organochlorine pesticides, Polychlorinated biphenyls and Polybrominated diphenyl ethers) and the amphiphilic POPs, Perfluoroalkylated substances (PFASs), were measured. We analysed the associations between maternal serum levels of POPs and birth weight, length, head circumference and GA using linear regression analysis. We found significant inverse associations between Perfluorooctanoic Acid (PFOA) and birth weight (adjusted ß = -119 g, 95% CI: -201; -36), birth length (adjusted ß = -0.37 cm, 95% CI: -0.76; 0.02, borderline significant) and head circumference (adjusted ß = -0.35 cm, 95% CI: -0.59; -0.10) and a positive association with GA (adjusted ß = 0.45 week, 95% CI: 0.17; 0.74). For the lipophilic POPs, we found an overall trend of inverse associations to foetal growth indices. In conclusion, we found that the amphiphilic PFOA had a significant inversely association with foetal growth indices, whereas GA was positively associated. The data indicate that POPs have a negative effect on foetal growth.

DNA methylation level in blood and relations to breast cancer, risk factors and environmental exposure in Greenlandic Inuit women.

Several studies have found aberrant DNA methylation levels in breast cancer cases, but factors influencing DNA methylation patterns and the mechanisms are not well understood. This case-control study evaluated blood methylation level of two repetitive elements and selected breast cancer-related genes in relation to breast cancer risk, and the associations with serum level of persistent organic pollutants (POPs) and breast cancer risk factors in Greenlandic Inuit. DNA methylation was determined using bisulphite pyrosequencing in blood from 74 breast cancer cases and 80 controls. Using first tertile as reference, the following was observed. Positive associations for ATM in second tertile (OR: 2.33, 95% CI: 1.04; 5.23) and ESR2 in third tertile (OR: 2.22, 95% CI: 0.97; 5.05) suggest an increased breast cancer risk with high DNA methylation. LINE-1 methylation was lower in cases than controls. In third tertile (OR: 0.42, 95% CI: 0.18; 0.98), associations suggest in accordance with the literature an increased risk of breast cancer with LINE-1 hypomethylation. Among controls, significant associations between methylation levels and serum level of POPs and breast cancer risk factors (age, body mass index, cotinine level) were found. Thus, breast cancer risk factors and POPs may alter the risk through changes in methylation levels; further studies are needed to elucidate the mechanisms.

Associations of Fetal Growth Outcomes with Measures of the Combined Xenoestrogenic Activity of Maternal Serum Perfluorinated Alkyl Acids in Danish Pregnant Women.

BACKGROUND: Higher concentrations of single perfluorinated alkyl acids (PFAAs) have been associated with lower birth weight (BW), but few studies have examined the combined effects of PFAA mixtures. PFAAs have been reported to induce estrogen receptor (ER) transactivity, and estrogens may influence human fetal growth. We hypothesize that mixtures of PFAAs may affect human fetal growth by disrupting the ER. OBJECTIVES: We aimed to study the associations between the combined xenoestrogenic activity of PFAAs in pregnant women's serum and offspring BW, length, and head circumference. METHODS: We extracted the actual mixture of PFAAs from the serum of 702 Danish pregnant women (gestational wk 11­13) enrolled in the Aarhus Birth Cohort (ABC) using solid phase extraction, high-performance liquid chromatography (HPLC), and weak anion exchange. PFAA-induced xenoestrogenic receptor transactivation (XER) was determined using the stable transfected MVLN cell line. Associations between XER and measures of fetal growth were estimated using multivariable linear regression with primary adjustment for maternal age, body mass index (BMI), educational level, smoking, and alcohol intake, and sensitivity analyses with additional adjustment for gestational age (GA) (linear and quadratic). RESULTS: On average, an interquartile range (IQR) increase in XER was associated with a [Formula: see text] [95% confidence interval (CI): [Formula: see text], [Formula: see text]] decrease in BW and a [Formula: see text] (95% CI: 0.1, 0.5) decrease in birth length. Upon additional adjustment for GA, the estimated mean differences were [Formula: see text] (95% CI: [Formula: see text], 4) and [Formula: see text] (95% CI: [Formula: see text], 0.0), respectively. CONCLUSION: Higher-serum PFAA-induced xenoestrogenic activities were associated with lower BW and length in offspring, suggesting that PFAA mixtures may affect fetal growth by disrupting ER function. https://doi.org/10.1289/EHP1884.