Profiling the endocrine disrupting properties of triazines, triazoles and short-chain PFAS.

Persistent, mobile and toxic (PMT) compounds released to the environment are likely to pollute drinking water sources due to their slow environmental degradation (persistency) and high water solubility (mobility). The aim of the present study was to create in vitro hazard profiles for sixteen triazoles, nine triazines and eleven PFAS based on their agonistic and antagonistic effects in estrogen receptor (ER), androgen receptor (AR) and thyroid hormone receptor (TR) reporter gene assays, their ability to bind human transthyretin (TTR), and their effects on steroidogenesis. The triazole fungicides tetraconazole, bitertanol, fenbuconazole, tebuconazole, cyproconazole, difenoconazole, propiconazole, paclobutrazol and triadimenol had agonistic or antagonistic effects on the ER and AR. Difenoconazole, propiconazole and triadimenol were also found to be TR antagonists. The triazine herbicide ametryn was an ER, AR and TR antagonist. The same nine triazole fungicides and the triazines atrazine, deethyl-atrazine and ametryn affected the secretion of steroid hormones. Furthermore, PFAS compounds PFBS, PFHxS, PFHxA, PFOS, PFOA and GenX and the triazoles bitertanol, difenoconazole and 4-methyl benzotriazole were found to displace T4 from TTR. These results are in line with earlier in vitro and in vivo studies on the endocrine disrupting properties of triazines, triazoles and PFAS. The present study demonstrates that this battery of in vitro bioassays can be used to profile compounds from different classes based on their endocrine disrupting properties as a first step to prioritize them for further research, emission reduction, environmental remediation and regulatory purposes.

Investigating Exposure and Hazards of Micro- and Nanoplastics During Pregnancy and Early Life (AURORA Project): Protocol for an Interdisciplinary Study.

BACKGROUND: Micro- and nanoplastics (MNPs) are emerging pollutants of concern with ubiquitous presence in global ecosystems. MNPs pose potential implications for human health; however, the health impacts of MNP exposures are not yet understood. Recent evidence suggests that MNPs can cross the placental barrier, underlying the urgent need to understand their impact on reproductive health and development. OBJECTIVE: The Actionable eUropean ROadmap for early-life health Risk Assessment of micro- and nanoplastics (AURORA) project will investigate MNP exposures and their biological and health effects during pregnancy and early life, which are critical periods due to heightened vulnerability to environmental stressors. The AURORA project will enhance exposure assessment capabilities for measuring MNPs, MNP-associated chemicals, and plastic additives in human tissues, including placenta and blood. METHODS: In this interdisciplinary project, we will advance methods for in-depth characterization and scalable chemical analytical strategies, enabling high-resolution and large-scale toxicological, exposure assessment, and epidemiological studies. The AURORA project performs observational studies to investigate determinants and health impacts of MNPs by including 800 mother-child pairs from 2 existing birth cohorts and 110 women of reproductive age from a newly established cohort. This will be complemented by toxicological studies using a tiered-testing approach and epidemiological investigations to evaluate associations between maternal and prenatal MNP exposures and health perturbations, such as placental function, immune-inflammatory responses, oxidative stress, accelerated aging, endocrine disruption, and child growth and development. The ultimate goal of the AURORA project is to create an MNP risk assessment framework and identify the remaining knowledge gaps and priorities needed to comprehensively assess the impact of MNPs on early-life health. RESULTS: In the first 3 years of this 5-year project (2021-2026), progress was made toward all objectives. This includes completion of recruitment and data collection for new and existing cohorts, development of analytical methodological protocols, and initiation of the toxicological tiered assessments. As of September 2024, data analysis is ongoing and results are expected to be published starting in 2025. CONCLUSIONS: As plastic pollution increases globally, it is imperative to understand the impact of MNPs on human health, particularly during vulnerable developmental stages such as early life. The contributions of the AURORA project will inform future risk assessment. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/63176.

Effects of polystyrene micro- and nanoplastics on androgen- and estrogen receptor activity and steroidogenesis in vitro.

While many plastic additives show endocrine disrupting properties, this has not been studied for micro- and nanoplastics (MNPs) particles despite their ubiquitous presence in humans. The objective of this study was to determine the effects of various sizes and concentrations of polystyrene (PS)-MNPs (50-10,000 nm, 0.01-100 µg/mL) on estrogen- and androgen receptor (ER and AR) activity and steroidogenesis in vitro. Fluorescent (F)PS-MNPs of ≤1000 nm were internalized in VM7 and H295R cells and FPS-MNPs ≤200 nm in AR-ecoscreen cells. H295R cells displayed the highest uptake and particles were closer to the nucleus than other cell types. None of the sizes and concentrations PS-MNPs tested affected ER or AR activity. In H295R cells, PS-MNPs caused some statistically significant changes in hormone levels, though these showed no apparent concentration or size-dependent patterns. Additionally, PS-MNPs caused a decrease in estriol (E3) with a maximum of 37.5 % (100 µg/mL, 50 nm) and an increase in gene expression of oxidative stress markers GPX1 (1.26-fold) and SOD1 (1.23-fold). Taken together, our data show limited endocrine-disrupting properties of PS-MNPs in vitro. Nevertheless the importance of E3 in the placenta warrants further studies in the potential effects of MNPs during pregnancy.

Minor changes to circulating steroid hormones in female rats after perinatal exposure to diethylstilbestrol or ketoconazole.

Current chemical test strategies lack sensitive markers for detecting female reproductive toxicity caused by endocrine disrupting chemicals (EDCs). In search of a potentially sensitive readout, the steroidogenic disrupting effects of the well-known EDCs ketoconazole (KTZ) and diethylstilbestrol (DES) were investigated in vitro and on circulating steroid hormones in perinatally exposed female Sprague-Dawley rats. Twenty-one steroid hormones were analysed using LC-MS/MS in plasma from female rat offspring at postnatal day (PD) 6, 14, 22, 42 and 90. Most circulating steroid hormone levels increased with age except for estrone (E1), estradiol (E2) and backdoor pathway androsterone (ANDROST), which decreased after PD 22. Perinatal exposure to DES did not affect circulating steroid hormone levels at any dose or age compared to controls. KTZ exposure resulted in dose-dependent increase of corticosterone (CORTICO) at PD 6 and PD 14, with statistical significance only at PD 14. In the in vitro gold standard H295R steroidogenesis assay, twenty-one steroid hormones were measured instead of only T and E2. DES had subtle effects on steroidogenesis, whereas KTZ decreased most steroid hormones, but increased CORTICO, progesterone (P4), estriol (E3) initially (around 0.1-1 µM) before decreasing. Our data suggests that circulating steroidomic profiling may not be a sensitive readout for EDC-induced female reproductive toxicity. Further studies are needed to associate H295R assay steroidomic profiles with in vivo profiles, especially in target tissues such as adrenals or gonads. Expanding the H295R steroidogenic assay to include a comprehensive steroidomic profile may enhance its regulatory applicability.

Reduced ovarian cholesterol and steroid biosynthesis along with increased inflammation are associated with high DEHP metabolite levels in human ovarian follicular fluids.

The plasticizer di(2-ethylhexyl) phthalate (DEHP) is known to have endocrine-disrupting properties mediated by its many metabolites that form upon exposure in biological systems. In a previous study, we reported an inverse association between DEHP metabolites in the human ovarian follicular fluid (FF) and the responsiveness of the follicles to controlled ovarian stimulation during in vitro fertilization (IVF) treatments. Here, we explored this association further through molecular analysis of the ovarian FF samples. Ninety-six IVF patients from Swedish (N = 48) and Estonian (N = 48) infertility clinics were selected from the previous cohort (N = 333) based on the molar sum of DEHP metabolites in their FF samples to arrive at "high" (mean 7.7 ± SD 2.3 nM, N = 48) and "low" (0.8 ± 0.4 nM, N = 48) exposure groups. Extracellular miRNA levels and concentrations of 15 steroid hormones were measured across FF samples. In addition, FF somatic cells, available for the Estonian patients, were used for RNA sequencing. Differential expression (DE) and interactions between miRNA and mRNA networks revealed that the expression levels of genes in the cholesterol biosynthesis and steroidogenesis pathways were significantly decreased in the high compared to the low DEHP group. In addition, the DE miRNAs were predicted to target key enzymes within these pathways (FDR < 0.05). A decreased 17-OH-progesterone to progesterone ratio was observed in the FF of the high DEHP group (p < 0.05). Additionally, the expression levels of genes associated with inflammatory processes were elevated in the FF somatic cells, and a computational cell-type deconvolution analysis suggested an increased immune cell infiltration into the high DEHP follicles (p < 0.05). In conclusion, elevated DEHP levels in FF were associated with a significantly altered follicular milieu within human ovaries, involving a pro-inflammatory environment and reduced cholesterol metabolism, including steroid synthesis. These results contribute to our understanding of the molecular mechanisms of female reprotoxic effects of DEHP.

AOP Report: An Upstream Network for Reduced Androgen Signaling Leading to Altered Gene Expression of Androgen Receptor-Responsive Genes in Target Tissues.

Adverse outcome pathways (AOPs) can aid with chemical risk assessment by providing plausible links between chemical activity at the molecular level and effect outcomes in intact organisms. Because AOPs can be used to infer causality between upstream and downstream events in toxicological pathways, the AOP framework can also facilitate increased uptake of alternative methods and new approach methodologies to help inform hazard identification. However, a prevailing challenge is the limited number of fully developed and endorsed AOPs, primarily due to the substantial amount of work required by AOP developers and reviewers. Consequently, a more pragmatic approach to AOP development has been proposed where smaller units of knowledge are developed and reviewed independent of full AOPs. In this context, we have developed an upstream network comprising key events (KEs) and KE relationships related to decreased androgen signaling, converging at a nodal KE that can branch out to numerous adverse outcomes (AOs) relevant to androgen-sensitive toxicological pathways. Androgen signaling represents an extensively studied pathway for endocrine disruption. It is linked to numerous disease outcomes and can be affected by many different endocrine-disrupting chemicals. Still, pathways related to disrupted androgen signaling remain underrepresented in the AOP-wiki, and endorsed AOPs are lacking. Given the pivotal role of androgen signaling in development and function across vertebrate taxa and life stages of both sexes, this upstream AOP network serves as a foundational element for developing numerous AOPs. By connecting the upstream network with various downstream AOs, encompassing different species, it can also facilitate cross-species extrapolations for hazard and risk assessment of chemicals. Environ Toxicol Chem 2024;43:2329-2337. © 2024 The Author(s). Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Enhanced identification of endocrine disruptors through integration of science-based regulatory practices and innovative methodologies: The MERLON Project.

The prevalence of hormone-related health issues caused by exposure to endocrine disrupting chemicals (EDCs) is a significant, and increasing, societal challenge. Declining fertility rates together with rising incidence rates of reproductive disorders and other endocrine-related diseases underscores the urgency in taking more action. Addressing the growing threat of EDCs in our environment demands robust and reliable test methods to assess a broad variety of endpoints relevant for endocrine disruption. EDCs also require effective regulatory frameworks, especially as the current move towards greater reliance on non-animal methods in chemical testing puts to test the current paradigm for EDC identification, which requires that an adverse effect is observed in an intact organism. Although great advances have been made in the field of predictive toxicology, disruption to the endocrine system and subsequent adverse health effects may prove particularly difficult to predict without traditional animal models. The MERLON project seeks to expedite progress by integrating multispecies molecular research, new approach methodologies (NAMs), human clinical epidemiology, and systems biology to furnish mechanistic insights and explore ways forward for NAM-based identification of EDCs. The focus is on sexual development and function, from foetal sex differentiation of the reproductive system through mini-puberty and puberty to sexual maturity. The project aims are geared towards closing existing knowledge gaps in understanding the effects of EDCs on human health to ultimately support effective regulation of EDCs in the European Union and beyond.

Effects of chemical in vitro activation versus fragmentation on human ovarian tissue and follicle growth in culture.

STUDY QUESTION: What is the effect of the chemical in vitro activation (cIVA) protocol compared with fragmentation only (Frag, also known as mechanical IVA) on gene expression, follicle activation and growth in human ovarian tissue in vitro? SUMMARY ANSWER: Although histological assessment shows that cIVA significantly increases follicle survival and growth compared to Frag, both protocols stimulate extensive and nearly identical transcriptomic changes in cultured tissue compared to freshly collected ovarian tissue, including marked changes in energy metabolism and inflammatory responses. WHAT IS KNOWN ALREADY: Treatments based on cIVA of the phosphatase and tensin homolog (PTEN)-phosphatidylinositol 3-kinase (PI3K) pathway in ovarian tissue followed by auto-transplantation have been administered to patients with refractory premature ovarian insufficiency (POI) and resulted in live births. However, comparable effects with mere tissue fragmentation have been shown, questioning the added value of chemical stimulation that could potentially activate oncogenic responses. STUDY DESIGN SIZE DURATION: Fifty-nine ovarian cortical biopsies were obtained from consenting women undergoing elective caesarean section (C-section). The samples were fragmented for culture studies. Half of the fragments were exposed to bpV (HOpic)+740Y-P (Frag+cIVA group) during the first 24 h of culture, while the other half were cultured with medium only (Frag group). Subsequently, both groups were cultured with medium only for an additional 6 days. Tissue and media samples were collected for histological, transcriptomic, steroid hormone, and cytokine/chemokine analyses at various time points. PARTICIPANTS/MATERIALS SETTING METHODS: Effects on follicles were evaluated by counting and scoring serial sections stained with hematoxylin and eosin before and after the 7-day culture. Follicle function was assessed by quantification of steroids by ultra-performance liquid chromatography tandem-mass spectrometry at different time points. Cytokines and chemokines were measured by multiplex assay. Transcriptomic effects were measured by RNA-sequencing (RNA-seq) of the tissue after the initial 24-h culture. Selected differentially expressed genes (DEGs) were validated by quantitative PCR and immunofluorescence in cultured ovarian tissue as well as in KGN cell (human ovarian granulosa-like tumor cell line) culture experiments. MAIN RESULTS AND THE ROLE OF CHANCE: Compared to the Frag group, the Frag+cIVA group exhibited a significantly higher follicle survival rate, increased numbers of secondary follicles, and larger follicle sizes. Additionally, the tissue in the Frag+cIVA group produced less dehydroepiandrosterone compared to Frag. Cytokine measurement showed a strong inflammatory response at the start of the culture in both groups. The RNA-seq data revealed modest differences between the Frag+cIVA and Frag groups, with only 164 DEGs identified using a relaxed cut-off of false discovery rate (FDR) <0.1. Apart from the expected PI3K-protein kinase B (Akt) pathway, cIVA also regulated pathways related to hypoxia, cytokines, and inflammation. In comparison to freshly collected ovarian tissue, gene expression in general was markedly affected in both the Frag+cIVA and Frag groups, with a total of 3119 and 2900 DEGs identified (FDR < 0.001), respectively. The top enriched gene sets in both groups included several pathways known to modulate follicle growth such as mammalian target of rapamycin (mTOR)C1 signaling. Significant changes compared to fresh tissue were also observed in the expression of genes encoding for steroidogenesis enzymes and classical granulosa cell markers in both groups. Intriguingly, we discovered a profound upregulation of genes related to glycolysis and its upstream regulator in both Frag and Frag+cIVA groups, and these changes were further boosted by the cIVA treatment. Cell culture experiments confirmed glycolysis-related genes as direct targets of the cIVA drugs. In conclusion, cIVA enhances follicle growth, as expected, but the mechanisms may be more complex than PI3K-Akt-mTOR alone, and the impact on function and quality of the follicles after the culture period remains an open question. LARGE SCALE DATA: Data were deposited in the GEO data base, accession number GSE234765. The code for sequencing analysis can be found in https://github.com/tialiv/IVA_project. LIMITATIONS REASONS FOR CAUTION: Similar to the published IVA protocols, the first steps in our study were performed in an in vitro culture model where the ovarian tissue was isolated from the regulation of hypothalamic-pituitary-ovarian axis. Further in vivo experiments will be needed, for example in xeno-transplantation models, to explore the long-term impacts of the discovered effects. The tissue collected from patients undergoing C-section may not be comparable to tissue of patients with POI. WIDER IMPLICATIONS OF THE FINDINGS: The general impact of fragmentation and short (24 h) in vitro culture on gene expression in ovarian tissue far exceeded the effects of cIVA. Yet, follicle growth was stimulated by cIVA, which may suggest effects on specific cell populations that may be diluted in bulk RNA-seq. Nevertheless, we confirmed the impact of cIVA on glycolysis using a cell culture model, suggesting impacts on cellular signaling beyond the PI3K pathway. The profound changes in inflammation and glycolysis following fragmentation and culture could contribute to follicle activation and loss in ovarian tissue culture, as well as in clinical applications, such as fertility preservation by ovarian tissue auto-transplantation. STUDY FUNDING/COMPETING INTERESTS: This study was funded by research grants from European Union's Horizon 2020 Research and Innovation Programme (Project ERIN No. 952516, FREIA No. 825100), Swedish Research Council VR (2020-02132), StratRegen funding from Karolinska Institutet, KI-China Scholarship Council (CSC) Programme and the Natural Science Foundation of Hunan (2022JJ40782). International Iberian Nanotechnology Laboratory Research was funded by the European Union's H2020 Project Sinfonia (857253) and SbDToolBox (NORTE-01-0145-FEDER-000047), supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund. No competing interests are declared.

The profile of steroid hormones in human fetal and adult ovaries.

BACKGROUND: Reproduction in women is at risk due to exposure to chemicals that can disrupt the endocrine system during different windows of sensitivity throughout life. Steroid hormone levels are fundamental for the normal development and function of the human reproductive system, including the ovary. This study aims to elucidate steroidogenesis at different life-stages in human ovaries. METHODS: We have developed a sensitive and specific LC-MS/MS method for 21 important steroid hormones and measured them at different life stages: in media from cultures of human fetal ovaries collected from elective terminations of normally progressing pregnancy and in media from adult ovaries from Caesarean section patients, and follicular fluid from women undergoing infertility treatment. Statistically significant differences in steroid hormone levels and their ratios were calculated with parametric tests. Principal component analysis (PCA) was applied to explore clustering of the ovarian-derived steroidogenic profiles. RESULTS: Comparison of the 21 steroid hormones revealed clear differences between the various ovarian-derived steroid profiles. Interestingly, we found biosynthesis of both canonical and "backdoor" pathway steroid hormones and corticosteroids in first and second trimester fetal and adult ovarian tissue cultures. 17α-estradiol, a less potent naturally occurring isomer of 17ß-estradiol, was detected only in follicular fluid. PCA of the ovarian-derived profiles revealed clusters from: adult ovarian tissue cultures with relatively high levels of androgens; first trimester and second trimester fetal ovarian tissue cultures with relatively low estrogen levels; follicular fluid with the lowest androgens, but highest corticosteroid, progestogen and estradiol levels. Furthermore, ratios of specific steroid hormones showed higher estradiol/ testosterone and estrone/androstenedione (indicating higher CYP19A1 activity, p < 0.01) and higher 17-hydroxyprogesterone/progesterone and dehydroepiandrosterone /androstenedione (indicating higher CYP17A1 activity, p < 0.01) in fetal compared to adult ovarian tissue cultures. CONCLUSIONS: Human ovaries demonstrate de novo synthesis of non-canonical and "backdoor" pathway steroid hormones and corticosteroids. Elucidating the steroid profiles in human ovaries improves our understanding of physiological, life-stage dependent, steroidogenic capacity of ovaries and will inform mechanistic studies to identify endocrine disrupting chemicals that affect female reproduction.

The 2022 world health organization reevaluation of human and mammalian toxic equivalency factors for polychlorinated dioxins, dibenzofurans and biphenyls.

In October 2022, the World Health Organization (WHO) convened an expert panel in Lisbon, Portugal in which the 2005 WHO TEFs for chlorinated dioxin-like compounds were reevaluated. In contrast to earlier panels that employed expert judgement and consensus-based assignment of TEF values, the present effort employed an update to the 2006 REP database, a consensus-based weighting scheme, a Bayesian dose response modeling and meta-analysis to derive "Best-Estimate" TEFs. The updated database contains almost double the number of datasets from the earlier version and includes metadata that informs the weighting scheme. The Bayesian analysis of this dataset results in an unbiased quantitative assessment of the congener-specific potencies with uncertainty estimates. The "Best-Estimate" TEF derived from the model was used to assign 2022 WHO-TEFs for almost all congeners and these values were not rounded to half-logs as was done previously. The exception was for the mono-ortho PCBs, for which the panel agreed to retain their 2005 WHO-TEFs due to limited and heterogenous data available for these compounds. Applying these new TEFs to a limited set of dioxin-like chemical concentrations measured in human milk and seafood indicates that the total toxic equivalents will tend to be lower than when using the 2005 TEFs.

Associations between lifestyle factors and levels of per- and polyfluoroalkyl substances (PFASs), phthalates and parabens in follicular fluid in women undergoing fertility treatment.

BACKGROUND: Concerns have been raised whether exposure to endocrine-disrupting chemicals (EDCs) can alter reproductive functions and play a role in the aetiology of infertility in women. With increasing evidence of adverse effects, information on factors associated with exposure is necessary to form firm recommendations aiming at reducing exposure. OBJECTIVE: Our aim was to identify associations between lifestyle factors including the home environment, use of personal care products (PCP), and dietary habits and concentrations of EDCs in ovarian follicular fluid. METHODS: April-June 2016, 185 women undergoing ovum pick-up for in vitro fertilisation in Sweden were recruited. Correlation analyses were performed between self-reported lifestyle factors and concentration of EDCs analysed in follicular fluid. Habits related to cleaning, PCPs, and diet were assessed together with concentration of six per- and polyfluoroalkyl substances (PFASs) [PFHxS, PFOA, PFOS, PFNA, PFDA and PFUnDA], methyl paraben and eight phthalate metabolites [MECPP, MEHPP, MEOHP, MEHP, cxMinCH, cxMiNP, ohMiNP, MEP, MOHiBP]. Spearman's partial correlations were adjusted for age, parity and BMI. RESULTS: Significant associations were discovered between multiple lifestyle factors and concentrations of EDCs in ovarian follicular fluid. After correcting p values for multiple testing, frequent use of perfume was associated with MEP (correlation ρ = 0.41 (confidence interval 0.21-0.47), p < 0.001); hens' egg consumption was positively associated with PFOS (ρ = 0.30 (0.15-0.43), p = 0.007) and PFUnDA (ρ = 0.27 (0.12-0.40), p = 0.036). White fish consumption was positively associated with PFUnDA (ρ = 0.34 (0.20-0.47), p < 0.001) and PFDA (ρ = 0.27 (0.13-0.41), p = 0.028). More correlations were discovered when considering the raw uncorrected p values. Altogether, our results suggest that multiple lifestyle variables affect chemical contamination of follicular fluid. IMPACT STATEMENT: This study shows how lifestyle factors correlate with the level of contamination in the ovary by both persistent and semi-persistent chemicals in women of reproductive age. Subsequently, these data can be used to form recommendations regarding lifestyle to mitigate possible negative health outcomes and fertility problems associated with chemical exposure, and to inform chemical policy decision making. Our study can also help form the basis for the design of larger observational and intervention studies to examine possible effects of lifestyle changes on exposure levels, and to unravel the complex interactions between biological factors, lifestyle and chemical exposures in more detail.

New approach methods to improve human health risk assessment of thyroid hormone system disruption-a PARC project.

Current test strategies to identify thyroid hormone (TH) system disruptors are inadequate for conducting robust chemical risk assessment required for regulation. The tests rely heavily on histopathological changes in rodent thyroid glands or measuring changes in systemic TH levels, but they lack specific new approach methodologies (NAMs) that can adequately detect TH-mediated effects. Such alternative test methods are needed to infer a causal relationship between molecular initiating events and adverse outcomes such as perturbed brain development. Although some NAMs that are relevant for TH system disruption are available-and are currently in the process of regulatory validation-there is still a need to develop more extensive alternative test batteries to cover the range of potential key events along the causal pathway between initial chemical disruption and adverse outcomes in humans. This project, funded under the Partnership for the Assessment of Risk from Chemicals (PARC) initiative, aims to facilitate the development of NAMs that are specific for TH system disruption by characterizing in vivo mechanisms of action that can be targeted by in embryo/in vitro/in silico/in chemico testing strategies. We will develop and improve human-relevant in vitro test systems to capture effects on important areas of the TH system. Furthermore, we will elaborate on important species differences in TH system disruption by incorporating non-mammalian vertebrate test species alongside classical laboratory rat species and human-derived in vitro assays.

Identification of biomarkers and outcomes of endocrine disruption in human ovarian cortex using In Vitro Models.

Endocrine disrupting chemicals (EDCs) are raising concerns about adverse effects on fertility in women. However, there is a lack of information regarding mechanisms and effects in humans. Our study aims to identify mechanisms of endocrine disruption using two EDCs, diethylstilbestrol (DES) and ketoconazole (KTZ)1. Human ovarian cortical tissue obtained from Caesarean section patients was exposed to 10-9 M - 10-5 M KTZ and 10-10 M - 10-6 M DES in vitro for 6 days. Follicle survival and growth were studied via histology analysis and liquid-chromatography-mass spectrometry-based steroid quantification. RNA-sequencing was performed on COV434, KGN, and primary ovarian cells that were exposed for 24 h. Significantly lower unilaminar follicle densities were observed in DES 10-10 M group, whereas low KTZ exposure reduced secondary follicle density. KTZ 10-5 M reduced levels of pregnenolone and progesterone. RNA-sequencing revealed that 445 and 233 differentially expressed genes (false discovery rate < 0.1) altogether in DES and KTZ exposed groups. Gene set variation analysis showed that both chemicals modulated pathways that are important for folliculogenesis and steroidogenesis. We selected stearoyl-CoA desaturase (SCD) and 7-dehydrocholesterol reductase (DHCR7) for further validation. Up-regulation of both genes in response to KTZ was confirmed by qPCR and in situ RNA hybridization. Further validation with immunofluorescence focused on the expression of SCD in growing follicles in exposed ovarian tissue. In conclusion, SCD may serve as a potential novel human-relevant biomarker of EDC exposure and effects on ovaries.

Experimental human placental models for studying uptake, transport and toxicity of micro- and nanoplastics.

Micro- and nanoplastics (MNPs) are ubiquitous in the environment and have recently been found in human lungs, blood and placenta. However, data on the possible effects of MNPs on human health is extremely scarce. The potential toxicity of MNPs during pregnancy, a period of increased susceptibility to environmental insults, is of particular concern. The placenta provides a unique interface between maternal and fetal circulation which is essential for in utero survival and healthy pregnancy. Placental toxicokinetics and toxicity of MNPs are still largely unexplored and the limited studies performed up to now focus mainly on polystyrene particles. Practical and ethical considerations limit research options in humans, and extrapolation from animal studies is challenging due to marked differences between species. Nevertheless, diverse in vitro and ex vivo human placental models exist e.g., plasma membrane vesicles, mono-culture and co-culture of placental cells, placenta-on-a-chip, villous tissue explants, and placental perfusion that can be used to advance this research area. The objective of this concise review is to recapitulate different human placental models, summarize the current understanding of placental uptake, transport and toxicity of MNPs and define knowledge gaps. Moreover, we provide perspectives for future research urgently needed to assess the potential hazards and risks of MNP exposure to maternal and fetal health.

Association between chemical mixtures and female fertility in women undergoing assisted reproduction in Sweden and Estonia.

OBJECTIVE: Women of reproductive age are exposed to ubiquitous chemicals such as phthalates, parabens, and per- and polyfluoroalkyl substances (PFAS), which have potential endocrine disrupting properties and might affect fertility. Our objective was to investigate associations between potential endocrine-disrupting chemicals (EDCs) and female fertility in two cohorts of women attending fertility clinics. METHODS: In a total population of 333 women in Sweden and Estonia, we studied the associations between chemicals and female fertility, evaluating ovarian sensitivity index (OSI) as an indicator of ovarian response, as well as clinical pregnancy and live birth from fresh and frozen embryo transfers. We measured 59 chemicals in follicular fluid samples and detected 3 phthalate metabolites, di-2-ethylhexyl phthalate (DEHP) metabolites, 1 paraben, and 6 PFAS in >90% of the women. Associations were evaluated using multivariable-adjusted linear or logistic regression, categorizing EDCs into quartiles of their distributions, as well as with Bayesian Kernel Machine Regression. RESULTS: We observed statistically significant lower OSI at higher concentrations of the sum of DEHP metabolites in the Swedish cohort (Q4 vs Q1, ß = -0.21, 95% CI: -0.38, -0.05) and methylparaben in the Estonian cohort (Q3 vs Q1, ß = -0.22, 95% CI: -0.44, -0.01). Signals of potential associations were also observed at higher concentrations of PFUnDA in both the combined population (Q2 vs. Q1, ß = -0.16, 95% CI -0.31, -0.02) and the Estonian population (Q2 vs. Q1, ß = -0.27, 95% CI -0.45, -0.08), and for PFOA in the Estonian population (Q4 vs. Q1, ß = -0.31, 95% CI -0.61, -0.01). Associations of chemicals with clinical pregnancy and live birth presented wide confidence intervals. CONCLUSIONS: Within a large chemical mixture, we observed significant inverse associations levels of DEHP metabolites and methylparaben, and possibly PFUnDA and PFOA, with OSI, suggesting that these chemicals may contribute to altered ovarian function and infertility in women.

A Putative Adverse Outcome Pathway Network for Disrupted Female Pubertal Onset to Improve Testing and Regulation of Endocrine Disrupting Chemicals.

The average age for pubertal onset in girls has declined over recent decades. Epidemiological studies in humans and experimental studies in animals suggest a causal role for endocrine disrupting chemicals (EDCs) that are present in our environment. Of concern, current testing and screening regimens are inadequate in identifying EDCs that may affect pubertal maturation, not least because they do not consider early-life exposure. Also, the causal relationship between EDC exposure and pubertal timing is still a matter of debate. To address this issue, we have used current knowledge to elaborate a network of putative adverse outcome pathways (pAOPs) to identify how chemicals can affect pubertal onset. By using the AOP framework, we highlight current gaps in mechanistic understanding that need to be addressed and simultaneously point towards events causative of pubertal disturbance that could be exploited for alternative test methods. We propose 6 pAOPs that could explain the disruption of pubertal timing by interfering with the central hypothalamic trigger of puberty, GnRH neurons, and by so doing highlight specific modes of action that could be targeted for alternative test method development.

Putative adverse outcome pathways for female reproductive disorders to improve testing and regulation of chemicals.

Modern living challenges female reproductive health. We are witnessing a rise in reproductive disorders and drop in birth rates across the world. The reasons for these manifestations are multifaceted and most likely include continuous exposure to an ever-increasing number of chemicals. The cause-effect relationships between chemical exposure and female reproductive disorders, however, have proven problematic to determine. This has made it difficult to assess the risks chemical exposures pose to a woman's reproductive development and function. To address this challenge, this review uses the adverse outcome pathway (AOP) concept to summarize current knowledge about how chemical exposure can affect female reproductive health. We have a special focus on effects on the ovaries, since they are essential for lifelong reproductive health in women, being the source of both oocytes and several reproductive hormones, including sex steroids. The AOP framework is widely accepted as a new tool for toxicological safety assessment that enables better use of mechanistic knowledge for regulatory purposes. AOPs equip assessors and regulators with a pragmatic network of linear cause-effect relationships, enabling the use of a wider range of test method data in chemical risk assessment and regulation. Based on current knowledge, we propose ten putative AOPs relevant for female reproductive disorders that can be further elaborated and potentially be included in the AOPwiki. This effort is an important step towards better safeguarding the reproductive health of all girls and women.

Remote sensing and signaling in kidney proximal tubules stimulates gut microbiome-derived organic anion secretion.

Membrane transporters and receptors are responsible for balancing nutrient and metabolite levels to aid body homeostasis. Here, we report that proximal tubule cells in kidneys sense elevated endogenous, gut microbiome-derived, metabolite levels through EGF receptors and downstream signaling to induce their secretion by up-regulating the organic anion transporter-1 (OAT1). Remote metabolite sensing and signaling was observed in kidneys from healthy volunteers and rats in vivo, leading to induced OAT1 expression and increased removal of indoxyl sulfate, a prototypical microbiome-derived metabolite and uremic toxin. Using 2D and 3D human proximal tubule cell models, we show that indoxyl sulfate induces OAT1 via AhR and EGFR signaling, controlled by miR-223. Concomitantly produced reactive oxygen species (ROS) control OAT1 activity and are balanced by the glutathione pathway, as confirmed by cellular metabolomic profiling. Collectively, we demonstrate remote metabolite sensing and signaling as an effective OAT1 regulation mechanism to maintain plasma metabolite levels by controlling their secretion.

Imatinib mesylate does not counteract ovarian tissue fibrosis in postnatal rat ovary.

Chemotherapy may result in ovarian atrophy, a depletion of the primordial follicle pool, diminished ovarian weight, cortical and stromal fibrosis. Imatinib mesylate is an anticancer agent that inhibits competitively several receptor tyrosine kinases (RTKs). RTKs play important roles in cell metabolism, proliferation, and apoptosis. In clinic, imatinib mesylate is also known as an anti-fibrotic medicine. In the present study, the impact of imatinib on the ovarian tissue was investigated by assessing ovarian tissue fibrosis in postnatal rat administered with or without imatinib for three days. Fibrosis in the ovarian tissue was determined by histology (Picrosirius and Masson's trichrome staining) and the protein expression of vimentin and alpha-smooth muscle actin (α-SMA). Furthermore, mRNA expression of Forkhead box transcription factor O1 and O3 (FOXO1 and FOXO3), which are markers of cell proliferation was quantified. A short-term exposure to imatinib showed to increase tissue fibrosis in ovaries. This was observed by Masson's trichrome staining. Exposure to imatinib led also to a down-regulation of vimentin protein expression and up-regulation mRNA expression of FOXO3. This may indicate a role of FOXO3 in ovarian tissue fibrosis in postnatal rat ovaries.

Anti-tumor properties of methoxylated analogues of resveratrol in malignant MCF-7 but not in non-tumorigenic MCF-10A mammary epithelial cell lines.

Resveratrol is a plant-derived polyphenol that is known for its anti-inflammatory and anti-tumorigenic properties in in vitro and in vivo models. Recent studies show that some resveratrol analogues might be more potent anti-tumor agents, which may partly be attributed to their ability to activate the aryl hydrocarbon receptor (AHR). Here, the anti-tumorigenic properties of resveratrol and structural analogues oxyresveratrol, pinostilbene, pterostilbene and tetramethoxystilbene (TMS) were studied in vitro, using in the malignant human MCF-7 breast cancer cell line and non-tumorigenic breast epithelial cell line MCF-10A. Cell viability and migration assays showed that methoxylated analogues of resveratrol are more potent anti-tumorigenic compounds than resveratrol and its hydroxylated analogue oxyresveratrol, with 2,3',4,5'-tetramethoxy-trans-stilbene (TMS) being the most potent compound. TMS decreased MCF-7 tumor cell viability with 50% at 3.6 µM and inhibited migration with 37.5 ± 14.8% at 3 µM. In addition, TMS activated the AHR more potently (EC50 in a reporter gene assay 2.0 µM) and induced AHR-mediated induction of cytochrome P450 1A1 (CYP1A1) activity (EC50 value of 0.7 µM) more than resveratrol and the other analogues tested. Cell cycle analysis showed that TMS induced a shift in cell cycle status from the G1 to the G2/M phase causing a cell cycle arrest in the MCF-7 cells, while no effect of TMS was observed in the non-tumorigenic MCF-10A mammary epithelial cell line. Gene expression analysis showed that 3 µM TMS increased gene expression of CYP1A1 (289-fold), CYP1B1 (5-fold) and Nqo1 (2-fold), and decreased gene expression of IL-8 (3-fold) in MCF-7 cells. In MCF-10A cells, 10 µM TMS also increased gene expression of CYP1A1 (5-fold) and CYP1B1 (2-fold), but decreased gene expression of Nqo1 (1.4-fold) in contrast to MCF-7 cells. TMS displays more potent anti-tumorigenic properties and activates the AHR more effectively than resveratrol. In addition, this is the first study to show that TMS, but not resveratrol, selectively inhibits the cell cycle of breast tumor cells and not the non-tumorigenic cells. Our study provides more insight in the anti-tumor properties of the methoxylated analogues of resveratrol in breast cells in vitro.