Molecular Assessment of Pro-Adipogenic Effects for Common-Use Contraceptives and their Mixtures.

Hormonal contraceptives are widely prescribed due to their effectiveness and convenience and have become an integral part of family planning strategies worldwide. In the US, ∼65% of reproductive aged women are estimated to be using contraceptive options, with ∼33% using one or a combination of hormonal contraceptives. While these methods have undeniably contributed to improved reproductive health, recent studies have raised concerns regarding their potential impact on metabolic health. Despite widespread anecdotal reports, epidemiological research has been mixed as to whether hormonal contraceptives contribute to metabolic health effects. As such, the goals of this study were to assess the adipogenic activity of common hormonal contraceptive chemicals and their mixtures. Five different models of adipogenesis were utilized to provide a rigorous assessment of metabolism disrupting effects. Interestingly, every individual contraceptive (both estrogens and progestins) and each mixture promoted significant adipogenesis (e.g., triglyceride accumulation and/or pre-adipocyte proliferation). These effects appeared to be mediated in part through estrogen receptor signaling, particularly for the contraceptive mixtures, as co-treatment with fulvestrant acted to inhibit contraceptive-mediated pro-adipogenic effects on triglyceride accumulation. In conclusion, this research provides valuable insights into the complex interactions between hormonal contraceptives and adipocyte development. The results suggest that both progestins and estrogens within these contraceptives can influence adipogenesis, and the specific effects may vary based on the receptor activation profiles. Further research is warranted to establish translation of these findings to in vivo models and to further assess causal mechanisms underlying these effects.

The Conflict between Regulatory Agencies over the 20,000-Fold Lowering of the Tolerable Daily Intake (TDI) for Bisphenol A (BPA) by the European Food Safety Authority (EFSA).

BACKGROUND: The European Food Safety Authority (EFSA) recommended lowering their estimated tolerable daily intake (TDI) for bisphenol A (BPA) 20,000-fold to 0.2 ng/kg body weight (BW)/day. BPA is an extensively studied high production volume endocrine disrupting chemical (EDC) associated with a vast array of diseases. Prior risk assessments of BPA by EFSA as well as the US Food and Drug Administration (FDA) have relied on industry-funded studies conducted under good laboratory practice protocols (GLP) requiring guideline end points and detailed record keeping, while also claiming to examine (but rejecting) thousands of published findings by academic scientists. Guideline protocols initially formalized in the mid-twentieth century are still used by many regulatory agencies. EFSA used a 21st century approach in its reassessment of BPA and conducted a transparent, but time-limited, systematic review that included both guideline and academic research. The German Federal Institute for Risk Assessment (BfR) opposed EFSA's revision of the TDI for BPA. OBJECTIVES: We identify the flaws in the assumptions that the German BfR, as well as the FDA, have used to justify maintaining the TDI for BPA at levels above what a vast amount of academic research shows to cause harm. We argue that regulatory agencies need to incorporate 21st century science into chemical hazard identifications using the CLARITY-BPA (Consortium Linking Academic and Regulatory Insights on BPA Toxicity) nonguideline academic studies in a collaborative government-academic program model. DISCUSSION: We strongly endorse EFSA's revised TDI for BPA and support the European Commission's (EC) apparent acceptance of this updated BPA risk assessment. We discuss challenges to current chemical risk assessment assumptions about EDCs that need to be addressed by regulatory agencies to, in our opinion, become truly protective of public health. Addressing these challenges will hopefully result in BPA, and eventually other structurally similar bisphenols (called regrettable substitutions) for which there are known adverse effects, being eliminated from all food-related and many other uses in the EU and elsewhere. https://doi.org/10.1289/EHP13812.

Characterizing azobenzene disperse dyes and related compounds in house dust and their correlations with other organic contaminant classes.

Azobenzene disperse dyes are the fastest-growing category of commercial dyestuffs and are implicated in the literature as potentially allergenic. In the indoor environment, these dyes may be shed from various textiles, including clothing and upholstery and accumulate in dust particles potentially leading to exposure in young children who have higher exposure to chemicals associated with dust due to their crawling and mouthing behaviors. Children may be more vulnerable to dye exposure due to their developing immune systems, and therefore, it is critical to characterize azobenzene disperse dyes in children's home environments. Here, we investigate azobenzene disperse dyes and related compounds in house dust samples (n = 124) that were previously analyzed for flame retardants, phthalates, pesticides and per- and polyfluoroalkyl substances (PFAS). High-resolution mass spectrometry was used to support both targeted and suspect screening of dyes in dust. Statistical analyses were conducted to determine if dye concentrations were related to demographic information. Detection frequencies for 12 target dyes ranged from 11% to 89%; of the dyes that were detected in at least 50% of the samples, geometric mean levels ranged from 32.4 to 360 ng/g. Suspect screening analysis identified eight additional high-abundance azobenzene compounds in dust. Some dyes were correlated to numerous flame retardants and several antimicrobials, and statistically higher levels of some dyes were observed in homes of non-Hispanic Black mothers than in homes of non-Hispanic white mothers. To our knowledge, this is the most comprehensive study of azobenzene disperse dyes in house dust to date. Future studies are needed to quantify additional dyes in dust and to examine exposure pathways of dyes in indoor environments where children are concerned.

The regulation of endocrine-disrupting chemicals to minimize their impact on health.

Endocrine-disrupting chemicals (EDCs) are substances generated by human industrial activities that are detrimental to human health through their effects on the endocrine system. The global societal and economic burden posed by EDCs is substantial. Poorly defined or unenforced policies can increase human exposure to EDCs, thereby contributing to human disease, disability and economic damage. Researchers have shown that policies and interventions implemented at both individual and government levels have the potential to reduce exposure to EDCs. This Review describes a set of evidence-based policy actions to manage, minimize or even eliminate the widespread use of these chemicals and better protect human health and society. A number of specific challenges exist: defining, identifying and prioritizing EDCs; considering the non-linear or non-monotonic properties of EDCs; accounting for EDC exposure effects that are latent and do not appear until later in life; and updating testing paradigms to reflect 'real-world' mixtures of chemicals and cumulative exposure. A sound strategy also requires partnering with health-care providers to integrate strategies to prevent EDC exposure in clinical care. Critical next steps include addressing EDCs within global policy frameworks by integrating EDC exposure prevention into emerging climate policy.

Complex Mixtures and Multiple Stressors: Evaluating Combined Chemical Exposures and Cumulative Toxicity.

The problem of chemical mixtures in the environment encompasses biological, analytical, logistical, and regulatory challenges, among others [...].

Cetyl Alcohol Polyethoxylates Disrupt Metabolic Health in Developmentally Exposed Zebrafish.

Alcohol polyethoxylates (AEOs), such as cetyl alcohol ethoxylates (CetAEOs), are high-production-volume surfactants used in laundry detergents, hard-surface cleaners, pesticide formulations, textile production, oils, paints, and other products. AEOs have been suggested as lower toxicity replacements for alkylphenol polyethoxylates (APEOs), such as the nonylphenol and octylphenol polyethoxylates. We previously demonstrated that nonylphenol polyethoxylates induced triglyceride accumulation in several in vitro adipogenesis models and promoted adiposity and increased body weights in developmentally exposed zebrafish. We also demonstrated that diverse APEOs and AEOs were able to increase triglyceride accumulation and/or pre-adipocyte proliferation in a murine pre-adipocyte model. As such, the goals of this study were to assess the potential of CetAEOs to promote adiposity and alter growth and/or development (toxicity, length, weight, behavior, energy expenditure) of developmentally exposed zebrafish (Danio rerio). We also sought to expand our understanding of ethoxylate chain-length dependent effects through interrogation of varying chain-length CetAEOs. We demonstrated consistent adipogenic effects in two separate human bone-marrow-derived mesenchymal stem cell models as well as murine pre-adipocytes. Immediately following chemical exposures in zebrafish, we reported disrupted neurodevelopment and aberrant behavior in light/dark activity testing, with medium chain-length CetAEO-exposed fish exhibiting hyperactivity across both light and dark phases. By day 30, we demonstrated that cetyl alcohol and CetAEOs disrupted adipose deposition in developmentally exposed zebrafish, despite no apparent impacts on standard length or gross body weight. This research suggests metabolic health concerns for these common environmental contaminants, suggesting further need to assess molecular mechanisms and better characterize environmental concentrations for human health risk assessments.

Adipogenic and endocrine disrupting mixture effects of organic and inorganic pollutant mixtures.

Chronic health conditions are rapidly increasing in prevalence and cost to society worldwide: in the US, >42 % of adults aged 20 and older are currently classified as obese. Exposure to endocrine disrupting chemicals (EDCs) has been implicated as a causal factor; some EDCs, termed "obesogens", can increase weight and lipid accumulation and/or perturb metabolic homeostasis. This project aimed to assess the potential combination effects of diverse inorganic and organic contaminant mixtures, which more closely reflect environmentally realistic exposures, on nuclear receptor activation/inhibition and adipocyte differentiation. Herein, we focused on two polychlorinated biphenyls (PCB-77 and 153), two perfluoroalkyl substances (PFOA and PFOS), two brominated flame retardants (PBB-153 and BDE-47), and three inorganic contaminants (lead, arsenic, and cadmium). We examined adipogenesis using human mesenchymal stem cells and receptor bioactivities using luciferase reporter gene assays in human cell lines. We observed significantly greater effects for several receptor bioactivities by various contaminant mixtures relative to individual components. All nine contaminants promoted triglyceride accumulation and/or pre-adipocyte proliferation in human mesenchymal stem cells. Comparing simple component mixtures to individual components at 10 % and 50 % effect levels revealed putative synergistic effects for each of the mixtures for at least one of the concentrations relative to the individual component chemicals, some of which also exhibited significantly greater effects than the component contaminants. Our results support further testing of more realistic and complex contaminant mixtures that better reflect environmental exposures, in order to more conclusively define mixture responses both in vitro and in vivo.

Systematic evidence on migrating and extractable food contact chemicals: Most chemicals detected in food contact materials are not listed for use.

Food packaging is important for today's globalized food system, but food contact materials (FCMs) can also be a source of hazardous chemicals migrating into foodstuffs. Assessing the impacts of FCMs on human health requires a comprehensive identification of the chemicals they contain, the food contact chemicals (FCCs). We systematically compiled the "database on migrating and extractable food contact chemicals" (FCCmigex) using information from 1210 studies. We found that to date 2881 FCCs have been detected, in a total of six FCM groups (Plastics, Paper & Board, Metal, Multi-materials, Glass & Ceramic, and Other FCMs). 65% of these detected FCCs were previously not known to be used in FCMs. Conversely, of the more than 12'000 FCCs known to be used, only 1013 are included in the FCCmigex database. Plastic is the most studied FCM with 1975 FCCs detected. Our findings expand the universe of known FCCs to 14,153 chemicals. This knowledge contributes to developing non-hazardous FCMs that lead to safer food and support a circular economy.

The Pediatrician's Role in Protecting Children from Environmental Hazards.

Children suffer disproportionately from disease and disability due to environmental hazards, for reasons rooted in their biology. The contribution is substantial and increasingly recognized, particularly due to ever-increasing awareness of endocrine disruption. Regulatory actions can be traced directly to reductions in toxic exposures, with tangible benefits to society. Deep flaws remain in the policy framework in industrialized countries, failing to offer sufficient protection, but are even more limited in industrializing nations where the majority of chemical production and use will occur by 2030. Evidence-based steps for reducing chemical exposures associated with adverse health outcomes exist and should be incorporated into anticipatory guidance.

Exposure to a mixture of per-and polyfluoroalkyl substances modulates pulmonary expression of ACE2 and circulating hormones and cytokines.

Genetic and environmental factors impact on the interindividual variability of susceptibility to communicable and non-communicable diseases. A class of ubiquitous chemicals, Per- and polyfluoroalkyl substances (PFAS) have been linked in epidemiological studies to immunosuppression and increased susceptibility to viral infections, but possible mechanisms are not well elucidated. To begin to gain insight into the role of PFAS in susceptibility to one such viral infection, Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), male and female C57BL/6 J mice were exposed to control water or a mixture of 5 PFAS (PFOS, PFOA, PFNA, PFHxS, Genx) for 12 weeks and lungs were isolated for examination of expression of SARS-CoV-2-related receptors Angiotensin-Converting Enzyme 2 (ACE2) and others. Secondary analyses included circulating hormones and cytokines which have been shown to directly or indirectly impact on ACE2 expression and severity of viral infections. Changes in mRNA and protein expression were analyzed by RT-qPCR and western blotting and circulating hormones and cytokines were determined by ELISA and MESO QuickPlex. The PFAS mixture decreased Ace2 mRNA 2.5-fold in male mice (p < 0.0001), with no significant change observed in females. In addition, TMPRSS2, ANPEP, ENPEP and DPP4 (other genes implicated in COVID-19 infection) were modulated due to PFAS. Plasma testosterone, but not estrogen were strikingly decreased due to PFAS which corresponded to PFAS-mediated repression of 4 representative pulmonary AR target genes; hemoglobin, beta adult major chain (Hbb-b1), Ferrochelatase (Fech), Collagen Type XIV Alpha 1 Chain (Col14a1), 5'-Aminolevulinate Synthase 2 (Alas2). Finally, PFAS modulated circulating pro and anti-inflammatory mediators including IFN-γ (downregulated 3.0-fold in females; p = 0.0301, 2.1-fold in males; p = 0.0418) and IL-6 (upregulated 5.6-fold in males; p = 0.030, no change in females). In conclusion, our data indicate long term exposure to a PFAS mixture impacts mechanisms related to expression of ACE2 in the lung. This work provides a mechanistic rationale for important future studies of PFAS exposure and subsequent viral infection.

Correction: Kassotis et al. Nonylphenol Polyethoxylates Enhance Adipose Deposition in Developmentally Exposed Zebrafish. Toxics 2022, 10, 99.

In the original publication [...].

On the Utility of ToxCast-Based Predictive Models to Evaluate Potential Metabolic Disruption by Environmental Chemicals.

BACKGROUND: Research suggests environmental contaminants can impact metabolic health; however, high costs prohibit in vivo screening of putative metabolic disruptors. High-throughput screening programs, such as ToxCast, hold promise to reduce testing gaps and prioritize higher-order (in vivo) testing. OBJECTIVES: We sought to a) examine the concordance of in vitro testing in 3T3-L1 cells to a targeted literature review for 38 semivolatile environmental chemicals, and b) assess the predictive utility of various expert models using ToxCast data against the set of 38 reference chemicals. METHODS: Using a set of 38 chemicals with previously published results in 3T3-L1 cells, we performed a metabolism-targeted literature review to determine consensus activity determinations. To assess ToxCast predictive utility, we used two published ToxPi models: a) the 8-Slice model published by Janesick et al. (2016) and b) the 5-Slice model published by Auerbach et al. (2016). We examined the performance of the two models against the Janesick in vitro results and our own 38-chemical reference set. We further evaluated the predictive performance of various modifications to these models using cytotoxicity filtering approaches and validated our best-performing model with new chemical testing in 3T3-L1 cells. RESULTS: The literature review revealed relevant publications for 30 out of the 38 chemicals (the remaining 8 chemicals were only examined in our previous 3T3-L1 testing). We observed a balanced accuracy (average of sensitivity and specificity) of 0.86 comparing our previous in vitro results to the literature-derived calls. ToxPi models provided balanced accuracies ranging from 0.55 to 0.88, depending on the model specifications and reference set. Validation chemical testing correctly predicted 29 of 30 chemicals as per 3T3-L1 testing, suggesting good adipogenic prediction performance for our best adapted model. DISCUSSION: Using the most recent ToxCast data and an updated ToxPi model, we found ToxCast performed similarly to that of our own 3T3-L1 testing in predicting consensus calls. Furthermore, we provide the full ranked list of largely untested chemicals with ToxPi scores that predict adipogenic activity and that require further investigation. https://doi.org/10.1289/EHP6779.

Obesity II: Establishing causal links between chemical exposures and obesity.

Obesity is a multifactorial disease with both genetic and environmental components. The prevailing view is that obesity results from an imbalance between energy intake and expenditure caused by overeating and insufficient exercise. We describe another environmental element that can alter the balance between energy intake and energy expenditure: obesogens. Obesogens are a subset of environmental chemicals that act as endocrine disruptors affecting metabolic endpoints. The obesogen hypothesis posits that exposure to endocrine disruptors and other chemicals can alter the development and function of the adipose tissue, liver, pancreas, gastrointestinal tract, and brain, thus changing the set point for control of metabolism. Obesogens can determine how much food is needed to maintain homeostasis and thereby increase the susceptibility to obesity. The most sensitive time for obesogen action is in utero and early childhood, in part via epigenetic programming that can be transmitted to future generations. This review explores the evidence supporting the obesogen hypothesis and highlights knowledge gaps that have prevented widespread acceptance as a contributor to the obesity pandemic. Critically, the obesogen hypothesis changes the narrative from curing obesity to preventing obesity.

Obesity III: Obesogen assays: Limitations, strengths, and new directions.

There is increasing evidence of a role for environmental contaminants in disrupting metabolic health in both humans and animals. Despite a growing need for well-understood models for evaluating adipogenic and potential obesogenic contaminants, there has been a reliance on decades-old in vitro models that have not been appropriately managed by cell line providers. There has been a quick rise in available in vitro models in the last ten years, including commercial availability of human mesenchymal stem cell and preadipocyte models; these models require more comprehensive validation but demonstrate real promise in improved translation to human metabolic health. There is also progress in developing three-dimensional and co-culture techniques that allow for the interrogation of a more physiologically relevant state. While diverse rodent models exist for evaluating putative obesogenic and/or adipogenic chemicals in a physiologically relevant context, increasing capabilities have been identified for alternative model organisms such as Drosophila, C. elegans, zebrafish, and medaka in metabolic health testing. These models have several appreciable advantages, including most notably their size, rapid development, large brood sizes, and ease of high-resolution lipid accumulation imaging throughout the organisms. They are anticipated to expand the capabilities of metabolic health research, particularly when coupled with emerging obesogen evaluation techniques as described herein.

Nonylphenol Polyethoxylates Enhance Adipose Deposition in Developmentally Exposed Zebrafish.

Alkylphenol polyethoxylates (APEOs), such as nonylphenol ethoxylates (NPEOs), are high-production-volume surfactants used in laundry detergents, hard-surface cleaners, pesticide formulations, textile production, oils, paints, and other products. NPEOs comprise -80% of the total production of APEOs and are widely reported across diverse environmental matrices. Despite a growing push for replacement products, APEOs continue to be released into the environment through wastewater at significant levels. Research into related nonionic surfactants from varying sources has reported metabolic health impacts, and we have previously demonstrated that diverse APEOs and alcohol polyethoxylates promote adipogenesis in the murine 3T3-L1 pre-adipocyte model. These effects appeared to be independent of the base alkylphenol and related to the ethoxylate chain length, though limited research has evaluated NPEO exposures in animal models. The goals of this study were to assess the potential of NPEOs to promote adiposity (Nile red fluorescence quantification) and alter growth and/or development (toxicity, length, weight, and energy expenditure) of developmentally exposed zebrafish (Danio rerio). We also sought to expand our understanding of the ability to promote adiposity through evaluation in human mesenchymal stem cells. Herein, we demonstrated consistent adipogenic effects in two separate human bone-marrow-derived mesenchymal stem cell models, and that nonylphenol and its ethoxylates promoted weight gain and increased adipose deposition in developmentally exposed zebrafish. Notably, across both cell and zebrafish models we report increasing adipogenic/obesogenic activity with increasing ethoxylate chain lengths up to maximums around NPEO-6 and then decreasing activity with the longest ethoxylate chain lengths. This research suggests metabolic health concerns for these common obesogens, suggesting further need to assess molecular mechanisms and better characterize environmental concentrations for human health risk assessments.

Characterization of Per- and Polyfluorinated Alkyl Substances Present in Commercial Anti-fog Products and Their In Vitro Adipogenic Activity.

Anti-fog sprays and solutions are used on eyeglasses to minimize the condensation of water vapor, particularly while wearing a mask. Given their water-repellent properties, we sought to characterize per- and polyfluorinated alkyl substance (PFAS) compounds in four anti-fog spray products, five anti-fog cloth products, and two commercial fluorosurfactant formulations suspected to be used in preparing anti-fog products. Fluorotelomer alcohols (FTOHs) and fluorotelomer ethoxylates (FTEOs) were detected in all products and formulations. While 6:2 FTOH and the 6:2 FTEO polymeric series were predominant, one anti-fog cloth and one formulation contained 8:2, 10:2, 12:2, 14:2, and 16:2 FTOH and FTEO polymeric series. PFAS concentrations varied in samples and were detected at levels up to 25,000 µg/mL in anti-fog sprays and 185,000 µg (g cloth)-1 in anti-fog cloth products. The total organic fluorine (TOF) measurements of anti-fog products ranged from 190 to 20,700 µg/mL in sprays and 44,200 to 131,500 µg (g cloth)-1 in cloths. Quantified FTOHs and FTEOs accounted for 1-99% of TOF mass. In addition, all four anti-fog sprays and both commercial formulations exhibited significant cytotoxicity and adipogenic activity (either triglyceride accumulation and/or pre-adipocyte proliferation) in murine 3T3-L1 cells. Results suggest that FTEOs are a significant contributor to the adipogenic activity exhibited by the anti-fog sprays. Altogether, these results suggest that FTEOs are present in commercial products at toxicologically relevant levels, and more research is needed to fully understand the health risks from using these PFAS-containing products.

Endocrine disruptor global policy.

Over the past several decades, scientific consensus has grown around the concept and evidence for human health impacts from exposure to endocrine disrupting chemicals (EDCs). A series of publications have now demonstrated considerable economic costs of EDC exposure-induced adverse health outcomes. This research has suggested economic burdens in the hundreds of billions, even considering only a small subset of EDCs and health. As of yet, regulatory efforts and policies to protect and decrease human exposure to most EDCs have been insufficient and have not kept pace with the science. Given the overwhelming scientific evidence, referenced throughout this collection, as well as the economic costs of inaction, described here, regulations are clearly needed. The EU and some other countries have taken promising steps towards protective regulation of EDCs, though the response of the US and many other countries has been limited or altogether lacking. Regulatory bodies that have and continue to apply risk-based approaches to regulating EDCs have also failed to consider the complete economic impacts of EDC-related health impacts. In this chapter, we will discuss broad strategies taken to regulate EDCs, examine the approaches currently taken to regulate EDCs in a global context (discussing the strengths and weaknesses of these regulations), discuss the economic costs of EDC exposures (detailing where consideration of health and economic costs could improve regulations), and discuss next steps and novel approaches to adapting existing regulatory frameworks to this class of chemicals.

Reproducibility of adipogenic responses to metabolism disrupting chemicals in the 3T3-L1 pre-adipocyte model system: An interlaboratory study.

The 3T3-L1 murine pre-adipocyte line is an established cell culture model for screening Metabolism Disrupting Chemicals (MDCs). Despite a need to accurately identify MDCs for further evaluation, relatively little research has been performed to comprehensively evaluate reproducibility across laboratories, assess factors that might contribute to varying degrees of differentiation between laboratories (media additives, plastics, cell source, etc.), or to standardize protocols. As such, the goals of this study were to assess interlaboratory variability of efficacy and potency outcomes for triglyceride accumulation and pre-adipocyte proliferation using the mouse 3T3-L1 pre-adipocyte cell assay to test chemicals. Ten laboratories from five different countries participated. Each laboratory evaluated one reference chemical (rosiglitazone) and three blinded test chemicals (tributyltin chloride, pyraclostrobin, and bisphenol A) using: 1) their Laboratory-specific 3T3-L1 Cells (LC) and their Laboratory-specific differentiation Protocol (LP), 2) Shared 3T3-L1 Cells (SC) with LP, 3) LC with a Shared differentiation Protocol (SP), and 4) SC with SP. Blinded test chemical responses were analyzed by the coordinating laboratory. The magnitude and range of bioactivities reported varied considerably across laboratories and test conditions, though the presence or absence of activity for each tested chemical was more consistent. Triglyceride accumulation activity determinations for rosiglitazone ranged from 90 to 100% across test conditions, but 30-70 % for pre-adipocyte proliferation; this was 40-80 % for triglyceride accumulation induced by pyraclostrobin, 80-100 % for tributyltin, and 80-100 % for bisphenol A. Consistency was much lower for pre-adipocyte proliferation, with 30-70 % active determinations for pyraclostrobin, 30-50 % for tributyltin, and 20-40 % for bisphenol A. Greater consistency was observed for the SC/SP assessment. As such, working to develop a standardized adipogenic differentiation protocol represents the best strategy for improving consistency of adipogenic responses using the 3T3-L1 model to reproducibly identify MDCs and increase confidence in reported outcomes.