Investigating the applicability domain of the hiPSC-based PluriLum assay: an embryotoxicity assessment of chemicals and drugs.

To meet the growing demand for developmental toxicity assessment of chemicals, New Approach Methodologies (NAMs) are needed. Previously, we developed two 3D in vitro assays based on human-induced pluripotent stem cells (hiPSC) and cardiomyocyte differentiation: the PluriBeat assay, based on assessment of beating differentiated embryoid bodies, and the PluriLum assay, a reporter gene assay based on the expression of the early cardiac marker NKX2.5; both promising assays for predicting embryotoxic effects of chemicals and drugs. In this work, we aimed to further describe the predictive power of the PluriLum assay and compare its sensitivity with PluriBeat and similar human stem cell-based assays developed by others. For this purpose, we assessed the toxicity of a panel of ten chemicals from different chemical classes, consisting of the known developmental toxicants 5-fluorouracil, all-trans retinoic acid and valproic acid, as well as the negative control compounds ascorbic acid and folic acid. In addition, the fungicides epoxiconazole and prochloraz, and three perfluoroalkyl substances (PFAS), PFOS, PFOA and GenX were tested. Generally, the PluriLum assay displayed higher sensitivity when compared to the PluriBeat assay. For several compounds the luminescence readout of the PluriLum assay showed effects not detected by the PluriBeat assay, including two PFAS compounds and the two fungicides. Overall, we find that the PluriLum assay has the potential to provide a fast and objective detection of developmental toxicants and has a level of sensitivity that is comparable to or higher than other in vitro assays also based on human stem cells and cardiomyocyte differentiation for assessment of developmental toxicity.

An Exemestane Derivative, Oxymestane-D1, as a New Multi-Target Steroidal Aromatase Inhibitor for Estrogen Receptor-Positive (ER+) Breast Cancer: Effects on Sensitive and Resistant Cell Lines.

Around 70-85% of all breast cancer (BC) cases are estrogen receptor-positive (ER+). The third generation of aromatase inhibitors (AIs) is the first-line treatment option for these tumors. Despite their therapeutic success, they induce several side effects and resistance, which limits their efficacy. Thus, it is crucial to search for novel, safe and more effective anti-cancer molecules. Currently, multi-target drugs are emerging, as they present higher efficacy and lower toxicity in comparison to standard options. Considering this, this work aimed to investigate the anti-cancer properties and the multi-target potential of the compound 1α,2α-epoxy-6-methylenandrost-4-ene-3,17-dione (Oxy), also designated by Oxymestane-D1, a derivative of Exemestane, which we previously synthesized and demonstrated to be a potent AI. For this purpose, it was studied for its effects on the ER+ BC cell line that overexpresses aromatase, MCF-7aro cells, as well as on the AIs-resistant BC cell line, LTEDaro cells. Oxy reduces cell viability, impairs DNA synthesis and induces apoptosis in MCF-7aro cells. Moreover, its growth-inhibitory properties are inhibited in the presence of ERα, ERß and AR antagonists, suggesting a mechanism of action dependent on these receptors. In fact, Oxy decreased ERα expression and activation and induced AR overexpression with a pro-death effect. Complementary transactivation assays demonstrated that Oxy presents ER antagonist and AR agonist activities. In addition, Oxy also decreased the viability and caused apoptosis of LTEDaro cells. Therefore, this work highlights the discovery of a new and promising multi-target drug that, besides acting as an AI, appears to also act as an ERα antagonist and AR agonist. Thus, the multi-target action of Oxy may be a therapeutic advantage over the three AIs applied in clinic. Furthermore, this new multi-target compound has the ability to sensitize the AI-resistant BC cells, which represents another advantage over the endocrine therapy used in the clinic, since resistance is a major drawback in the clinic.

Influence on the adult male Leydig cell biomarker insulin-like peptide 3 of maternal exposure to estrogenic and anti-androgenic endocrine disrupting compound mixtures: A retrospective study.

Insulin-like peptide 3 (INSL3) is a peptide biomarker secreted specifically by the mature Leydig cells of the testes. It is constitutive, has low within-individual variance, and effectively measures the functional capacity of Leydig cells to make testosterone. In young adult men there is a large 10-fold range of serum INSL3 concentration, persisting into old age, and implying that later hypogonadal status might be programmed in early life. To determine whether maternal exposure to environmental endocrine disrupting compounds (EDCs) influences adult serum INSL3 concentration, using a retrospective paradigm, INSL3 was measured in young adult male rats (80-90 days) from the F1 generation of females maternally exposed to varied doses of bisphenol A (BPA), butylparaben, epoxiconazole, and fludioxonil as single compounds, as well as estrogenic and anti-androgenic mixtures of BPA and butylparaben, and di(2-ethylhexyl) phthalate and procymidone respectively. A mixture of BPA and butylparaben significantly reduced circulating INSL3 concentration in adult male progeny. The remaining compounds or mixtures tested, though sufficient to induce other effects in the F1 generation were without significant effect. Maternal exposure to low concentrations of some EDCs may be a contributing factor to the variation in the Leydig cell biomarker INSL3 in young adulthood, though caution is warranted translating results from rats to humans.

Creating a human-induced pluripotent stem cell-based NKX2.5 reporter gene assay for developmental toxicity testing.

To test large numbers of chemicals for developmental toxicity, rapid in vitro tests with standardized readouts for automated data acquisition are needed. However, the most widely used assay, the embryonic stem cell test, relies on the counting of beating embryoid bodies by visual inspection, which is laborious and time consuming. We previously developed the PluriBeat assay based on differentiation of human induced pluripotent stem cells (hiPSC) that we demonstrated to be predictive for known teratogens at relevant concentrations using the readout of beating cardiomyocytes. Here, we report the development of a novel assay, which we term the PluriLum assay, where we have introduced a luciferase reporter gene into the locus of NKX2.5 of our hiPSC line. This enabled us to measure luminescence intensities instead of counting beating cardiomyocytes, which is less labor intensive. We established two NKX2.5 reporter cell lines and validated their pluripotency and genetic stability. Moreover, we confirmed that the genetically engineered NKX2.5 reporter cell line differentiated into cardiomyocytes with the same efficiency as the original wild-type line. We then exposed the cells to valproic acid (25-300 µM) and thalidomide (0.1-36 µM) and compared the PluriBeat readout of the cardiomyocytes with the luminescence intensity of the PluriLum assay. The results showed that thalidomide decreased luminescence intensity significantly with a higher potency and efficacy compared to the beating readout. With this, we have developed a novel hiPSC-based assay with a standardized readout that may have the potential for higher throughput screening for developmental toxicity.

Calretinin is a novel candidate marker for adverse ovarian effects of early life exposure to mixtures of endocrine disruptors in the rat.

Disruption of sensitive stages of ovary development during fetal and perinatal life can have severe and life-long consequences for a woman's reproductive life. Exposure to endocrine disrupting chemicals may affect ovarian development, leading to subsequent reproductive disorders. Here, we investigated the effect of early life exposure to defined mixtures of human-relevant endocrine disrupting chemicals on the rat ovary. We aimed to identify molecular events involved in pathogenesis of ovarian dysgenesis syndrome that have potential for future adverse outcome pathway development. We therefore focused on the ovarian proteome. Rats were exposed to a mixture of phthalates, pesticides, UV-filters, bisphenol A, butyl-paraben, and paracetamol during gestation and lactation. The chemicals were tested together or in subgroups of chemicals with anti-androgenic or estrogenic potentials at doses 450-times human exposure. Paracetamol was tested separately, at a dose of 360 mg/kg. Using shotgun proteomics on ovaries from pup day 17 offspring, we observed exposure effects on the proteomes. Nine proteins were affected in more than one exposure group and of these, we conclude that calretinin is a potential key event biomarker of early endocrine disruption in the ovary.

A novel human pluripotent stem cell-based assay to predict developmental toxicity.

There is a great need for novel in vitro methods to predict human developmental toxicity to comply with the 3R principles and to improve human safety. Human-induced pluripotent stem cells (hiPSC) are ideal for the development of such methods, because they are easy to retrieve by conversion of adult somatic cells and can differentiate into most cell types of the body. Advanced three-dimensional (3D) cultures of these cells, so-called embryoid bodies (EBs), moreover mimic the early developing embryo. We took advantage of this to develop a novel human toxicity assay to predict chemically induced developmental toxicity, which we termed the PluriBeat assay. We employed three different hiPSC lines from male and female donors and a robust microtiter plate-based method to produce EBs. We differentiated the cells into cardiomyocytes and introduced a scoring system for a quantitative readout of the assay-cardiomyocyte contractions in the EBs observed on day 7. Finally, we tested the three compounds thalidomide (2.3-36 µM), valproic acid (25-300 µM), and epoxiconazole (1.3-20 µM) on beating and size of the EBs. We were able to detect the human-specific teratogenicity of thalidomide and found the rodent toxicant epoxiconazole as more potent than thalidomide in our assay. We conclude that the PluriBeat assay is a novel method for predicting chemicals' adverse effects on embryonic development.

Anogenital distance as a toxicological or clinical marker for fetal androgen action and risk for reproductive disorders.

Male reproductive development is intricately dependent on fetal androgen action. Consequently, disrupted androgen action during fetal life can interfere with the development of the reproductive system resulting in adverse effects on reproductive function later in life. One biomarker used to evaluate fetal androgen action is the anogenital distance (AGD), the distance between the anus and the external genitalia. A short male AGD is strongly associated with genital malformations at birth and reproductive disorders in adulthood. AGD is therefore used as an effect readout in rodent toxicity studies aimed at testing compounds for endocrine activity and anti-androgenic properties, and in human epidemiological studies to correlate fetal exposure to endocrine disrupting chemicals to feminization of new-born boys. In this review, we have synthesized current data related to intrauterine exposure to xenobiotics and AGD measurements. We discuss the utility of AGD as a retrospective marker of in utero anti-androgenicity and as a predictive marker for male reproductive disorders, both with respect to human health and rodent toxicity studies. Finally, we highlight four areas that need addressing to fully evaluate AGD as a biomarker in both a regulatory and clinical setting.

Enniatin B and beauvericin are common in Danish cereals and show high hepatotoxicity on a high-content imaging platform.

Mycotoxins are fungi-born metabolites that can contaminate foods through mould-infected crops. They are a significant food/feed-safety issue across the globe and represent a substantial financial burden for the world economy. Moreover, with a changing climate and fungal biota, there is now much discussion about emerging mycotoxins that are measurable at significant levels in crops world-wide. Unfortunately, we still know very little about the bioavailability and toxic potentials of many of these less characterized mycotoxins, including the large family of enniatins. In this study, we present new occurrence data for enniatin A, A1, B, B1 and beauvericin in four Danish crops: oat, wheat, and barley from the 2010 harvest, and rye from 2011 harvest. The occurrence of the four enniatins were B > B1 > A1 > A. Enniatin B was detected in 100% of tested samples regardless of crop type. In addition to occurrence data, we report a proof-of-concept study using a human-relevant high-content hepatotoxicity, or "quadroprobe," assay to screen mycotoxins for their cytotoxic potential. The assay was sensitive for most cytotoxic compounds in the 0.009-100 µM range. Among eight tested mycotoxins (enniatin B, beauvericin, altenariol, deoxynivalenol, aflatoxin B1, andrastin A, citrinin, and penicillic acid), enniatin B and beauvericin showed significant cytotoxicity at a concentration lower than that for aflatoxin B1, which is the archetypal acute hepatotoxic and liver-carcinogenic mycotoxin. Hence, the quadroprobe hepatotoxicity assay may become a valuable assessment tool for toxicity assessment of mycotoxins in the future. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1658-1664, 2017.

Perfluorononanoic acid in combination with 14 chemicals exerts low-dose mixture effects in rats.

Humans are simultaneously exposed to several chemicals that act jointly to induce mixture effects. At doses close to or higher than no-observed adverse effect levels, chemicals usually act additively in experimental studies. However, we are lacking knowledge on the importance of exposure to complex real-world mixtures at more relevant human exposure levels. We hypothesised that adverse mixture effects occur at doses approaching high-end human exposure levels. A mixture (Mix) of 14 chemicals at a combined dose of 2.5 mg/kg bw/day was tested in combination with perfluorononanoic acid (PFNA) at doses of 0.0125 (Low PFNA), 0.25 (Mid PFNA) and 5 (High PFNA) mg/kg bw/day by oral administration for 14 days in juvenile male rats. Indication of a toxicokinetic interaction was found, as simultaneous exposure to PFNA and the Mix caused a 2.8-fold increase in plasma PFNA concentrations at Low PFNA. An increase in testosterone and dihydrotestosterone plasma concentrations was observed for Low PFNA + Mix. This effect was considered non-monotonic, as higher doses did not cause this effect. Reduced LH plasma concentrations together with increased androgen concentrations indicate a disturbed pituitary-testis axis caused by the 15-chemical mixture. Low PFNA by itself increased the corticosterone plasma concentration, an effect which was normalised after simultaneous exposure to Mix. This combined with affected ACTH plasma concentrations and down-regulation of 11ß HSD mRNA in livers indicates a disturbed pituitary-adrenal axis. In conclusion, our data suggest that mixtures of environmental chemicals at doses approaching high-end human exposure levels can cause a hormonal imbalance and disturb steroid hormones and their regulation. These effects may be non-monotonic and were observed at low doses. Whether this reflects a more general phenomenon that should be taken into consideration when predicting human mixture effects or represents a rarer phenomenon remains to be shown.

Perinatal exposure to mixtures of anti-androgenic chemicals causes proliferative lesions in rat prostate.

BACKGROUND: Elevated levels of endogenous or exogenous estrogens during fetal life can induce permanent disturbances in prostate growth and predispose to precancerous lesions. Recent studies have indicated that also early anti-androgen exposure may affect prostate cancer risk. METHODS: We examined the influence of perinatal exposure to mixtures of anti-androgenic and estrogenic chemicals on prostate development. Wistar rats were exposed from gestation day 7 to postnatal day 22 to a mixture of 8 anti-androgenic compounds (AAMix), a mixture of four estrogenic compounds (EMix), or paracetamol or a mixture of all 13 compounds (TotalMix) in mixture ratios reflecting human exposure levels. RESULTS: Ventral prostate weights were reduced by the TotalMix and AAMix in pre-pubertal rats. Histological changes in prostate appeared with increasing age and indicated a shift from the normal age-dependent epithelial atrophy towards hyperplasia. These lesions showed similarities to pre-cancerous lesions in humans. Increased proliferation was observed already in pre-puberty and it was hypothesized that this could be associated with reduced ERß signaling, but no clear conclusions could be made from gene expression studies on ERß-related pathways. The influences of the estrogenic chemicals and paracetamol on prostate morphology were minor, but in young adulthood the estrogen mixture reduced ventral prostate mRNA levels of Igf1 and paracetamol reduced the mRNA level ofPbpc3. CONCLUSIONS: Mixtures of endocrine disrupters relevant for human exposure was found to elicit persistent effects on the rat prostate following perinatal exposure, suggesting that human perinatal exposure to environmental chemicals may increase the risk of prostate cancer later in life.

Low-dose effects of bisphenol A on early sexual development in male and female rats.

Bisphenol A (BPA) is widely detected in human urine and blood. BPA has been reported to impair many endpoints for reproductive and neurological development; however, it is controversial whether BPA has effects in the microgram per kilogram dose range. The aim of the current study was to examine the influence of BPA on early sexual development in male and female rats at dose levels covering both regulatory no observed adverse effect levels (NOAELs) (5 and 50 mg/kg bw per day) as well as doses in the microgram per kilogram dose range (0.025 and 0.25 mg/kg bw per day). Time-mated Wistar rats (n=22) were gavaged during pregnancy and lactation from gestation day 7 to pup day 22 with 0, 0.025, 0.25, 5 or 50 mg/kg bw per day BPA. From 0.250 mg/kg and above, male anogenital distance (AGD) was significantly decreased, whereas decreased female AGD was seen from 0.025 mg/kg bw per day and above. Moreover, the incidence of nipple retention in males appeared to increase dose relatedly and the increase was statistically significant at 50 mg/kg per day. No significant changes in reproductive organ weights in the 16-day-old males and females and no signs of maternal toxicity were seen. The decreased AGD at birth in both sexes indicates effects on prenatal sexual development and provides new evidence of low-dose adverse effects of BPA in rats in the microgram per kilogram dose range. The NOAEL in this study is clearly below 5 mg/kg for BPA, which is used as the basis for establishment of the current tolerable daily intake (TDI) by EFSA; thus a reconsideration of the current TDI of BPA appears warranted.

Late-life effects on rat reproductive system after developmental exposure to mixtures of endocrine disrupters.

This study examined late-life effects of perinatal exposure of rats to a mixture of endocrine-disrupting contaminants. Four groups of 14 time-mated Wistar rats were exposed by gavage from gestation day 7 to pup day 22 to a mixture of 13 anti-androgenic and estrogenic chemicals including phthalates, pesticides, u.v.-filters, bisphenol A, parabens, and the drug paracetamol. The groups received vehicle (control), a mixture of all 13 chemicals at 150-times (TotalMix150) or 450-times (TotalMix450) high-end human exposure, or 450-times a mixture of nine predominantly anti-androgenic chemicals (AAMix450). Onset of puberty and estrous cyclicity at 9 and 12 months of age were assessed. Few female offspring showed significantly regular estrus cyclicity at 12 months of age in the TotalMix450 and AAMix450 groups compared with controls. In 19-month-old male offspring, epididymal sperm counts were lower than controls, and in ventral prostate an overrepresentation of findings related to hyperplasia was observed in exposed groups compared with controls, particularly in the group dosed with anti-androgens. A higher incidence of pituitary adenoma at 19 months of age was found in males and females in the AAMix450 group. Developmental exposure of rats to the highest dose of a human-relevant mixture of endocrine disrupters induced adverse effects late in life, manifested as earlier female reproductive senescence, reduced sperm counts, higher score for prostate atypical hyperplasia, and higher incidence of pituitary tumors. These delayed effects highlight the need for further studies on the role of endocrine disrupters in hormone-related disorders in aging humans.

In vitro antiandrogenic effects of the herbicide linuron and its metabolites.

Although the herbicide linuron is banned for use in the EU due to its reproductive and developmental toxicity, it can still be found in randomly sampled foods grown in and outside the EU. It is not clear if metabolites of linuron can contribute to the endocrine disrupting effects following exposure to the parent compound. To address this gap, we analysed linuron and the metabolites 1-(3,4-dichlorophenyl) urea (DCU), 3,4-dichloroaniline (DCA) and 1-(3,4-dichlorophenyl)-3-methoxyurea (DCXU) for androgen receptor (AR) activities and effects on steroidogenesis. Generally, linuron and the metabolites showed qualitatively similar antiandrogenic profiles, but potencies varied. All compounds were AR antagonists, with linuron showing highest potency (IC50 of 2.8 µM). The overall picture of effects on steroidogenesis showed that linuron and metabolites increased the levels of estrogens and corticosteroids, whereas the synthesis of androgens was inhibited. The metabolite DCU was by far the most potent inhibitor of testosterone synthesis (IC50 of 6.7 µM compared to IC50 of 51.1 µM for linuron). We suggest that it is likely that the metabolites contribute to the antiandrogenic effects of linuron in vivo, especially by inhibiting testosterone synthesis.

In vitro-in vivo correlations for endocrine activity of a mixture of currently used pesticides.

Two pesticide mixtures were investigated for potential endocrine activity. Mix 3 consisted of bitertanol, propiconazole, and cypermethrin, and Mix 5 included malathion and terbuthylazine in addition to the three pesticides in Mix 3. All five single pesticides and the two mixtures were investigated for their ability to affect steroidogenesis in vitro in H295R cells. The pesticides alone and both mixtures affected steroidogenesis with both mixtures causing increase in progesterone and decrease in testosterone. For Mix 5 an increase in estradiol was seen as well, indicating increased aromatase activity. The two mixtures were also investigated in pregnant rats dosed from gestational day 7 to 21, followed by examination of dams and fetuses. Decreased estradiol and reduced placental testosterone were seen in dams exposed to Mix 5. Also a significant increase in aromatase mRNA-levels in female adrenal glands was found for Mix5. However, either of the two mixtures showed any effects on fetal hormone levels in plasma or testis, or on anogenital distance. Overall, potential aromatase induction was found for Mix 5 both in vitro and in vivo, but not for Mix 3, an effect likely owed to terbuthylazine in Mix 5. However, the hormonal responses in vitro were only partly reflected in vivo, probably due to some toxicokinetic issues, as the pesticide levels in the amniotic fluid also were found to be negatively affected by the number of compounds present in the mixtures. Nonetheless, the H295R assay gives hints on conceivable interference with steroidogenesis, thus generating hypotheses on in vivo effects.

Science and policy on endocrine disrupters must not be mixed: a reply to a "common sense" intervention by toxicology journal editors.

The "common sense" intervention by toxicology journal editors regarding proposed European Union endocrine disrupter regulations ignores scientific evidence and well-established principles of chemical risk assessment. In this commentary, endocrine disrupter experts express their concerns about a recently published, and is in our considered opinion inaccurate and factually incorrect, editorial that has appeared in several journals in toxicology. Some of the shortcomings of the editorial are discussed in detail. We call for a better founded scientific debate which may help to overcome a polarisation of views detrimental to reaching a consensus about scientific foundations for endocrine disrupter regulation in the EU.

Cannabidiol as a Promising Adjuvant Therapy for Estrogen Receptor-Positive Breast Tumors: Unveiling Its Benefits with Aromatase Inhibitors.

BACKGROUND: Estrogen receptor-positive (ER+) breast cancer is the most diagnosed subtype, with aromatase inhibitors (AIs) being one of the therapeutic drug types used in the clinic. However, endocrine resistance may develop after prolonged treatment, and different approaches, such as combining endocrine and targeted therapies, have been applied. Recently, we demonstrated that cannabidiol (CBD) induces anti-tumor actions in ER+ breast cancer cells by targeting aromatase and ERs. Considering this, we studied, in vitro, whether CBD when combined with AIs could improve their effectiveness. METHODS: MCF-7aro cells were used and the effects on cell viability and on the modulation of specific targets were investigated. RESULTS: CBD when combined with anastrozole (Ana) and letrozole (Let) caused no beneficial effect in comparison to the isolated AIs. In contrast, when combined with the AI exemestane (Exe), CBD potentiated its pro-cell death effects, abolished its estrogen-like effect, impaired ERα activation, and prevented its oncogenic role on the androgen receptor (AR). Moreover, this combination inhibited ERK1/2 activation, promoting apoptosis. The study of the hormonal microenvironment suggests that this combination should not be applied in early stages of ER+ breast tumors. CONCLUSIONS: Contrary to Ana and Let, this study highlights the potential benefits of combining CBD with Exe to improve breast cancer treatment and opens up the possibility of new therapeutic approaches comprising the use of cannabinoids.

Human risk associated with exposure to mixtures of antiandrogenic chemicals evaluated using in vitro hazard and human biomonitoring data.

BACKGROUND: Scientific evidence for underestimated toxicity from unintentional exposure to chemical mixtures is mounting. Yet, harmonized approaches on how to assess the actual risk of mixtures is lacking. As part of the European Joint programme 'Human Biomonitoring for Europe' we explored a novel methodology for mixture risk assessment of chemicals affecting male reproductive function. METHODOLOGY: We explored a methodology for chemical mixture risk assessment based on human in vitro data combined with human exposure data, thereby circumventing the drawbacks of using hazard data from rodents and estimated exposure intake levels. Human androgen receptor (hAR) antagonism was selected as the most important molecular initiating event linked to adverse outcomes on male reproductive health. RESULTS: Our work identified 231 chemicals able to interfere with hAR activity. Among these were 61 finally identified as having both reliable hAR antagonist and human biomonitoring data. Calculation of risk quotients indicated that PCBs (118, 138, 157), phthalates (BBP, DBP, DIBP), benzophenone-3, PFOS, methylparaben, triclosan, some pesticides (i.e cypermethrin, ß-endosulfan, methylparathion, p,p-DDE), and a PAH metabolite (1-hydroxypyrene) contributed to the mixture effect. The major chemical mixture drivers were PCB 118, BBP, PFOS, DBP, and the UV filter benzophenone-3, together contributing with 75% of the total mixture effect that was primarily driven by high exposure values. CONCLUSIONS: This viable way forward for mixture risk assessment of chemicals has the advantages of (1) being a more comprehensive mixture risk assessment also covering data-poor chemicals, and (2) including human data only. However, the approach is subjected to uncertainties in terms of in vitro to in vivo extrapolation, it is not ready for decision making, and needs further development. Still, the results indicate a concern for adverse effects on reproductive function in highly exposed boys, especially when considering additional exposure to data-poor chemicals and chemicals acting by other mechanisms of action.

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.

How to use human biomonitoring in chemical risk assessment: Methodological aspects, recommendations, and lessons learned from HBM4EU.

One of the aims of the European Human Biomonitoring Initiative, HBM4EU, was to provide examples of and good practices for the effective use of human biomonitoring (HBM) data in human health risk assessment (RA). The need for such information is pressing, as previous research has indicated that regulatory risk assessors generally lack knowledge and experience of the use of HBM data in RA. By recognising this gap in expertise, as well as the added value of incorporating HBM data into RA, this paper aims to support the integration of HBM into regulatory RA. Based on the work of the HBM4EU, we provide examples of different approaches to including HBM in RA and in estimations of the environmental burden of disease (EBoD), the benefits and pitfalls involved, information on the important methodological aspects to consider, and recommendations on how to overcome obstacles. The examples are derived from RAs or EBoD estimations made under the HBM4EU for the following HBM4EU priority substances: acrylamide, o-toluidine of the aniline family, aprotic solvents, arsenic, bisphenols, cadmium, diisocyanates, flame retardants, hexavalent chromium [Cr(VI)], lead, mercury, mixture of per-/poly-fluorinated compounds, mixture of pesticides, mixture of phthalates, mycotoxins, polycyclic aromatic hydrocarbons (PAHs), and the UV-filter benzophenone-3. Although the RA and EBoD work presented here is not intended to have direct regulatory implications, the results can be useful for raising awareness of possibly needed policy actions, as newly generated HBM data from HBM4EU on the current exposure of the EU population has been used in many RAs and EBoD estimations.

Developmental exposure to the brominated flame retardant DE-71 reduces serum thyroid hormones in rats without hypothalamic-pituitary-thyroid axis activation or neurobehavioral changes in offspring.

Polybrominated diphenyl ethers (PBDEs) are legacy flame retardants for which human exposure remains ubiquitous. This is of concern since these chemicals can perturb development and cause adverse health effects. For instance, DE-71, a technical mixture of PBDEs, can induce liver toxicity as well as reproductive and developmental toxicity. DE-71 can also disrupt the thyroid hormone (TH) system which may induce developmental neurotoxicity indirectly. However, in developmental toxicity studies, it remains unclear how DE-71 exposure affects the offspring's thyroid hormone system and if this dose-dependently relates to neurodevelopmental effects. To address this, we performed a rat toxicity study by exposing pregnant dams to DE-71 at 0, 40 or 60 mg/kg/day during perinatal development from gestational day 7 to postnatal day 16. We assessed the TH system in both dams and their offspring, as well as potential hearing and neurodevelopmental effects in prepubertal and adult offspring. DE-71 significantly reduced serum T4 and T3 levels in both dams and offspring without a concomitant upregulation of TSH, thus inducing a hypothyroxinemia-like effect. No discernible effects were observed on the offspring's brain function when assessed in motor activity boxes and in the Morris water maze, or on offspring hearing function. Our results, together with a thorough review of the literature, suggest that DE-71 does not elicit a clear dose-dependent relationship between low serum thyroxine (T4) and effects on the rat brain in standard behavioral assays. However, low serum TH levels are in themselves believed to be detrimental to human brain development, thus we propose that we lack assays to identify developmental neurotoxicity caused by chemicals disrupting the TH system through various mechanisms.