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.

Exposure to the pesticides linuron, dimethomorph and imazalil alters steroid hormone profiles and gene expression in developing rat ovaries.

Inhibition of androgen signaling during critical stages of ovary development can disrupt folliculogenesis with potential consequences for reproductive function later in life. Many environmental chemicals can inhibit the androgen signaling pathway, which raises the question if developmental exposure to anti-androgenic chemicals can negatively impact female fertility. Here, we report on altered reproductive hormone profiles in prepubertal female rats following developmental exposure to three pesticides with anti-androgenic potential: linuron (25 and 50 mg/kg bw/d), dimethomorph (60 and 180 mg/kg bw/d) and imazalil (8 and 24 mg/kg bw/d). Dams were orally exposed from gestational day 7 (dimethomorph and imazalil) or 13 (linuron) until birth, then until end of dosing at early postnatal life. Linuron and dimethomorph induced dose-related reductions to plasma corticosterone levels, whereas imazalil mainly suppressed gonadotropin levels. In the ovaries, expression levels of target genes were affected by linuron and dimethomorph, suggesting impaired follicle growth. Based on our results, we propose that anti-androgenic chemicals can negatively impact female reproductive development. This highlights a need to integrate data from all levels of the hypothalamic-pituitary-gonadal axis, as well as the hypothalamic-pituitary-adrenal axis, when investigating the potential impact of endocrine disruptors on female reproductive development and function.

The anti-androgenic fungicide triticonazole induces region-specific transcriptional changes in the developing rat perineum and phallus.

Intrauterine exposure to endocrine disrupting chemicals can interfere with male reproductive development. This can lead to male reproductive disorders such as hypospadias, cryptorchidism and reduced fertility, as well as shorter anogenital distance (AGD) - a biomarker for incomplete androgen-dependent fetal masculinization. However, it remains challenging to predict adverse in vivo outcomes based on in vitro effect patterns for many chemicals. This is a challenge for modern toxicology, which aims to reduce animal testing for chemical safety assessments. To enable the transition towards higher reliance on alternative test methods, we need to better map underlying mechanisms leading to adverse effects. Herein, we have analyzed the transcriptome of the perineum and phallus of male fetal rats and defined the impacts of exposure to an anti-androgenic fungicide, triticonazole. Previously we have shown that developmental exposure to triticonazole can induce short male AGD, but without a marked effect on the transcriptome of the fetal testes. In contrast, we report here significant changes to the transcriptional landscape of the perineum and phallus, including regional differences between these adjacent tissues. This highlights the importance of analyzing the correct tissue when characterizing mechanisms of complex in vivo effect outcomes. Our results provide a rich resource for the spatiotemporal gene networks that are involved in the development of male external genitalia, and that can be disrupted upon exposure to chemicals that prevent normal masculinization of the perineum and phallus. Such data will be critical in the development of novel alternative test methods to determine the endocrine disrupting potential of existing and emerging chemicals.

AOP key event relationship report: Linking decreased androgen receptor activation with decreased granulosa cell proliferation of gonadotropin-independent follicles.

We recently proposed to formally recognize Key Event Relationships (KERs) as building blocks of Adverse Outcome Pathways (AOPs) that can be independently developed and peer-reviewed. Here, we follow this approach and provide an independent KER from AOP345, which describes androgen receptor (AR) antagonism leading to decreased female fertility. This KER connects AR antagonism to reduced granulosa cell proliferation of gonadotropin-independent follicles (KER2273). We have developed both the KER and the two adjacent Key Events (KEs). A systematic approach was used to ensure that all relevant supporting evidence for KER2273 was retrieved. Supporting evidence for the KER highlights the importance of AR action during the early stages of follicular development. Both biological plausibility and empirical evidence are presented, with the latter also assessed for quality. We believe that tackling isolated KERs instead of whole AOPs will accelerate the AOP development. Faster AOP development will lead to the development of simple test methods that will aid screening of chemicals, endocrine disruptor identification, risk assessment, and subsequent regulation.

Azole Fungicides and Their Endocrine Disrupting Properties: Perspectives on Sex Hormone-Dependent Reproductive Development.

Azoles are antifungal agents used in both agriculture and medicine. They typically target the CYP51 enzyme in fungi and, by so doing, disrupt cell membrane integrity. However, azoles can also target various CYP enzymes in mammals, including humans, which can disrupt hormone synthesis and signaling. For instance, several azoles can inhibit enzymes of the steroidogenic pathway and disrupt steroid hormone biosynthesis. This is of particular concern during pregnancy, since sex hormones are integral to reproductive development. In other words, exposure to azole fungicides during fetal life can potentially lead to reproductive disease in the offspring. In addition, some azoles can act as androgen receptor antagonists, which can further add to the disrupting potential following exposure. When used as pharmaceuticals, systemic concentrations of the azole compounds can become significant as combatting fungal infections can be very challenging and require prolonged exposure to high doses. Although most medicinal azoles are tightly regulated and used as prescription drugs after consultations with medical professionals, some are sold as over-the-counter drugs. In this review, we discuss various azole fungicides known to disrupt steroid sex hormone biosynthesis or action with a focus on what potential consequences exposure during pregnancy can have on the life-long reproductive health of the offspring.

Using alternative test methods to predict endocrine disruption and reproductive adverse outcomes: do we have enough knowledge?

Endocrine disrupting chemicals (EDCs) are a matter of great concern. They are ubiquitous in the environment, are considered harmful to humans and wildlife, yet remain challenging to identify based on current international test guidelines and regulatory frameworks. For a compound to be identified as an EDC within the EU regulatory system, a plausible link between an endocrine mode-of-action and an adverse effect outcome in an intact organism must be established. This requires in-depth knowledge about molecular pathways regulating normal development and function in animals and humans in order to elucidate causes for disease. Although our knowledge about the role of the endocrine system in animal development and function is substantial, it remains challenging to predict endocrine-related disease outcomes in intact animals based on non-animal test data. A main reason for this is that our knowledge about mechanism-of-action are still lacking for essential causal components, coupled with the sizeable challenge of mimicking the complex multi-organ endocrine system by methodological reductionism. Herein, we highlight this challenge by drawing examples from male reproductive toxicity, which is an area that has been at the forefront of EDC research since its inception. We discuss the importance of increased focus on characterizing mechanism-of-action for EDC-induced adverse health effects. This is so we can design more robust and reliable testing strategies using non-animal test methods for predictive toxicology; both to improve chemical risk assessment in general, but also to allow for considerable reduction and replacement of animal experiments in chemicals testing of the 21st Century.

A Pragmatic Approach to Adverse Outcome Pathway Development and Evaluation.

The adverse outcome pathway (AOP) framework provides a practical means for organizing scientific knowledge that can be used to infer cause-effect relationships between stressor events and toxicity outcomes in intact organisms. It has reached wide acceptance as a tool to aid chemical safety assessment and regulatory toxicology by supporting a systematic way of predicting adverse health outcomes based on accumulated mechanistic knowledge. A major challenge for broader application of the AOP concept in regulatory toxicology, however, has been developing robust AOPs to a level where they are peer reviewed and accepted. This is because the amount of work required to substantiate the modular units of a complete AOP is considerable, to the point where it can take years from start to finish. To help alleviate this bottleneck, we propose a more pragmatic approach to AOP development whereby the focus becomes on smaller blocks. First, we argue that the key event relationship (KER) should be formally recognized as the core building block of knowledge assembly within the AOP knowledge base (AOP-KB), albeit framing them within full AOPs to ensure regulatory utility. Second, we argue that KERs should be developed using systematic review approaches, but only in cases where the underlying concept does not build on what is considered canonical knowledge. In cases where knowledge is considered canonical, rigorous systematic review approaches should not be required. It is our hope that these approaches will contribute to increasing the pace at which the AOP-KB is populated with AOPs with utility for chemical safety assessors and regulators.

Human-relevant concentrations of the antifungal drug clotrimazole disrupt maternal and fetal steroid hormone profiles in rats.

Clotrimazole is a non-prescription and broad-spectrum antifungal drug sold under brand names such as Canesten® and Lotrimin®. It is used to treat different types of fungal infections, from oral thrush to athlete's foot and vaginal mycosis. The level of exposure to clotrimazole is uncertain, as the exact usage amongst self-medicating patients is unclear. Recent studies have raised potential concern about the unsupervised use of clotrimazole during pregnancy, especially since it is a potent inhibitor of CYP enzymes of the steroidogenesis pathway. To address some of these concerns, we have assessed the effects of intrauterine exposure to clotrimazole on developing rat fetuses. By exposing pregnant rats to clotrimazole 25 or 75 mg/kg bw/day during gestation days 7-21, we obtained internal fetal concentrations close to those observed in humans. These in vivo data are in strong agreement with our physiologically-based pharmacokinetic (PBK)-modelled levels. At these doses, we observed no obvious morphological changes to the reproductive system, nor shorter male anogenital distance; a well-established morphometric marker for anti-androgenic effects in male offspring. However, steroid hormone profiles were significantly affected in both maternal and fetal plasma, in particular pronounced suppression of estrogens was seen. In fetal testes, marked up-concentration of hydroxyprogesterone was observed, which indicates a specific action on steroidogenesis. Since systemic clotrimazole is rapidly metabolized in humans, relevant exposure levels may not in itself cause adverse changes to the reproductive systems. Its capacity to significantly alter steroid hormone concentrations, however, suggests that clotrimazole should be used with caution during pregnancy.

Classical toxicity endpoints in female rats are insensitive to the human endocrine disruptors diethylstilbestrol and ketoconazole.

Developmental exposure to endocrine disrupting chemicals can have negative consequences for reproductive health in both men and women. Our knowledge about how chemicals can cause adverse health outcomes in females is, however, poorer than our knowledge in males. This is possibly due to lack of sensitive endpoints to evaluate endocrine disruption potential in toxicity studies. To address this shortcoming we carried out rat studies with two well-known human endocrine disruptors, diethylstilbestrol (DES) and ketoconazole (KTZ), and evaluated the sensitivity of a series of endocrine related endpoints. Sprague-Dawley rats were exposed orally from gestational day 7 until postnatal day 22. In a range-finding study, disruption of pregnancy-related endpoints was seen from 0.014 mg/kg bw/day for DES and 14 mg/kg bw/day for KTZ, so doses were adjusted to 0.003; 0.006; and 0.0012 mg/kg bw/day DES and 3; 6; or 12 mg/kg bw/day KTZ in the main study. We observed endocrine disrupting effects on sensitive endpoints in male offspring: both DES and KTZ shortened anogenital distance and increased nipple retention. In female offspring, 0.0012 mg/kg bw/day DES caused slightly longer anogenital distance. We did not see effects on puberty onset when comparing average day of vaginal opening; however, we saw a subtle delay after exposure to both chemicals using a time-curve analysis. No effects on estrous cycle were registered. Our study shows a need for more sensitive test methods to protect the reproductive health of girls and women from harmful chemicals.

Transcriptome analysis of fetal rat testis following intrauterine exposure to the azole fungicides triticonazole and flusilazole reveals subtle changes despite adverse endocrine effects.

Azoles are used in agriculture and medicine to combat fungal infections. We have previously examined the endocrine disrupting properties of the agricultural azole fungicides triticonazole and flusilazole. Triticonazole displayed strong androgen receptor (AR) antagonism in vitro, whereas in utero exposure resulted in anti-androgenic effects in vivo evidenced by shorter anogenital distance (AGD) in fetal male rats. Flusilazole displayed strong AR antagonism, but less potent than triticonazole, and disrupted steroidogenesis in vitro, whereas in utero exposure disrupted fetal male plasma hormone levels. To elaborate on how these azole fungicides can disrupt male reproductive development by different mechanisms, and to investigate whether feminization effects such as short AGD in males can also be detected at the transcript level in fetal testes, we profiled fetal testis transcriptomes after in utero exposure to triticonazole and flusilazole by 3'Digital Gene Expression (3'DGE). The analysis revealed few transcriptional changes after exposure to either compound at gestation day 17 and 21. This suggests that the observed influence of flusilazole on hormone production may be by directly targeting steroidogenic enzyme activity in the testis at the protein level, whereas observations of shorter AGD by triticonazole may primarily be due to disturbed androgen signaling in androgen-sensitive tissues. Expression of Calb2 and Gsta2 was altered by flusilazole but not triticonazole and may pinpoint novel pathways of disrupted testicular steroid synthesis. Our findings have wider implication for how we integrate omics data in chemical testing frameworks, including selection of non-animal test methods and building of Adverse Outcome Pathways for regulatory purposes.

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.

Developmental biology meets toxicology: contributing reproductive mechanisms to build adverse outcome pathways.

An adverse outcome pathway (AOP) is a simplified description of the sequence of mechanistic events that lead to a particular toxicological effect, from initial trigger to adverse outcome. Although designed to inform regulatory risk assessors, the AOP framework also provides a platform for innovative collaborations between experts from relevant research fields and the regulatory community. The underpinning for any AOP is basic knowledge about molecular and developmental processes; such knowledge can only be attained by solid bioscientific research. Starting with this fundamental knowledge, the objective is to devise novel testing strategies that focus on key events in a causative pathway. It is anticipated that such a knowledge-based approach will ultimately alleviate many of the burdens associated with classical chemical testing strategies that typically involve large-scale animal toxicity regimens. This hails from the notion that a solid understanding of the underlying mechanisms will allow the development and use of alternative test methods, including both in vitro and in silico approaches. This review is specifically targeted at professionals working in bioscientific fields, such as developmental and reproductive biology, and aims to (i) inform on the existence of the AOP framework and (ii) encourage new cross-disciplinary collaborations. It is hoped that fundamental biological knowledge can thus be better exploited for applied purposes: firstly, an improved understanding of how our perpetual exposure to environmental chemicals is causing human reproductive disease and, secondly, new approaches to screen for harmful chemicals more efficiently. This is not an instructional manual on how to create AOPs; rather, we discuss how to harness fundamental knowledge from the biosciences to assist regulatory toxicologists in their efforts to protect humans against chemicals that harm human reproductive development and function.

In vitro and in vivo endocrine disrupting effects of the azole fungicides triticonazole and flusilazole.

Azoles are effective antifungal agents used in both medicine and agriculture. They typically work by inhibiting cytochrome P450 enzymes, primarily CYP51 of the ergosterol biosynthesis pathway, thus damaging the fungal cell membrane. However, apart from their desired antifungal properties, several azoles also exhibit endocrine disrupting properties in mammals, both in vitro and in vivo. Here, we have tested two currently used agricultural azole fungicides, triticonazole and flusilazole, for their in vitro anti-androgenic activity and potential effects on reproductive parameters. Both fungicides showed strong androgen receptor (AR) antagonism and disruption of steroid biosynthesis in vitro. Following gestational exposure to flusilazole (15 or 45 mg/kg bw/day) or triticonazole (150 or 450 mg/kg bw/day) in time-mated Sprague Dawley rats, triticonazole induced shorter male anogenital distance (AGD). Flusilazole exposure did not affect the AGD, but altered fetal male blood hormone profile, with increased androstenedione and decreased estrone levels. Flusilazole and triticonazole have dissimilar effects on reproductive parameters in vivo, but both show endocrine disrupting activities.