Transcriptomic profiling reveals thyroid hormone-mediated and compound-specific effects of methimazole and amitrole on testis development Ida W Strand, Monica K Draskau, Sofie Frank Rising, Bertrand Evrard, Louise Ramhøj, et al. Toxicological Sciences, 2026 Thyroid hormones (THs) influence testis development, with early life hypothyroidism resulting in smaller testes. Developmental exposure to thyroperoxidase (TPO)-inhibiting drugs such as propylthiouracil (PTU) and methimazole (MMI) also impair testis development in rodents by reducing TH levels, leading to smaller testes in pups due to, for instance, disrupted Sertoli cell proliferation and maturation. Comparable effects are seen following exposure to the TPO-inhibiting pesticide amitrole, one of many environmental chemicals with TH-disrupting properties. Despite this phenotype, the molecular underpinnings of hypothyroid-induced testis effects are less clear, complicating mechanism-based chemical toxicity testing relying on alternative test methods and omics approaches. Here, we report on transcriptomics profiling of testes from hypothyroid rats induced by chemical exposures. Pregnant Sprague-Dawley rat dams were exposed by oral gavage to 2 doses of MMI (8 or 16 mg/kg body weight/day) or amitrole (25 or 50 mg/kg bw/day) from gestational day (GD) 7 to pup day (PD) 16, with BRB-seq performed for both life stages, specifically GD21 and PD16. Both MMI and amitrole caused significant changes to the testis transcriptome, seen particularly at PD16, with 313 differentially expressed genes (DEGs) defining a shared TH-mediated profile. Additionally, amitrole exposure resulted in a distinct profile of 1,517 DEGs, suggesting compound-specific effects beyond TH disruption. This study underscores the potential sensitivity of transcriptomic profiling in detecting early tissue disruption under toxicological conditions, in this case, testis disruption under hypothyroid state, offering critical insights for chemical risk assessment beyond histopathological endpoints.
AOP report: adverse outcome pathway network for developmental androgen signaling inhibition leading to short anogenital distance in male offspring Terje Svingen, Emilie Elmelund, Marie L Holmer, Anna O Bindel, Henrik Holbech, et al. Environmental Toxicology and Chemistry, 2025 This report outlines an adverse outcome pathway network (AOPN) linking reduced androgen signaling during the fetal masculinization programming window to shortened anogenital distance (AGD) at birth. In mammals such as mice, rats, and humans, the AGD is approximately twice as long in males as in females, with the length driven by androgen-dependent differentiation of the male phenotype. Impaired androgen signaling during fetal development can lead to a shorter AGD in male offspring, a sexually dimorphic feature widely used in rodent toxicity studies and human epidemiological research to assess exposure to anti-androgenic substances. Measurement of the AGD is performed in several OECD Test Guideline studies for reproductive toxicity. Notably, androgen signaling can be perturbed by various molecular initiating events, and capturing these causal toxicological pathways may facilitate the use of alternative test data in predictive toxicology by describing the mechanistic knowledge required for hazard and risk assessment of chemicals. The AOPN includes three adverse outcome pathways (AOPs) converging on the same adverse outcome of “reduced AGD” but with distinct upstream pathways. The upstream portion of the AOPN includes more generalized key events (KEs) and KE relationships (KERs) with broad applicability domains, many of which are measurable by established in vitro methods. In contrast, the downstream events have a narrower applicability domain, focusing on development of the perineal region and AGD in male offspring. This report provides overall assessments of AOPs 305, 306, and 307, including one new KE (2298) and four new KERs (2127, 3448, 3449, and 3349) not previously reported. Overall confidence levels for all three AOPs are moderate to high, with few inconsistencies or uncertainties. The taxonomic domain of the AOPN is mammals, with most evidence coming from mouse, rat, and human studies. The upstream network has broad taxonomic applicability to all mammals and could likely be extended to some other vertebrates.
Androgen receptor antagonist flutamide modulates estrogen receptor alpha expression in distinct regions of the hypospadiac rat penis Emilie Elmelund, Monica K. Draskau, Marie Berg, Ida W. Strand, Jay R. Black, et al. Frontiers in Endocrinology, 2025 IntroductionIntrauterine exposure to endocrine disrupting chemicals (EDCs), particularly anti-androgens, has been implicated in hypospadias by disrupting fetal masculinization of the genital tubercle (GT). Other pathways, including estrogen signaling, may also contribute but remain poorly characterized, especially in rats – a key model in chemical toxicity testing. Estrogen signaling has also been linked to hypospadias in mice, raising questions about androgen-estrogen interactions in guiding GT differentiation. MethodsWe induced hypospadias in male rat offspring via intrauterine exposure to the antiandrogenic drug flutamide and characterized androgen and estrogen receptor expression.ResultsWe observed key structural and transcriptional changes in the developing penis, including altered estrogen receptor a (ERa, Esr1) expression. Notably, beyond this established androgen-estrogen relationship in hormone-sensitive tissues, anti-androgenic exposure also induced spatial changes in Esr1 expression in specific regions of the GT.DiscussionFuture toxicological testing using new approach methodologies (NAMs) should consider androgen-estrogen balance and crosstalk in reproductive tissues as a mechanism of action.
