Androgen Pathway Research Articles

Adverse events in men with advanced prostate cancer treated with androgen biosynthesis inhibitors and androgen receptor inhibitors.

The use of androgen biosynthesis and second-generation androgen receptor inhibitors for advanced prostate cancer is increasing. Because these therapies alter the androgen pathway, they have been associated with cardiometabolic and neurocognitive toxicities. Although their safety profiles have been assessed in clinical trials, real-world data are limited. A 20% sample of national Medicare claims was used to perform a retrospective cohort study of Medicare beneficiaries with advanced prostate cancer treated with androgen biosynthesis (ie, abiraterone) and second-generation androgen receptor inhibitors between 2012 and 2019. Outcomes were assessed after the first fill of either class of drug for the 12-month period after starting therapy. The primary outcome was a hospital admission or emergency department visit for a cardiometabolic event. Secondary outcomes included neurocognitive events and fractures. Multivariable regression was used to assess the association between the class of drug and occurrence of an adverse event. There were 3488 (60%) men started on an androgen biosynthesis inhibitor and 2361 (40%) started on an androgen receptor inhibitor for the first time. Cardiometabolic adverse events were more common in men managed with androgen biosynthesis inhibitor (9.2% vs 7.5%, P = .027). No difference between androgen biosynthesis and androgen receptor inhibitors was observed for neurocognitive events (3.3% vs 3.4%, respectively; P = .71) or fractures (4.2% vs 3.6%, respectively; P = .26). Men with advanced prostate cancer initiating an androgen biosynthesis inhibitor for the first time more commonly had cardiometabolic events than those started on androgen receptor inhibitors. Neurocognitive events and fractures did not differ by drug class.

Multiple androgen pathways contribute to the steroid signature of adrenarche

ContextAdrenarche is a normal developmental event in mid-childhood characterized by increasing adrenal androgen secretion. The role of the classic androgen pathway has been well described in adrenarche, but the role of newer active androgens and additional androgen pathways is less clear. ObjectiveTo study the contribution of novel androgens and related steroid biosynthesis pathways to the development of adrenarche, and to identify additional steroid biomarkers of adrenarche. DesignA longitudinal study of children aged 6–8 years at baseline, followed up at ages 8–10 and 14–16 years. A total of 34 children (20 girls) with clinical and/or biochemical signs of adrenarche (cases) and 24 children (11 girls) without these signs (controls) at age 8–10 years were included. Serum steroid profiling was performed by liquid chromatography high-resolution mass spectrometry. Main outcome measuresThirty-two steroids compartmentalized in progestagens, gluco- and mineralocorticoid pathways, and four androgen related pathways, including the classic, backdoor, 11-oxy, and 11-oxy backdoor pathways. ResultsThe classic and 11-oxy androgen pathways were more active, and serum concentrations of main androgens in the classic (dehydroepiandrosterone, dehydroepiandrosterone sulfate, androstenedione and androsterone) and 11-oxy (11β-hydroxyandrostenedione, 11β-hydroxytestosterone, 11-ketoandrostenedione, and 11-ketotestosterone) pathways were higher in cases at ages 6–8 and 8–10 years. Pregnenolone concentrations at adrenarchal age (8–10 years) and cortisol concentrations at adolescence (14–16 years) were higher in cases. 11β-hydroxyandrosterone and 11-ketoandrosterone tended to be higher in cases with clinical signs compared to cases who had only biochemical evidence of adrenarche, albeit they were detected at low levels. In biomarker analyses, calculated steroid ratios with cortisol, cortisone, or 11-deoxycortisone as dividers were better classifiers for adrenarche than single steroids. Among these ratios, androstenedione/cortisone was the best. ConclusionsThe classic and 11-oxy androgen pathways are active in adrenarche. Children with earlier timing of adrenarche have higher serum cortisol levels at late pubertal age, suggesting that early adrenarche might have long-term effects on adrenal steroidogenesis by increasing the activity of the glucocorticoid pathway. Future studies should employ comprehensive steroid profiling to define novel classifiers and biomarkers for adrenarche and premature adrenarche.

Efficacy of first line (1L) poly ADP-ribose polymerase inhibitors (PARPi) in combination with androgen pathway inhibitors (API) by homologous recombination repair (HRR) genes in metastatic castration-resistant prostate cancer (mCRPC): A living meta-analysis.

e17059 Background: It is unclear if patients with different HRR gene-mutated mCRPC derive consistent benefit from treatment with PARPi and API combination. Approval labels for PARPi+API are inconsistent across regulatory bodies in the US (Food and Drug Administration, FDA) and Europe (European Medical Agency, EMA), adding to confusion among practicing clinicians. Therefore, we aimed to assess the differential efficacy of PARP+API therapy by different genes using up-to-date trial evidence. Methods: We maintain a living meta-analysis on mCRPC treatments that is updated when new data becomes available. Here, we report data from phase III randomized control trials (RCTs) assessing PARPi+API in 1L mCRPC. The outcomes of interest included radiographic progression-free survival (rPFS) and overall survival (OS). Hazard ratios (HR) for rPFS and OS by different genes were summarized using random effects meta-analysis via inverse variance approach. Results: We have screened 27376 references since the inception of this living review. Here, we report data from three trials (15 references) with a total of 1618 patients. Frequently reported HRR gene mutations in the included trials were BRCA2 (35%), ATM (21%), CDK12 (12.2%), CHEK2 (12.2%), BRCA1 (4.6%), and PALB2 (3.4%). In terms of rPFS, statistical benefit was observed in BRCA (0.28; 95% CI: 0.13-0.59) and CDK12 (0.58; 95% CI: 0.35-0.95) subgroups, whereas no statistically significant benefit was seen in PALB2 (0.53; 95% CI: 0.21-1.32), ATM (0.93; 95% CI, 0.57-1.53), and CHEK2 (0.92; 95% CI: 0.53-1.61) subgroups. For OS, benefit was observed in BRCA (0.47; 95%CI: 0.31-0.71) subgroup. While not statistically significant, a signal of meaningful OS benefit was observed in PALB2 (0.33; 95%CI: 0.10-1.16) but no OS benefit was observed in ATM (0.97; 95% CI: 0.57-1.67), CDK12 (0.80; 95% CI: 0.36-1.78), and CHEK2 (0.81; 95% CI: 0.37-1.75) subgroups. Conclusions: Current results show that mCRPC patients with BRCA and CDK12 alterations had delayed disease progression with PARPi+API as 1L therapy. BRCA mutations are also associated with improved OS, while patients with CHEK2 and ATM derived no significant benefit with PARPi+API combination. [Table: see text]

Antifungal climbazole alters androgenic pathways in mammalian cells

Among antifungal agents used in pharmaceuticals and personal care products, the synthetic azole climbazole (CBZ; 1-(4-Chlorophenoxy)-1-(imidazol-1-yl)-3,3-dimethylbutan-2-one) acts on the fungus Malassezia. Despite concerns surrounding its effects on health, based on alterations to reproduction and steroidogenesis found in fish, little is known about its mechanism of action as an endocrine disrupting chemical (EDC) in mammalian cells.In this study, using OECD test guidelines, we investigated the effects of CBZ (i) in H295R cells, on the production of estradiol and testosterone, as well as intermediate metabolites in steroidogenesis pathway, and (ii) in HeLa9903 and AR-EcoScreen cell lines, on the transactivation of estrogen and androgen receptors.Our results are the first evidence in H295R cells, that CBZ treatment (from 0.3 μM) decreased secreted levels of testosterone and estradiol. This was associated with reduced 17α-hydroxypregnenolone and 17α-hydroxyprogesterone levels. The altered levels of these metabolites were associated with a decrease in cytochrome P450 17α-hydroxylase/17,20-lyase (Cyp17A1) activity without any effect on its protein level. CBZ was also found to exert antagonistic effects toward androgen and estrogen α receptors. These results give insights into the toxicological mechanism of action of CBZ. Many azoles share structural similarities; therefore, caution should be adopted due to their potential toxicity.

Gonadal hormone deprivation regulates response to tibolone in neurodegenerative pathways

Gonadal hormone deprivation (GHD) and decline such as menopause and bilateral oophorectomy are associated with an increased risk of neurodegeneration. Yet, hormone therapies (HTs) show varying efficacy, influenced by factors such as sex, drug type, and timing of treatment relative to hormone decline. We hypothesize that the molecular environment of the brain undergoes a transition following GHD, impacting the effectiveness of HTs. Using a GHD model in mice treated with Tibolone, we conducted proteomic analysis and identified a reprogrammed response to Tibolone, a compound that stimulates estrogenic, progestogenic, and androgenic pathways. Through a comprehensive network pharmacological workflow, we identified a reprogrammed response to Tibolone, particularly within "Pathways of Neurodegeneration", as well as interconnected pathways including "cellular respiration", "carbon metabolism", and "cellular homeostasis". Analysis revealed 23 proteins whose Tibolone response depended on GHD and/or sex, implicating critical processes like oxidative phosphorylation and calcium signalling. Our findings suggest the therapeutic efficacy of HTs may depend on these variables, suggesting a need for greater precision medicine considerations whilst highlighting the need to uncover underlying mechanisms.

Sex differences orchestrated by androgens at single-cell resolution.

Sex differences in mammalian complex traits are prevalent and are intimately associated with androgens1-7. However, a molecular and cellular profile of sex differences and their modulation by androgens is still lacking. Here we constructed a high-dimensional single-cell transcriptomic atlas comprising over 2.3 million cells from 17 tissues in Mus musculus and explored the effects of sex and androgens on the molecular programs and cellular populations. In particular, we found that sex-biased immune gene expression and immune cell populations, such as group 2 innate lymphoid cells, were modulated by androgens. Integration with the UK Biobank dataset revealed potential cellular targets and risk gene enrichment in antigen presentation for sex-biased diseases. This study lays the groundwork for understanding the sex differences orchestrated by androgens and provides important evidence for targeting the androgen pathway as a broad therapeutic strategy for sex-biased diseases.

Abstract 3440: Etiology of prostate cancer with the TMPRSS2:ERG fusion: A systematic review of risk factors

Abstract Background: The TMPRSS2:ERG gene fusion is the most common somatic alteration in primary prostate cancer and an early event in carcinogenesis. There are emerging data highlighting etiologic differences in prostate cancer by TMPRSS2:ERG status. This systematic review synthesized evidence from epidemiologic studies on prostate cancer risk factors for tumors with and without the TMPRSS2:ERG fusion. Methods: A systematic literature search was performed in Ovid MEDLINE, EMBASE, and Web of Science without language restriction for studies published by September 27, 2022. Studies that assessed associations between epidemiologic and genetic factors and prostate cancer risk by tumor TMPRSS2:ERG (ERG) fusion status in human populations were included. Results: Of 3,071 records identified, 20 publications comprising prospective cohort and case-control studies from five study populations (published 2009-2022) were included. Risk factors included germline genetic variants, circulating hormones, diet, lifestyle factors, and medications. The included studies suggested that taller height and higher total and free circulating testosterone levels tended to be associated with higher risk of ERG-positive, but not ERG-negative prostate cancer. CAG repeat length in the androgen receptor (AR) gene, related inversely to transcriptional activity, appeared to be associated with a lower risk of ERG-positive, but not ERG-negative prostate cancer. There was statistical evidence for etiologic differences for several germline single nucleotide polymorphisms (SNPs) by ERG status. Excess body weight, greater vigorous physical activity, greater lycopene intake, and calcium channel blocker use appeared to specifically be associated with a lower risk of ERG-positive tumors. Associations of other potential etiologic factors, including diabetes mellitus, baldness, aspirin use, circulating antioxidant levels, and sex hormones other than testosterone, with ERG-positive prostate cancer were null or inconclusive. Most associations had low precision, and the results were based on few distinct study populations, highlighting a need for replication. Conclusions: Prostate cancer with the TMPRSS2:ERG fusion may be an etiologically distinct subtype impacted by certain modifiable and hormonally-acting risk factors that align with the established mechanistic role of this gene fusion in androgen, insulin, and growth factor pathways. The findings suggest a potentially distinct genetic predisposition to prostate cancer with or without the TMPRSS2:ERG fusion. More broadly, these findings suggest that considering molecular subtypes of prostate cancer, beyond TMPRSS2:ERG, may strengthen etiologic understanding of prostate cancer and evidence for prevention. Citation Format: Colleen B. McGrath, Alaina H. Shreves, Megan R. Shanahan, Hannah E. Guard, Manelisi V. Nhliziyo, Kathryn L. Penney, Tamara L. Lotan, Michelangelo Fiorentino, Konrad H. Stopsack, Lorelei A. Mucci. Etiology of prostate cancer with the TMPRSS2:ERG fusion: A systematic review of risk factors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3440.

Androgen receptor blockade to activate NK cells and to upregulate the surface expression of HLA-E in prostate cancer cell lines.

190 Background: The blockade of the androgen receptor (AR) pathway is an effective treatment for prostate cancer (PCa), but most patients progress to castration-resistant prostate cancer (mCRPC). AR signaling modulates CD8+ T cytotoxic function, revealing the immune-modulatory function of the AR pathway. We have described the activation of NK cells by AR inhibitors and the potential synergistic effect with anti-NKG2a combination.1 Strategies to activate NK cells can be limited by the expression of HLA-E, a ligand of the inhibitory checkpoints on NK cells NKG2A. We investigated the mechanisms of AR-dependent modulation of HLA-E on tumor cells and the ADT-enhancing effect on patient-derived NK cells. Methods: PC cell lines were treated with second-generation androgen pathway inhibitors in vitro (enzalutamide 10uM, darolutamide 15 uM), and the expression of HLA-E was evaluated by flow cytometry. To evaluate the modulation of HLA-E by AR, the AR-negative cell lines (PC3 and DU145) were stably transduced with an inducible AR system. The pan HDAC inhibitors vorinostat (0.4 uM) and panobinostat (2.5 uM) were used to evaluate the regulation of HLA-E by epigenetics. We analyzed the activation status of paired peripheral blood NK cells isolated from patients with PCa prior to and post-androgen deprivation therapy (ADT). The patients (n=6) had a median time between collection of 26.3±2.3 days. Results: The AR-responsive LNCaP cell line displayed an increase in surface expression of HLA-E upon treatment with enzalutamide (Enza) or darolutamide (Daro) ([C]: 12.1±1.3%, Enza: 22.3±3.3%, Daro: 19.1±1.1%, p=0.004). AR blockade did not change the surface expression of HLA-E of AR negative PC3 ([C]: 10.5±2.7%, Daro: 13.1±2.3%, p=0.17) or DU145 cell lines ([C]: 3.5±1.7%, Daro: 2.5±0.6%, p=0.12). Transduction of AR in PC3 and DU145 lead to upregulation of HLA-E expression with AR blockade (PC3-AR+ [C]: 13.1±3.7%, Daro: 43.1±5.3%, DU145-AR+ [C]: 4.5±1.4%, Daro: 27.2±3.6%, p=0.001). The upregulation of HLA-E upon AR blockade was suppressed if cell lines were co-treated with vorinostat or panobinostat. Patient-derived peripheral blood NK cells displayed enhanced cytotoxic activity after ADT (expression of Granzyme B pre-ADT: 12.3±2.3%, post-ADT: 36.5±5.7% p=0.0017, Perforin pre-ADT: 3±0.6%, post-ADT: 26.5±3.7% p=0.001), and upregulation of the inhibitory checkpoint NKG2a (pre-ADT: 5.2±1.2%, post-ADT: 12.3±1.4%, p=0.04). Conclusions: Androgen inhibitors upregulate the expression of HLA-E in PCa cell lines by an AR-dependent mechanism regulated by epigenetics. ADT promotes peripheral blood patient-derived NK cell activation and upregulation of inhibitory NKG2A receptor. These findings support further investigative approaches targeting the HLA-E and NKG2A in mCRPC. 1. Schwermann, AACR 2023.

Abstract A021: Pre and post treatment transcriptomic analysis provide new insights on the mechanisms underlying the efficacy of PARPi and Androgen blockage in prostate cancer

Abstract Aim: Recent phase 3 trials (PROPEL, TALAPRO2) have demonstrated survival benefits when combining PARP inhibitors (PARPi) with androgen pathway inhibitors (API) in prostate cancer (PC) treatment. Importantly, this benefit is observed regardless of homologous repair deficiency (HRD) status, suggesting alternative underlying mechanisms beyond synthetic lethality. However, the complete identification of all the PC patients who can benefit from this combination therapy remains an unmet need. Therefore, further investigation into the distinct molecular pathways involved and identification of biomarkers of response is critical and is the aim of this study. Methods: We have performed an exploratory transcriptomic analysis including RNA-seq, differential expression analysis and gene set enrichment analyses (GSEA) of pre and post treatment biopsies of localized PC patients (pts) with higher risk of relapse and that were included in a window of opportunity (WoO) study. We have included pts recruited to the CANCAP-3 study that were exposed to either two weeks of olaparib alone or in combination with degarelix (1:1 randomization). A corresponding transcriptomic analysis of pre and post enzalutamide biopsies of patients recruited to the WoO study DARANA was also included. Results: GSEA results have shown p53 hallmark upregulation more pronounced in the combination cohort. In the combination cohort the androgen response genes were downregulated. Additionally, canonical cell cycle progression hallmarks (E2F and G2M checkpoint) were suppressed with a significant downregulation of the Prolaris signature. In the olaparib monotherapy cohort pts with the greatest PSA decline demonstrated large reductions in the Prolaris score. Furthermore, in the olaparib monotherapy cohort the two patients that relapsed within 4 years of follow up demonstrated greater Prolaris scores. Interestingly, a significant positive correlation between HR deficiency signatures and reduced prostate cancer proliferation index was observed across the study cohorts, although there was only minimal overlap in the genes constituting these two signatures. This correlation was maintained excluding HRD pts (n=2). Finally, this relationship was also observed following analysis of the DARANA trial. Conclusions: Tissue transcriptomic data analysis of PC pts can provide further insights on the possible biological mechanisms that underlie the effect of this drug combination. In this study the expression of HRD signature appeared to be in direct proportion to the reduction in cellular proliferation (Prolaris). This was seen in the combination treatment cohort, as well as in a separate API monotherapy study (DARANA). This suggests that androgen inhibition is the main driver for the reduction in HR signature. However, this phenomenon may represent a consequence of cell cycle arrest occurring more readily in cells with the greatest HR defect. Further studies are needed to further explain the unique upstream processes that contribute to an AR-supported DNA damage response, in the context of PARP inhibition. Citation Format: Ana Filipa Palma dos Reis, Toby Milne-Clark, Amit Dipak Amin, Liliya Nazlamova, Simon Pacey, Harveer Dev. Pre and post treatment transcriptomic analysis provide new insights on the mechanisms underlying the efficacy of PARPi and Androgen blockage in prostate cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr A021.

Update on Adrenarche-Still a Mystery.

Adrenarche marks the timepoint of human adrenal development when the cortex starts secreting androgens in increasing amounts, in healthy children at age 8-9 years, with premature adrenarche (PA) earlier. Because the molecular regulation and significance of adrenarche are unknown, this prepubertal event is characterized descriptively, and PA is a diagnosis by exclusion with unclear long-term consequences. We searched the literature of the past 5 years, including original articles, reviews, and meta-analyses from PubMed, ScienceDirect, Web of Science, Embase, and Scopus, using search terms adrenarche, pubarche, DHEAS, steroidogenesis, adrenal, and zona reticularis. Numerous studies addressed different topics of adrenarche and PA. Although basic studies on human adrenal development, zonation, and zona reticularis function enhanced our knowledge, the exact mechanism leading to adrenarche remains unsolved. Many regulators seem involved. A promising marker of adrenarche (11-ketotestosterone) was found in the 11-oxy androgen pathway. By current definition, the prevalence of PA can be as high as 9% to 23% in girls and 2% to 10% in boys, but only a subset of these children might face related adverse health outcomes. New criteria for defining adrenarche and PA are needed to identify children at risk for later disease and to spare children with a normal variation. Further research is therefore required to understand adrenarche. Prospective, long-term studies should characterize prenatal or early postnatal developmental pathways that modulate trajectories of birth size, early postnatal growth, childhood overweight/obesity, adrenarche and puberty onset, and lead to abnormal sexual maturation, fertility, and other adverse outcomes.

Rho GTPase activating protein 21-mediated regulation of prostate cancer associated 3 gene in prostate cancer cell.

The overexpression of the prostate cancer antigen 3 (PCA3) gene is well-defined as a marker for prostate cancer (PCa) diagnosis. Although widely used in clinical research, PCA3 molecular mechanisms remain unknown. Herein we used phage display technology to identify putative molecules that bind to the promoter region of PCA3 gene and regulate its expression. The most frequent peptide PCA3p1 (80%) was similar to the Rho GTPase activating protein 21 (ARHGAP21) and its binding affinity was confirmed using Phage Bead ELISA. We showed that ARHGAP21 silencing in LNCaP prostate cancer cells decreased PCA3 and androgen receptor (AR) transcriptional levels and increased prune homolog 2 (PRUNE2) coding gene expression, indicating effective involvement of ARHGAP21 in androgen-dependent tumor pathway. Chromatin immunoprecipitation assay confirmed the interaction between PCA3 promoter region and ARHGAP21. This is the first study that described the role of ARHGAP21 in regulating the PCA3 gene under the androgenic pathway, standing out as a new mechanism of gene regulatory control during prostatic oncogenesis.

ZNACZENIE RECEPTORA ANDROGENOWEGO W TERAPII RAK STERCZA

he androgen receptor, upon binding to a ligand, functions as a transcription factor, ultimately helping to maintain the proper structure and function of the prostate. In prostate cancer, the constant activation of the androgen pathway in the cell leads to the proliferation of cancer cells and the development of prostate cancer. The individual stages of androgen signaling have become the target of anti-androgen therapy, which ultimately results in a decrease in testosterone levels in the cell. Over time, the duration of therapy leads to the emergence of new mechanisms of resistance in prostate cancer cells, such as an increase in the number of androgen receptors or endogenous synthesis of testosterone. This ultimately leads to the evolution of cancer cells into castration-resistantprostate cancer. This article aims to review and compare contemporary anti-hormonal drugs used in prostate cancer. New generation drugs such as enzalutamide, apalutamide, and darolutamide bind to the androgen receptor and inhibit its translocation to the cell nucleus. Abiraterone acetate inhibits endogenous testosterone synthesis, while relugolix is the first oral gonadotropin-releasing hormone analogue that does not cause a “flare” effect. Understanding the molecular mechanisms and biology of the androgen receptor is crucial for future directions in the development of prostate cancer drug therapies and their appropriate use.

Comparative genomic survey and functional analysis of DKKL1 during spermatogenesis in the Chinese soft-shelled turtle (Pelodiscus sinensis)

A feature of the Chinese soft-shelled turtle (Pelodiscus sinensis) is seasonal spermatogenesis; however, the underlying molecular mechanism is not well clarified. Here, we firstly cloned and characterized P. sinensis DKKL1, and then performed comparative genomic studies, expression analysis, and functional validation. P. sinensis DKKL1 had 2 putative N-glycosylation sites and 16 phosphorylation sites. DKKL1 also had classic transmembrane structures that were extracellularly localized. DKKL1's genetic distance was close to turtles, followed by amphibians and mammals, but its genetic distance was far from fishes. DKKL1 genes from different species shared distinct genomic characteristics. Meanwhile, they were also relatively conserved among themselves, at least from the perspective of classes. Notably, the transcription factors associated with spermatogenesis were also identified, containing CTCF, EWSR1, and FOXL2. DKKL1 exhibited sexually dimorphic expression only in adult gonads, which was significantly higher than that in other somatic tissues (P < 0.001), and was barely expressed in embryonic gonads. DKKL1 transcripts showed a strong signal in sperm, while faint signals were detected in other male germ cells. DKKL1 in adult testes progressively increased per month (P < 0.05), displaying a seasonal expression trait. DKKL1 was significantly downregulated in testes cells after the sex hormones (17β-estradiol and 17α-methyltestosterone) and Wnt/β-catenin inhibitor treatment (P < 0.05). Likewise, the Wnt/β-catenin inhibitor treatment dramatically repressed CTCF, EWSR1, and FOXL2 expression. Conversely, they were markedly upregulated after the 17β-estradiol and 17α-methyltestosterone treatment, suggesting that the three transcription factors might bind to different promoter regions, thereby negatively regulating DKKL1 transcription in response to the changes in the estrogen and androgen pathways, and positively controlling DKKL1 transcription in answer to the alterations in the Wnt/β-catenin pathway. Knockdown of DKKL1 significantly reduced the relative expression of HMGB2 and SPATS1 (P < 0.01), suggesting that it may be involved in seasonal spermatogenesis of P. sinensis through a positive regulatory interaction with these two genes. Overall, our findings provide novel insights into the genome evolution and potential functions of seasonal spermatogenesis of P. sinensis DKKL1.

Toxicological investigation of lilial

Lilial (also called lysmeral) is a fragrance ingredient presented in many everyday cosmetics and household products. The concentrations of lilial in the final products is rather low. Its maximum concentration in cosmetics was limited and recently, its use in cosmetics products was prohibited in the EU due to the classification as reproductive toxicant. Additionally, according to the European Chemicals Agency, it was under assessment as one of the potential endocrine disruptors, i.e. a substance that may alter the function of the endocrine system and, as a result, cause health problems. Its ability to act as an androgen receptor agonist and the estrogenic and androgenic activity of its metabolites, to the best of our knowledge, have not yet been tested. The aim of this work was to determine the intestinal absorption, cytotoxicity, nephrotoxicity, mutagenicity, activation of cellular stress-related signal pathways and, most importantly, to test the ability to disrupt the endocrine system of lilial and its Phase I metabolites. This was tested using set of in vitro assays including resazurin assay, the CHO/HPRT mutation assay, γH2AX biomarker-based genotoxicity assay, qPCR and in vitro reporter assays based on luminescence of luciferase for estrogen, androgen, NF-κB and NRF2 signalling pathway. It was determined that neither lilial nor its metabolites have a negative effect on cell viability in the concentration range from 1 nM to 100 µM. Using human cell lines HeLa9903 and MDA-kb2, it was verified that this substance did not have agonistic activity towards estrogen or androgen receptor, respectively. Lilial metabolites, generated by incubation with the rat liver S9 fraction, did not show the ability to bind to estrogen or androgen receptors. Neither lilial nor its metabolites showed a nephrotoxic effect on human renal tubular cells (RPTEC/TERT1 line) and at the same time they were unable to activate the NF-κB and NRF2 signalling pathway at a concentration of 50 µM (HEK 293/pGL4.32 or pGL4.37). Neither lilial nor its metabolites showed mutagenic activity in the HPRT gene mutation test in CHO-K1 cells, nor were they able to cause double-strand breaks in DNA (γH2AX biomarker) in CHO-K1 and HeLa cells. In our study, no negative effects of lilial or its in vitro metabolites were observed up to 100 µM using different in vitro tests.

An international cross-laboratory survey on fish vitellogenin analysis: Methodological challenges and opportunities for best practice

Vitellogenin (VTG) is a biomarker for possible endocrine activity of chemicals acting via the estrogen, androgen, or steroidogenesis pathways. VTG is assessed in standardised fish guideline studies conducted for regulatory safety assessment of chemicals. VTG data can be highly variable leading to concerns for potential equivocal, false positive and/or negative outcomes. Consequently, additional fish testing may be required to address uncertainties in the VTG response, and possibly erroneous/missed identification of endocrine activity. To better understand the technical challenges of VTG assessment and reporting for regulatory purposes, a survey was sent to 27 testing laboratories performing these analyses. The survey results from 16 respondents (6 from the UK, 3 from the USA, and 7 from the EU) were analysed and discussed in a follow-up webinar. High variability in background VTG concentrations was widely acknowledged and thought to be associated with fish batch, husbandry, laboratory practices, and several methodological aspects. These include sample collection and storage, VTG quantification, data handling, and the benchmarks used for data acceptability. Information gathered in the survey provides a basis for improving and harmonizing the measurement of VTG in fish, and an opportunity to reassess the suitability of current acceptability criteria in test guidelines.

Comparative effects of finasteride and minoxidil on the male reproductive organs: A systematic review of in vitro and in vivo evidence

Finasteride and minoxidil are medicaments commonly prescribed for treating benign prostatic hyperplasia (BPA), hypertension, and/or androgenetic alopecia (AGA). The mechanism of action of finasteride is based on the interference in androgenic pathways, which may lead to fertility-related disorders in men. Minoxidil, however, can act in multiple ways, and there is no consensus that its use can adversely affect male fertility. Since finasteride and minoxidil could be risk factors for male fertility, we aimed to compare their impact on the two reproductive organs testis and epididymis of adult murine models, besides testis/epididymis-related cells, and describe the mechanism of action involved. For such, we used the PRISMA guideline. We included 31 original studies from a structured search on PubMed/MEDLINE, Scopus, and Web of Science databases. For in vivo studies, the bias analysis and the quality of the studies were assessed as described by SYRCLE (Systematic Review Centre for Laboratory Animal Experimentation). We concluded that finasteride and minoxidil act as hormone disruptors, causing oxidative stress and morphological changes mainly in the testis. Our results also revealed that finasteride treatment could be more harmful to male reproductive health because it was more associated with reproductive injuries, including damage to the epididymis, erectile dysfunction, decreased libido, and reduced semen volume. Thus, this study contributes to the global understanding of the mechanisms by which medicaments used for alopecia might lead to male reproductive disorders. We hope that our critical analysis expedites clinical research and reduces methodological bias. The registration number on the Prospero platform is CRD42022313347.

FRI382 High Androgen And Progesterone Metabolite Concentrations In Naturally Matured Follicles Are Concurrent With High Serum Anti-Müllerian Hormone Concentrations

Abstract Disclosure: T. Du Toit: None. M. Von Wolff: None. A.C. Swart: None. M. Groessl: None. C.E. Flueck: None. Infertility in women is routinely evaluated with the assessment of circulating steroid levels. Profiling steroid levels in follicular fluid (FF) would provide a focused evaluation of the ovarian endocrine milieu and is therefore better suited to identify key hormones potentially involved in dysfunctional follicular growth. The anti-Müllerian hormone (AMH) level in serum is a well-established clinical marker to estimate the ovarian reserve in assisted reproductive technology, while the AMH level in FF serves as a new prognostic marker for oocyte developmental potential. However, AMH-linked changes in the follicular steroid milieu have not been fully explored. We conducted an AMH-dependent analysis of the FF endocrine milieu. Hormones were quantified using LC-MS (n=42) and ELISA (n=4) in FF of 83 women undergoing natural cycle in vitro fertilisation. Patients were age-matched and classified into 4 statistically different (p&amp;lt;0.0001) groups according to serum AMH levels: group A (&amp;lt;1.1 pmol/L, n=20); group B (1 - 7.14 pmol/L, n=21); group C (7.14 - 35.5 pmol/L, n=21); and group D (&amp;gt;35.5 pmol/L, n=21). Group D was designated as our polycystic ovary syndrome (PCOS)-like group, as high serum AMH is a marker for PCOS. We characterised androgen and progesterone metabolic pathways in FF. Androstenedione (A4) and testosterone (T) levels differed significantly between the groups (p&amp;lt;0.01) and were highest in group D at 60 nmol/L and 4.7 nmol/L, respectively. The levels of 11-ketoandrostenedione (11KA4) and 11β-hydroxyandrosterone (adrenal-origin) also differed between the groups (p&amp;lt;0.05), with higher 11KA4 levels (±2.7-fold) quantified in group D. In parallel with serum AMH, FF AMH differed between the groups (p&amp;lt;0.0001), together with luteinizing hormone/FSH, A4/dehydroepiandrosterone, estradiol(E2)/T and E2/A4 ratios. In comparison to groups A, B and C combined, the PCOS-like group D showed significantly increased downstream progesterone metabolite levels –11α-hydroxyprogesterone [OHP4], 17α,20α-diOHP4, 16α-OHP4 and 5α-pregnanolone, with lower FSH levels. Our results show that the follicular milieu is significantly different when high AMH levels are measured compared to when AMH levels are low, marked by: (i) higher progesterone metabolite levels in follicles and (ii) higher A4 and T levels, concomitant with unchanged E2 levels suggesting that androgens were not fully converted to estrogens in these follicles. Our study furthermore corroborates other studies reporting an intrafollicular hyperandrogenic state in women with increased circulatory AMH levels. In summary, our study not only provides data based on a broad steroid profile that the follicular androgen endocrine milieu is affected in women with high serum AMH levels, but it also provides a model for the premature progesterone increase in PCOS women which leads to a reduction in the embryo implantation rate. Presentation: Friday, June 16, 2023

Uncovering the complex relationship between balding, testosterone and skin cancers in men

Male-pattern baldness (MPB) is related to dysregulation of androgens such as testosterone. A previously observed relationship between MPB and skin cancer may be due to greater exposure to ultraviolet radiation or indicate a role for androgenic pathways in the pathogenesis of skin cancers. We dissected this relationship via Mendelian randomization (MR) analyses, using genetic data from recent male-only meta-analyses of cutaneous melanoma (12,232 cases; 20,566 controls) and keratinocyte cancers (KCs) (up to 17,512 cases; >100,000 controls), followed by stratified MR analysis by body-sites. We found strong associations between MPB and the risk of KC, but not with androgens, and multivariable models revealed that this relationship was heavily confounded by MPB single nucleotide polymorphisms involved in pigmentation pathways. Site-stratified MR analyses revealed strong associations between MPB with head and neck squamous cell carcinoma and melanoma, suggesting that sun exposure on the scalp, rather than androgens, is the main driver. Men with less hair covering likely explains, at least in part, the higher incidence of melanoma in men residing in countries with high ambient UV.

Current Approaches to the Preclinical Assessment of Endocrine Toxicity

Scientific relevance. Endocrine disruptors affect the functioning of endocrine organs, which leads to adverse drug reactions. Endocrine toxicity requires special attention in preclinical studies of candidate medicinal products.Aim. The study aimed to review international guidelines and approaches to assessing the risk of endocrine toxicity associated with medicinal products.Discussion. This review covers documents that provide a methodological framework for identifying and classifying a chemical compound as an endocrine disruptor. These documents include the following: Revised Guidance Document 150 on Standardised Test Guidelines for Evaluating Chemicals for Endocrine Disruption (Organisation for Economic Cooperation and Development, 2018), Nonclinical Evaluation of Endocrine-Related Drug Toxicity (Food and Drug Administration, 2015), and Guidance for the Identification of Endocrine Disruptors in the Context of Regulations (EU) No. 528/2012 and (EC) No. 1107/2009. The proposed algorithm for endocrine toxicity assessment involves collecting all available data on the test compound, such as the literature and previously obtained experimental data, including acute and subchronic toxicity data, and in silico predictions. Particular attention should be paid to the standard battery of preclinical chronic toxicity studies, which can identify most side effects associated with the endocrine system. The main endpoints for endocrine toxicity include changes in the mass and histopathology of the major endocrine organs (adrenal glands, testes, epididymides, ovaries, and the thyroid gland), oestrous cycle effects, reproductive toxicity, and transplacental action. A thorough assessment of the data obtained provides for the determination of unfavourable endocrine activity that requires further studies.Conclusions. The OECD guidelines offer a set of validated in vivo and in vitro tests that characterise the most important mechanisms of endocrine toxicity (oestrogen, androgen, thyroid, and steroidogenic endocrine pathways) by identified toxic effects. This approach allows researchers to identify potential endocrine disorders early in the drug development process and to optimise the scope of the required studies accordingly.

P09-21: Antifungal Climbazole alters androgenic pathways in mammalian cells

P09-21: Antifungal Climbazole alters androgenic pathways in mammalian cells