?Background. Endocrine disruption is the Mode of Action of chemicals having a hormone-like role, interfering with the homeostasis of cells, organs and/or tissues via their hormone signaling. Such chemicals, known as Endocrine Disruptors (EDs), are defined as an exogenous substance or a mixture, that alter function(s) of the endocrine system, causing adverse health effects in an intact organism, or its progeny, or (sub)populations. Following EU regulations (REACH and CLP regulations) and internationally accepted Test Guidelines and Guidances, endocrine disruption is experimentally assessed either in in vivo assays or via mechanistic-based in vitro assays.
Objectives. In order to measure the occurrence of endocrine disruption in effect-based in vitro assays, we investigated the secretion of proteins biologically characterized as endocrine-dependent, cell-specific, functional (or toxicological) biomarkers of clinical relevance in cell lines representing key cells of human reproductive tissues. In such effect-based in vitro assays, a cell viability assay (MTS assay) is combined with the measurement (by ELISAassay) of a secreted protein usually employed in clinical practice as an oncological biomarker.
Methods. As a cell model system, the hormone-responsive prostate epithelial cell line LNCaP expressing the androgen-regulated Prostate-Specific Antigen (PSA) has been applied as a toxicological biomarkers and, at the same time, as an hormone (endocrine)-dependent, cell-specific, functional (or toxicological) biomarkers of clinical relevance. LNCaP grown in a static, bidimensional (2D) set up were either treated with sex steroids (e.g., the androgens testosterone or DHT, the anti-androgens bicalutamide and flutamide or the estrogen E2) or with dietary and environmental contaminants, such as the biocide vinclozolin and its metabolites M1 and M2. Cell viability (by MTS assay) and protein secretion (PSA measurement in LNCaP cell cultures by ELISA-based assay) were assessed in a concentration-dependent manner ranging broadly from 1 pM to 100 mM.
Results. Similarly to anti-androgens, vinclozolin and its two metabolites significantly reduced DHT-induced PSA secretion and gene expression, M2 showing the strongest downregulation (at ≥ 100 nM for PSA secretion, at 10 nM for PSA gene expression), at all tested concentrations within the 1 pM -100 ?M range. In absence of androgens, vinclozolin and its metabolites had an androgen-like effect inducing proliferation and PSA secretion at concentrations ≥ 100 nM, thus supporting the view that endogenous hormone levels are relevant to determine the role of external chemicals also as carcinogens.
Conclusions/Implications for practice or policy. The different behave of dietary and environmental contaminants in presence or ansence of endogenous hormones would allow to determine their behave as carcinogens as recently shown for vinclozolin and its metabolites M1 and M2 in the carcinogenic assessment of vinclozolin “as possibly carcinogenic to humans” (Group 2B) based on “sufficient” evidence for cancer in experimental animals and on “strong” mechanistic evidence in experimental systems.
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