Benzo[a]pyrene disrupts LH/hCG-dependent mouse Leydig cell steroidogenesis through receptor/Gαs protein targeting.

Steroidogenesis of gonadal cells is tightly regulated by gonadotropins. However, certain polycyclic aromatic hydrocarbons, including Benzo[a]pyrene (BaP), induce reproductive toxicity. Several existing studies have considered higher than environmentally relevant concentrations of BaP on male and female steroidogenesis following long-term exposure. Also, the impact of short-term exposure to BaP on gonadotropin-stimulated cells is understudied. Therefore, we evaluated the effect of 1 nM and 1 µM BaP on luteinizing hormone/choriogonadotropin (LH/hCG)-mediated signalling in two steroidogenic cell models, i.e. the mouse tumor Leydig cell line mLTC1, and the human primary granulosa lutein cells (hGLC) post 8- and 24-h exposure. Cell signalling studies were performed by homogeneous time-resolved fluorescence (HTRF) assay, bioluminescence energy transfer (BRET) and Western blotting, while immunostainings and immunoassays were used for intracellular protein expression and steroidogenesis analyses, respectively. BaP decreased cAMP production in gonadotropin-stimulated mLTC1 interfering with Gαs activation. Therefore, decrease in gonadotropin-mediated CREB phosphorylation in mLTC1 treated with 1 µM BaP was observed, while StAR protein levels in gonadotropin-stimulated mLTC1 cells were unaffected by BaP. Further, BaP decreased LH- and hCG-mediated progesterone production in mLTC1. Contrastingly, BaP failed to mediate any change in cAMP, genes and proteins of steroidogenic machinery and steroidogenesis of gonadotropin-treated hGLC. Our results indicate that short-term exposure to BaP significantly impairs steroidogenic signalling in mLTC1 interfering with Gαs. These findings could have a significant impact on our understanding of the mechanism of reproductive toxicity by endocrine disruptors.

Intracellular cGMP increase is not involved in thyroid cancer cell death.

INTRODUCTION: Type 5 phosphodiesterase (PDE5) inhibitors (PDE5i) lead to intracellular cyclic-guanosine monophosphate (cGMP) increase and are used for clinical treatment of erectile dysfunction. Studies found that cGMP may up/downregulate the growth of certain endocrine tumor cells, suggesting that PDE5i could impact cancer risk. AIM: We evaluated if PDE5i may modulate thyroid cancer cell growth in vitro. MATERIALS AND METHODS: We used malignant (K1) and benign (Nthy-ori 3-1) thyroid cell lines, as well as the COS7 cells as a reference model. Cells were treated 0-24 h with the PDE5i vardenafil or the cGMP analog 8-br-cGMP (nM-µM range). cGMP levels and caspase 3 cleavage were evaluated by BRET, in cGMP or caspase 3 biosensor-expressing cells. Phosphorylation of the proliferation-associated extracellularly-regulated kinases 1 and 2 (ERK1/2) was evaluated by Western blotting, while nuclear fragmentation by DAPI staining. Cell viability was investigated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. RESULTS: Both vardenafil and 8-br-cGMP effectively induced dose-dependent cGMP BRET signals (p≤0.05) in all the cell lines. However, no differences in caspase 3 activation occurred comparing PDE5i-treated vs untreated cells, at all concentrations and time-points tested (p>0.05). These results match those obtained upon cell treatment with 8-br-cGMP, which failed in inducing caspase 3 cleavage in all the cell lines (p>0.05). Moreover, they reflect the lack of nuclear fragmentation. Interestingly, the modulation of intracellular cGMP levels with vardenafil or the analog did not impact cell viability of both malignant and benign thyroid tumor cell lines, nor the phosphorylation of ERK1/2 (p>0.05). CONCLUSIONS: This study demonstrates that increased cGMP levels are not linked to cell viability or death in K1 and Nthy-ori 3-1 cell lines, suggesting that PDE5i do not impact the growth of thyroid cancer cells. Since different results were previously published, further investigations are recommended to clarify the impact of PDE5i on thyroid cancer cells.

Short-Term Exposure to Bisphenol A Does Not Impact Gonadal Cell Steroidogenesis In Vitro.

Bisphenol A (BPA) is a ubiquitous, synthetic chemical proven to induce reproductive disorders in both men and women. The available studies investigated the effects of BPA on male and female steroidogenesis following long-term exposure to the compound at relatively high environmental concentrations. However, the impact of short-term exposure to BPA on reproduction is poorly studied. We evaluated if 8 and 24 h exposure to 1 nM and 1 µM BPA perturbs luteinizing hormone/choriogonadotropin (LH/hCG)-mediated signalling in two steroidogenic cell models, i.e., the mouse tumour Leydig cell line mLTC1, and human primary granulosa lutein cells (hGLC). Cell signalling studies were performed using a homogeneous time-resolved fluorescence (HTRF) assay and Western blotting, while gene expression analysis was carried out using real-time PCR. Immunostainings and an immunoassay were used for intracellular protein expression and steroidogenesis analyses, respectively. The presence of BPA leads to no significant changes in gonadotropin-induced cAMP accumulation, alongside phosphorylation of downstream molecules, such as ERK1/2, CREB and p38 MAPK, in both the cell models. BPA did not impact STARD1, CYP11A1 and CYP19A1 gene expression in hGLC, nor Stard1 and Cyp17a1 expression in mLTC1 treated with LH/hCG. Additionally, the StAR protein expression was unchanged upon exposure to BPA. Progesterone and oestradiol levels in the culture medium, measured by hGLC, as well as the testosterone and progesterone levels in the culture medium, measured by mLTC1, did not change in the presence of BPA combined with LH/hCG. These data suggest that short-term exposure to environmental concentrations of BPA does not compromise the LH/hCG-induced steroidogenic potential of either human granulosa or mouse Leydig cells.