Fluorene-9-bisphenol regulates steroidogenic hormone synthesis in H295R cells through the AC/cAMP/PKA signaling pathway

Abstract

Fluorene-9-bisphenol (BHPF), which has been used as a substitute for bisphenol A (BPA) in consumer goods and industrial products, can be detected in environmental media and human urine. BHPF has been reported to have endocrine-disrupting effects, whereas deleterious effects on steroidogenesis in H295R cells and underlying mechanisms are still unclear. Here, we investigated effects of BHPF on steroidogenesis using human adrenocortical carcinoma cells (H295R). Cytotoxicity was initially assessed and half-maximal inhibitory concentration (IC₅₀) was determined based on proliferation of cells. Responses of four steroid hormones, aldosterone, cortisol, testosterone and 17β-estradiol (E₂), and ten critical genes, StAR, HMGR, CYP11A1, CYP11B1, CYP11B1, HSD3B2, CYP21, CYP17, 17β-HSD, and CYP19, involved in steroidogenesis after exposure to non-cytotoxic concentrations of BHPF were determined in the presence or absence of 100 μM dbcAMP. Adenylate cyclase (AC) activity, intracellular concentrations of cAMP, PKA activity and amounts of steroidogenic factor-1 (SF-1) gene and expressions of proteins were determined to elucidate underlying mechanisms of effects on steroidogenesis. BHPF was cytotoxic to H295R cells in a dose- and time-dependent manner. Effects on production of hormones results demonstrated that exposure to greater concentrations of BHPF inhibited productions of aldosterone, cortisol, testosterone and E₂ by down-regulation of steroidogenic genes. Inhibition of AC activity, intercellular cAMP content and PKA activity after exposure to BHPF implied that the AC/cAMP/PKA signaling pathway was involved in BHPF-induced suppression of steroidogenesis in H295R cells. Additionally, BHPF inhibited steroidogenesis and expressions of steroidogenic genes via decreasing expression of SF-1 protein, both in basal and dbcAMP-induced treatment. These results contributed to understanding molecular mechanisms of BHPF-induced effects on steroidogenesis and advancing the comprehensive risk assessment of BPs.

Keywords: BHPF; Endocrine disruption; In vitro; Signal transduction; Steroidogenesis.