In vitro evaluation for estrogenic mechanisms of the disinfectant benzalkonium chloride as an emerging contaminant

This study aimed to evaluate in vitro the possible mechanisms underlying the estrogenic potential of benzalkonium chloride (BAC) as a disinfectant emerging contaminant. Effects of BAC at the environmentally-relevant concentrations on estrogen synthesis and estrogen receptor (ER) signaling were assessed using the H295R steroidogenesis assay and the MCF-7 proliferation assay, respectively. Results showed that exposure to BAC at concentrations of 1.0-1.5 mg/L for 48 h significantly increased estradiol production of H295R cells in a concentration-dependent manner. Transcription of steroidogenic genes 3β‐HSD2, 17β‐HSD1, 17β‐HSD4, and CYP19A were significantly enhanced by BAC. In ER-positive MCF-7 cells, exposure to 0.5-1.5 mg/L BAC for 48 h significantly promoted cell proliferation and increased the expressions of ERα and G-protein coupled estrogen receptor 1. Flow cytometry analysis showed that 0.5-1.5 mg/L BAC significantly decreased the percentage of cells in G0/G1 phase, increased the percentage in S phase, and BAC at concentrations of 1.0 and 1.5 mg/L increased the G2/M phase cells. Findings of the study suggested that BAC at environmentally-relevant concentrations might act as a xenoestrogen through its inhibitory effect on steroidogenesis and ER-mediated mechanism.

Effect of aspirin on the proliferation and apoptosis of human adrenal cortical cancer cells / 中华内分泌代谢杂志

Objective To observe the effect of aspirin on the proliferation inhibition and apoptosis of human adrenal cortical cancer cells, and to explore preliminarily the related mechanisms. Methods The human adrenal cortical cancer cells were cultured in vitro. The experimental group was DEME/F-12 complete medium which contained different concentrations of aspirin (final concentration of 0.125, 0.25, 0.5, 1 mg/ml). The control group was DEME/F-12 complete medium which had no aspirin but 1%anhydrous ethanol instead. After treatment by aspirin at different concentrations for different durations (24, 48, 72 hours), we detected the proliferation inhibition of SW-13 cells and H295R cells by methyl thiazolyl tetrazolium (MTT) method, observed the changes of cell morphology with the inverted microscope; Observed and counted apoptotic cells through Hoechst33258 fluorescent staining, tested the cell apoptosis by flow cytometry, and detected the expression of Bcl-2 and Bax by western blotting. Results The date of MTT showed that after treated by different concentrations of aspirin,the growth inhibition ratio of SW-13 cells and H295R cells were higher than the control group (P＜0.05), and at the same time, the inhibition ratio would increase when the drug concentration increased ( P＜0. 05 ), with a dose-dependent tendency. When the drug concentration was constant, the inhibition ratio increased with the duration of the drug (P＜0.05), which showed a time-dependent tendency. The number of apoptosis cells and the cell apoptosis rate of both SW-13 and H295R which were treated by different concentrations of aspirin for 48 hours were higher than the control group ( P＜0. 01). According to the analysis of grayscale value of western blotting, Aspirin can increase the expression of Bax ( P＜0.05). On the contrary, the expression of bcl-2 in the experimental group was lower than that in the control group (P＜0.05). Conclusion Aspirin may inhibit the growth of human adrenal cortical cancer cell in vitro and induce its apoptosis, and the possible mechanism may be correlated with the up-regulation of the expression of Bax, and down regulation of Bcl-2 expression.

Effect of simvastatin on angiotensin Ⅱ-stimulated secretion and proliferation of adrenocortical carcinoma H295R cells / 基础医学与临床

Objective To investigate the potential effects of simvastatin on angiotensin Ⅱ-stimulated secretion and proliferation of adrenocortical carcinoma H295R cells.Methods The H295R cells were divided into control group, Angiotensin Ⅱgroup, simvastatin group and Angiotensin Ⅱ plus simvastatin group.Cortisol in medium was determined by chemiluminescent method , and aldosterone was determined by radioimmunoassay .The mRNA expression of 11 beta-hydroxylase ( CYP11B1 ) and aldosterone synthase ( CYP11B2 ) were examined by RT-qPCR.Cell proliferation was detected by MTS method.Results Compared with control group, angiotensin Ⅱincreased the secretion of cortisol and aldosterone, and the expression of CYP11B1 and CYP11B2.Simvastatin decreased cortisol secretion and CYP11B1 mRNA expression ( P<0.05 ) .Simvastatin also inhibited angiotensinⅡ-induced the secretion of cortisol and aldosterone , and the expression of CYP 11 B1 and CYP11 B2 compared with Angiotensin Ⅱgroup ( P<0.05 ) .Angiotensin Ⅱhad no effect on cell proliferation , while simvastatin significantly inhibited cell proliferation .The inhibitory effect of simvastatin on proliferation was enhanced when simvastatin was prescribed with angiotensin Ⅱ( P<0.05 ) .Conclusions Simvastatin inhibits angiotensin Ⅱ-induced secretion of cortisol and aldosterone in H295R cells.Simvastatin inhibits cell proliferation, which could be enhanced by angio-tensin Ⅱ.

ACTHR in regulating aldosterone secretion to angiotensin Ⅱ and potassium chloride stimulation / 中华内分泌外科杂志

Objective To study CYP11B2 mRNA and aldosterone secretion alteration in human adrenocortical carcinoma H295R cell after angiotensin Ⅱ (AT-Ⅱ) and potassium chloride stimulation,and to investigate the effect of adrenocorticotropic hormone receptor (ACTHR) on them.Methods Lentiviral vector was used to increase ACTHR expression.It was transfected into the H295R cells.Similarly,another H295R cells,without ACTHR vector,was used as the control group.ACTHR alteration was measured by Western blot and real-time polymerase chain reaction (RT-PCR).CYP11B2 mRNA was detected at 24 hours after 100 nmol/L AT-Ⅱ/16 mmol/L KCL stimulation,and the amplification of the two groups was compared.Aldosterone was measured by ELISA kit.Results Compared with those in control ceils,the protein and mRNA level of ACTHR in experimental cells were increased 2.4 times and 18 times respectively (P＜0.05).CYP11B2 mRNA of experimental cells was 1.7 times higher than control cells after 24 h stimulation of AT-Ⅱ.Aldosterone production was 121.98+8.31 and 104.05+6.88 ng/L respectively.The former amplification was 2.06 times higher than that of the latter (P＜0.05).Similarly,CYP11B2 mRNA of experimental cells was 19.2 times higher than control cells after 24 h stimulation of KCL.Aldosterone production was 137.67±10.35 and 104.05 ± 6.88 ng/L respectively.The former amplification was 3.13 times higher than that of the latter (P＜0.05).Conclusion Overexpression of ACTHR increases the sensitivity and response of CYP11B2 mRNA and aldosterone to AT-Ⅱ and KCL stimulation,and ACTHR is expected to become a key protein in understanding primary aldosteronism.

Effect and significance of steroidogenic factor-1 gene silencing on proliferation of human adrenocortical H295R cells / 中华泌尿外科杂志

Objective To study the influence of inhibited steroidogenic factor-1 on human adrenocortical H295R cells, and explore its role in the pathogenesis of adrenal tumors. Methods The plasmids pGenesil1-SF-1-shRNA which containing U6 promoter and SF-1-specific short hairpin RNA (shRNA) and pGenesil1-negative-shRNA containing unspecific shRNA were transfected into H295R cell. The expression of SF-1 was measured by Western blot and real-time polymerase chain reaction(RT-PCR). Cell proliferation was analyzed by WST-1 assay and cell count. Ki-67 expression was detected by immunohistochemistry and cell apoptosis was examined by TUNEL assay. Results Compared with those in control cells, the protein and mRNA level of SF-1- transfected cells were reduced by 69.7% and 71.2% (P＜0. 01). WST-1 and cell count method showed that SF-1 gene silencing obviously inhibited cell proliferation(P＜0. 01). By contrast, there was a 3. 7-fold increase in the percentage of apoptotic H295R cells in SF-1-inhibited group than that of control group (P＜0. 01). Immunohistochemistry showed that Ki-67 positive cells in SF-1-inhibited cells were lower than the negative control cells (16.90±2.17) % and (33. 48±3.16)%,(P＜0. 01). Conclusion SF-1 gene silencing can inhibit the proliferation of adrenocortical cells, and it is expected to become a key protein in understanding pathogenesis of adrenal tumors or treating them.

Research Progress of Aplication of H295R Cell Line for Screening Environmental Steroid Hormone Disruptors / 环境与健康杂志

H295R cells can express all the key enzymes involved in the synthesis of steroid hormones,and have the ability to produce the steroid hormones found in the adult human adrenal cortex. Good correlation of response to toxicity has been reported between H295R cell line and normal adult human adrenal cells. H295R cell line can be used to evaluate effects of chemicals on gene expression,enzymatic activities and hormone production,and to explore the mode of action of chemicals on steroidgenesis. H295R cell line has been suggested as a potentially useful tool for screening environmental steroid hormone disruptors in vitro. The application of H295R cell line for screening environmental steroid hormone disruptors in vitro and its current issues are reviewed in this article.