A sensitive and selective LC-MS/MS analysis coupled with an online sample enrichment technique for H295R steroidogenesis assay and its application in the investigation of the effect of sildenafil on steroidogenesis.

An in vitro steroidogenesis assay using H295R human adenocarcinoma cells is a useful tool for the fast identification of compounds that affect the production of testosterone and 17ß-estradiol. Selective and sensitive hormone measurement by liquid chromatography-tandem mass spectrometry (LC-MS/MS) can make this assay more reliable. Therefore, in the present study, a sensitive and selective method for the quantification of testosterone and 17ß-estradiol in the H295R steroidogenesis assay was developed and fully validated using LC-MS/MS coupled with an online sample enrichment technique. To prove its usefulness, the method developed was applied to investigate the effect of sildenafil on steroidogenesis. Cell medium samples were diluted and prepared using solid-phase extraction. The samples were prepared on ice and were not kept for more than 30 min to prevent degradation of hormones. The extracts were dried, reconstituted, filtered, and analyzed by LC-MS/MS with polarity switching electrospray ionization. The validation results for selectivity, matrix effect, recovery, linearity, precision, and accuracy were satisfactory. The limits of detection for testosterone and 17ß-estradiol were 5 and 10 pg/mL, respectively, and the limit of quantification for both testosterone and 17ß-estradiol was 10 pg/mL, which was in accordance with the OECD guideline. No degradation was observed under the storage conditions for 7 and 14 days at -80 °C as well as after three freeze-thaw cycles, whereas 17ß-estradiol was degraded after 1 h on ice during sample processing. The method developed was successfully used for the investigation of the effect of sildenafil on steroidogenesis. This method can be very useful for the initial selection of drugs with androgenic and/or estrogenic effects for specific purposes, e.g., in the selection of drugs that are used to reverse the effects of chemical castration.

Chemopreventive effects of Ginkgo biloba extract in estrogen-negative human breast cancer cells.

Excessive level of estrogen is considered as a main cause of breast cancer, therefore, many studies have focused on estrogen receptor (ER)-positive breast cancer, even though ER-negative cancer has a poor prognosis than ER-positive breast cancer. We evaluated the anti-cancer effects of Ginkgo biloba extract (GBE) in estrogen-independent breast cancer. GBE has been traditionally used as a platelet activating factor, a circulatory stimulant, a tonic, and anti-asthmatic drug, and anti-cancer agent. However, anti-cancer effects of GBE on ER-negative breast cancer have not been proved yet. In this study, we tested chemotherapeutic potential of GBE in the MDA-MB-231 (ER-negative) human breast cancer cell line. Our results showed that cytotoxicity effects of GBE in MDA-MB-231 lead to DNA fragmentation at high concentrations (500 and 1,000 µg/ml). Caspase-3 was significantly activated and mRNA levels of apoptosis-related genes (Bcl-2 and Bax) were altered. These results indicate that GBE induces apoptosis in MDA-MB-231 cells. It is presumed that GBE has chemopreventive effects in ER-independent breast cancer through anti-proliferation and apoptosis-inducing activities.

The inhibitory effects of the standardized extracts of Ginkgo biloba on Aromatase activity in JEG-3 human choriocarcinoma cells.

Breast cancer is the most common cancer in women worldwide. There are many endocrine adjuvant therapies for breast cancer patients that are categorized according to their mechanisms. Among them, aromatase inhibitors (AIs) that block the synthesis of estrogens have proven superiority compared with tamoxifen and have replaced it as a first-line hormonal therapy. However, AIs also have limitations due to their side effects - increased rate of bone loss and musculoskeletal complaints. We therefore need new candidate AIs with fewer side effects. The extracts of Ginkgo biloba (EGb), which contain phytochemicals from the tree, had biphasic effects for estrogens and osteoporosis-inhibiting activities in our previous experiments. In this study, we explored the possibility of EGb as an AI and their mechanisms. Aromatase activities were inhibited by EGb both in JEG-3 cells and in recombinant CYP19 microsomes. The results of polymerase chain reaction for aromatase from a coding sequence and specific promoter sequences (exon I.a, exon I.c) in JEG-3 cells as well as the results of reporter gene assays showed that EGb dose-dependently decreased the aromatase gene expression. The decreased protein levels were demonstrated by Western blotting. From these results, we concluded that EGb could act as an AI at both the enzyme and transcriptional levels.

Estrogenic effects and their action mechanism of the major active components of party pill drugs.

Benzylpiperazine (BZP) and trifluoromethylphenylpiperazine (TFMPP) are commonly used constituents of party pill drugs. They are reported to induce psychoactive effects such as euphoria and provide effects similar with other illicit drugs such as methylenedioxymethamphetamine (MDMA). A great deal of evidence has proven that party pills, as alternatives for MDMA, exert harmful effects on users. However, their toxicological effects have not been fully understood and endocrine disruptive effects are still unknown. In this study, we identified estrogenic effects of BZP and TFMPP by using in vitro and in vivo assays. BZP and TFMPP stimulated cell proliferation in a dose-dependent manner, while co-treatment with tamoxifen and BZP or TFMPP showed a decrease of E(2)-induced cell proliferation. In an estrogen sensitive reporter gene assay, BZP and TFMPP significantly increased transcriptional activities of party pill drugs. In addition, ER-related genes, PR and pS2, were significantly stimulated by BZP and TFMPP. These results indicated that BZP and TFMPP could have estrogenic activities related to the ER-mediated pathway. Unlike the in vitro assay results, BZP and TFMPP did not show significant effects on weight increase in a rodent uterotrophic assay. However, further studies would be necessary to verify the estrogenic activities of BZP and TFMPP by a chronic exposure animal study.

Genotoxic effects of silver nanoparticles stimulated by oxidative stress in human normal bronchial epithelial (BEAS-2B) cells.

Many classes of silver nanoparticles (Ag-NPs) have been synthesized and widely applied, but the genotoxicity of Ag-NPs and the factors leading to genotoxicity remain unknown. Therefore, the purpose of this study is to elucidate the genotoxic effects of Ag-NPs in lung and the role of oxidative stress on the genotoxic effects of Ag-NPs. For this, Ag-NPs were completely dispersed in medium by sonication and filtration. The Ag-NPs dispersed in medium were 43-260nm in size. We observed distinct uptake of Ag-NPs into BEAS-2B cells. The Ag-NPs aggregates were wrapped with an endocytic vesicle within the cytoplasm and nucleus of BEAS-2B cells. In the comet assay and micronucleus (MN) assay for BEAS-2B cells, Ag-NPs stimulated DNA breakage and MN formation in a dose-dependent manner. The genotoxic effect of Ag-NPs was partially blocked by scavengers. In particular, of the scavengers tested, superoxide dismutase most significantly blocked the genotoxic effects in both the cytokinesis-block MN assay and the comet assay. In the modified comet assay, Ag-NPs induced a significant increase in oxidative DNA damage. Furthermore, in the oxidative stress assay, Ag-NPs significantly increased the reactive oxygen radicals. These results suggest that Ag-NPs have genotoxic effects in BEAS-2B cells and that oxidative stress stimulated by Ag-NPs may be an important factor in their genotoxic effects.