Ginkgo biloba extract (EGb761) did not express estrogenic activity in an immature rat uterotrophic assay.

Ginkgo biloba is a dioecious tree that has been used in traditional Chinese medicine for about 5,000 years. In previous studies on ginkgo biloba extract (EGb761) using in vitro systems, we confirmed that EGb761 has biphasic effects on estrogenicity. In this study, we evaluated the agonistic and antagonistic activities of EGb761 using a uterotrophic assay in immature female rats. To evaluate agonistic and antagonistic effects of EGb761 on uterus, 21-day-old immature Sprague-Dawley (SD) female rats were treated with EGb761 (100, 200, or 400 mg/kg) by oral gavage, 10 µg/kg of estradiol (E2) or 1 mg/kg tamoxifen (TM) by subcutaneous injection, or with EGb761 plus E2 or TM for 3 consecutive days. At the end of the treatment period, animals were sacrificed and their body weights and organ weights (liver, lung, spleen and kidney) were measured. In addition, estrogen-related gene expressions (IGFBP-1 in liver and CaBP-9 in uterus) were determined. During the experiment, no animal showed clinical signs, a change in body weight or died. EGb761 treatment alone had no effect on absolute/relative uterine weight, luminal epithelial cell height (LECH, µm), or luminal circumference (LC, µm). In addition, uterine weights, LECHs, and LC induced by E2 or TM were not significantly changed by EGb761 at any dose. These results collectively suggested EGb761 has no agonistic/antagonistic effects in utero.

Morphological transformation induced by silver nanoparticles in a Balb/c 3T3 A31-1-1 mouse cell model to evaluate in vitro carcinogenic potential.

Carcinogenesis is a complex process involved in genotoxic and non-genotoxic pathways. The carcinogenic potential of silver nanoparticles (AgNPs) has been predicted by examining their genotoxic effects using several in vitro and in vivo models. However, there is no little information regarding the non-genotoxic effects of AgNPs related to carcinogenesis. The in vitro cell transformation assay (CTA) provides specific and sensitive evidence for predicting the tumorigenic potential of a chemical, which cannot be obtained by genotoxicity testing. Therefore, we carried out CTA in Balb/c 3T3 A31-1-1 cells to evaluate the carcinogenic potential of AgNPs. Colony-forming efficiency and crystal violet assays were carried out to determine the cytotoxicity of AgNPs. A cytokinesis-block micronucleus (CBMN) assay and CTA were performed using Balb/c 3T3 A31-1-1 cells to predict the in vitro carcinogenic potential of AgNPs. In the CBMN assay, AgNPs (10.6 µg/mL) induced a significant increase in micronucleus formation indicating a genotoxic effect. Thus, AgNPs could be an initiator of carcinogenesis. In the CTA, used to assess the carcinogenic potential of AgNPs, cells exposed to AgNPs for 72 hours showed significantly induced morphological neoplastic transformation at all tested doses (0.17, 0.66, 2.65, 5.30, and 10.60 µg/mL), and the transformation frequency was significantly increased in a dose-dependent manner. These results indicate that short-term exposure (72 hours) to AgNPs had in vitro carcinogenetic potency in Balb/c 3T3 A31-1-1 cells.

Long-term exposures to low doses of silver nanoparticles enhanced in vitro malignant cell transformation in non-tumorigenic BEAS-2B cells.

To predict carcinogenic potential of AgNPs on the respiratory system, BEAS-2B cells (human bronchial epithelial cells) were chronically exposed to low- and non-cytotoxic dose (0.13 and 1.33µg/ml) of AgNPs for 4months (#40 passages). To assess malignant cell transformation of chronic exposure to AgNPs, several bioassays including anchorage independent agar colony formation, cell migration/invasion assay, and epithelial-mesenchymal transition (EMT) were performed in BEAS-2B cells. Chronic exposure to AgNPs showed a significant increase of anchorage independent agar colony formation and cell migration/invasion. EMT, which is the loss of epithelial markers (E-Cadherin and Keratin) and the gain of mesenchymal marker (N-cadherin and Vimentin), was induced by chronic exposure to AgNPs. These responses indicated that chronic exposure to AgNPs could acquire characteristics of tumorigenic cells from normal BEAS-2B cells. In addition, caspase-3, p-p53, p-p38, and p-JNK were significantly decreased, while p-ERK1/2 was significantly increased. MMP-9 related to cell migration/invasion was upregulated, while a MMP-9 inhibitor, TIMP-1 was down-regulated. These results indicated that BEAS-2B cells exposed to AgNPs could induce anti-apoptotic response/anoikis resistance, and cell migration/invasion by complex regulation of MAPK kinase (p38, JNK, and ERK) and p53 signaling pathways. Therefore, we suggested that long-term exposure to low-dose of AgNPs could enhance malignant cell transformation in non-tumorigenic BEAS-2B cells. Our findings provide useful information needed to assess the carcinogenic potential of AgNPs.

The effects of the standardized extracts of Ginkgo biloba on steroidogenesis pathways and aromatase activity in H295R human adrenocortical carcinoma cells.

OBJECTIVES: Aromatase inhibitors that block estrogen synthesis are a proven first-line hormonal therapy for postmenopausal breast cancer. Although it is known that standardized extract of Ginkgo biloba (EGb761) induces anti-carcinogenic effects like the aromatase inhibitors, the effects of EGb761 on steroidogenesis have not been studied yet. Therefore, the effects of EGb761 on steroidogenesis and aromatase activity was studied using a H295R cell model, which was a good in vitro model to predict effects on human adrenal steroidogenesis. METHODS: Cortisol, aldosterone, testosterone, and 17ß-estradiol were evaluated in the H295R cells by competitive enzyme-linked immunospecific assay after exposure to EGb761. Real-time polymerase chain reaction were performed to evaluate effects on critical genes in steroid hormone production, specifically cytochrome P450 (CYP11/ 17/19/21) and the hydroxysteroid dehydrogenases (3ß-HSD2 and 17ß-HSD1/4). Finally, aromatase activities were measured with a tritiated water-release assay and by western blotting analysis. RESULTS: H295R cells exposed to EGb761 (10 and 100 µg/mL) showed a significant decrease in 17ß-estradiol and testosterone, but no change in aldosterone or cortisol. Genes (CYP19 and 17ß-HSD1) related to the estrogen steroidogenesis were significantly decreased by EGb761. EGb761 treatment of H295R cells resulted in a significant decrease of aromatase activity as measured by the direct and indirect assays. The coding sequence/ Exon PII of CYP19 gene transcript and protein level of CYP19 were significantly decreased by EGb761. CONCLUSIONS: These results suggest that EGb761 could regulate steroidogenesis-related genes such as CYP19 and 17ß-HSD1, and lead to a decrease in 17ß-estradiol and testosterone. The present study provides good information on potential therapeutic effects of EGb761 on estrogen dependent breast cancer.