Fig. 1. Effects of GBE in the presence of E₂ on the proliferation of ER-positive MCF-7 cells. The MCF-7 cells were incubated in DMEM supplemented with CDFBS 10% with a combination of E₂ and Ginkgo biloba extract (GBE) or tamoxifen (TM) for 144 h. Incubation with ethanol or DMSO alone was performed as the control and the final concentration of ethanol in the medium never exceeded 0.2%. After the cells were incubated for 144 h, a SRB assay was conducted to measure the level of cell proliferation. The proliferative effect of the compounds relative to E₂ (10⁻¹⁰ M, 100%) is represented as the relative proliferative effect (RPE). The results are expressed as the mean ± S.D. of three separate experiments for each data point. Comparisons were made using a Student's t-tests. Values are significantly different from each E₂, E₂ (10⁻¹¹ M, RPE = 92.18%) [p < 0.05, p < 0.01 (B)] or E₂ (10⁻⁷ M, RPE = 75.69%) [^#p < 0.05, ^##p < 0.01 (C)].

Fig. 2. Effects of GBE in the presence of E₂ on the luciferase activity (A), and pS2 gene expression (B) in MCF-7 cells. (A) The steroid-deprived MCF-7 cells were transiently transfected with the pERE-luc construct using lipofectamine. This cell was incubated in DMEM supplemented with CDFBS 10% with a combination of E₂ and GBE for 48 h. The total luciferase activity was measured using a Luciferase assay Reagent (Promega). (B) MCF-7 cells were incubated in DMEM supplemented with the steroid-free CDFBS 10% with E₂ or a combination of E₂ (10⁻¹⁰ M) and GBE for 48 h. After the cells were incubated for 48 h, the total RNA was extracted using a TRIzol reagent (Gibco BRL). The pS2 mRNA levels were measured by RT-PCR and normalized using α-actin mRNA as the internal standard. One microgram of the total RNA was applied to the RT-PCR reaction for 25 cycles with the pS2 (336 bp) and α-actin (240 bp) primers. The RT-PCR products were run on an ethidium bromide stained 1.5% agarose gel, which was then scanned using a Gel documentation & Analysis system. The results are expressed as the mean ± S.D. of three separate experiments for each data point. Comparisons were made using a Student's t-tests. Values are significantly different from E₂ (10⁻¹¹ M) [^**p < 0.01] or E₂ (10⁻¹⁰ M) [^#p < 0.05, ^##p < 0.01]. Con, 0.2% ethanol; GBE, Ginkgo biloba extract; TM, tamoxifen (10⁻⁶ M).

Fig. 3. Agonistic and antagonistic effect of EROD activity by GBE. The MCF-7 (black symbol) and AhR-deficient MCF-7 cells (white symbol) were incubated in DMEM supplemented with FBS 5% and various GBE concentrations with or without TCDD (10⁻⁹ M) for 24 h. Incubation with ethanol alone was performed as a negative control, and the final concentration of ethanol in the medium never exceeded 0.2%. After the cells were incubated for 24 h, the EROD activity was determined as described in Section 2. The EROD activity of the compounds relative to TCDD (10⁻⁹ M, 100%) is represented as the TCDD induction response (TIR). The results are expressed as a mean ± S.D. of three separate experiments for each data point. Comparisons were made using a Student's t-tests. The values are significantly different from TCDD 10⁻⁹ M (TIR 100%, ^**p < 0.01) or the vehicle control (TIR 0%, ^##p < 0.01).

Fig. 4. Agonistic (A) and antagonistic (B) effect of CYP1A1 mRNA expression by GBE. The MCF-7 cells were incubated in DMEM supplemented with FBS 5% with a combination of GBE and TCDD for 24 h. Ethanol was used as a negative control (Con), and TCDD was used as a positive control. The final concentration of ethanol in the medium never exceeded 0.2%. After the cells were incubated for 24 h, the total RNA was extracted using a TRIzol reagent. The CYP1A1 mRNA levels were measured by RT-PCR and normalized using α-actin mRNA as the internal standard. RT-PCR was conducted as described in Section 2. One microgram of the total RNA was applied to the RT-PCR reaction for 25 cycles with the CYP1A1 and α-actin primers. The RT-PCR products were run on ethidium bromide stained 1.5% agarose gel, which was then scanned using the Gel documentation & Analysis system. The results are expressed as a mean ± S.D. of three separate experiments for each data point. Comparisons were made using a Student's t-tests. The values are significantly different from the vehicle control (^**p < 0.01) or TCDD 10⁻⁹ M (^##p < 0.01).

Fig. 5. E₂ metabolism by GBE. MCF-7 cells were incubated in DMEM supplemented with CDFBS 10% with GBE or with a combination of GBE and TCDD for 72 h. Ethanol was used as a negative control (Con), and TCDD was used as a positive control. The final concentration of ethanol in the medium never exceeded 0.2%. After the cells were incubated for 72 h, they were treated with 10 nM [³H] E₂. The medium was collected after 24 h, and the [³H] E₂ was separated from the media by adsorption on charcoal. The radioactivity was determined using a liquid scintillation counter. The E₂ metabolism effect of the compounds relative to TCDD (10⁻⁹ M, 100%) are represented as a % of the control. The results are expressed as a means ± S.D. of three separate experiments for each data point. Comparisons were made using a Student's t-tests. Values are significantly different from Con (0.2% ethanol, ^*p < 0.05, ^**p < 0.01).

Fig. 6. Aromatase activity by GBE in the JEG-3 cells. JEG-3 cells were incubated in DMEM supplemented with CDFBS 3% with GBE for 8 h. Ethanol was used as a negative control (Con), and 4-hydroxyandrostenedione (HA, 10⁻⁷ M) was used as a positive control. The final concentration of ethanol in the medium never exceeded 0.2%. After the cells were incubated for 8 h, they were treated with 54 nM [1β-³H] androstenedione for 1 h. The aromatase activity was determined by a tritium release assay. ³H₂O formed by the aromatase activity was measured as described in Section 2. The aromatase activity is expressed as the amount of tritiated water (pmol) formed per hour per milligram of protein. The results are expressed as the mean ± S.D. of three separate experiments for each data point. Comparisons were made using a Student's t-test. Values are significantly different from Con (0.2% ethanol, ^*p < 0.05, ^**p < 0.01).