Can't get full text to see how high they dosed.
not quite the same without figures, but here is the text from the pdf
I don't have time to read it tonight but I'll check it out tomorrow morn
Introduction
Green tea (Camellia sinensis) is one of the most commonly consumed beverages worldwide. Its active components are reported to have several biological properties, including cancer chemoprevention, inhibition of tumour cell growth, antiviral and anti-inflammatory activities 1, antioxidant activity 2, 3 and inhibitory effects on several enzymes, such as aromatase 4, 5, angiotensin converting enzyme 6 and thyroid peroxidase 7. Dried leaves of C. sinensis contain polyphenols (30%–36%), principally flavanols, more commonly known as catechins 8. The predominant catechins are epigallocatechin-3-gallate (EGCG), epicatechin-3 gallate (ECG), epigallocatechin (EGC) and epicatechin (EC).
The effects of catechins on the male reproductive system have been described. Epidemiological and laboratory studies suggest an association between diet and androgens that can alter prostate cancer risk 9, 10, 11. It has been shown that parenteral injection of EGCG can suppress human prostate and breast tumour growth in athymic mice 12 and reduce the weight of testes and accessory reproductive organs, as well the circulating level of luteinizing hormone (LH) and testosterone in the intact rat 13. Although the antigonadotropic effect of catechins is explained as a secondary effect of EGCG on food intake 13 or on aromatase activity 4, 5, a modulatory function could be present even at the gonadal level.
Currently, there is no evidence for a direct effect of green tea catechins on testicular steroidogenesis or on the enzymes involved in androgen production. Although the involvement of the protein kinase A (PKA) and protein kinase C (PKC) signalling pathways in testicular androgen production is well known 14, 15, it is unclear whether green tea catechins modulate these pathways in Leydig cells. There is evidence that EGCG and other flavonoids can modulate the PKC 16, 17 and PKA signalling pathways in other animal models 16, 18. The aim of this study was to investigate the direct in vitro effects of green tea extract (GTE) and its purified catechins on the basal and the PKA- and PKC-stimulated testosterone production by rat Leydig cells.
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Materials and methods
Materials
Hank's balanced salt solution (HBSS) and Medium 199 were obtained from Gibco (Grand Island, NY, USA). Collagenase (Type I), soybean trypsin inhibitor, leupeptin, phorbol 12,13-dibutyrate (PDBu), human chorionic gonadotropin (hCG), N6,2'-O-dibutyryladenosine3':5'-cyclic monophosphate (dbcAMP), EC, EGCG from green tea, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), dimethylsulphoxide (DMSO), 22(R)-hydroxycholesterol and 4-androstene-3,17-dione were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Bovine serum albumin (BSA, fraction V) was obtained from Miles (Naperville, IL, USA). LH releasing hormone (LHRH) was purchased from Peninsula (San Carlos, CA, USA). Percoll was purchased from Pharmacia (Uppsala, Sweden).
Green tea extract preparation
Green tea extract was prepared according to Wang et al. 19 with slight modifications. Five grams of dry green tea leaves were infused for 5 min in 100 mL of boiling saline (90°C), allowed to cool to room temperature and then filtered. The resulting clear solution is similar to tea brews consumed by humans. The amount of solid matter present in this infusion was determined by drying samples (10 mL) in an oven overnight at 100°C and weighing the dry residue. The dry weight was determined to be 69.2 mg for 5 g of green tea leaves, making the concentration of this infusion 0.692% (w/v). The extracts were freshly prepared on a daily basis. This preparation is comprised of approximately 27% catechins, 8.0% caffeine and 0.4% theobromine, and EGCG represents greater than or equal to 50% of total catechins 19.
Animals and Leydig cell-enriched preparation
Adult (70–80 days old) male Wistar rats weighing 200–300 g were kept in a controlled environment (temperature 25–29°C; lights on from 05:00 to 18:00) with free access to standard laboratory chow and tap water. Isolation and purification of rat Leydig cell-enriched preparations were performed as described by Hedger and Eddy 15, 20. The rats were killed by ether anaesthesia and the testes were quickly removed and decapsulated. The decapsulated testes were incubated in an enzyme solution of 0.5 mg mL-1 collagenase, 0.2 mg mL-1 soybean trypsin inhibitor and 5 mug mL-1 leupeptin in Hank's balanced salt solution containing 0.1% BSA (HBSS/BSA), pH 7.4, in a shaking water bath (20 min, 90 Hz, 34°C). The dispersed testes were suspended in a final volume of 50 mL HBSS/BSA and the dissociated tubules were allowed to settle (5 min). The supernatant was filtered and washed with 5 mL HBSS/BSA. The filtered cell suspension was centrifuged (150 times g, 15 min, 20°C), and the pellet was re-suspended in 5 mL HBSS/BSA, loaded onto the top of a discontinuous Percoll density gradient (20%, 35%, 43%, 68% and 90%) and centrifuged at 800 times g for 30 min at 20°C. Cells in the 43%–68% interface (specific gravity: 1.0640–1.0960 g mL-1) were collected, washed twice with M199 containing 0.1% BSA, re-suspended in M199/0.1% BSA and used immediately for the experiments.
Cell viability
To assess the effects of GTE and EGCG on cell viability, the Trypan blue exclusion and MTT reduction assays were used. These assays are widely used screening methods to measure plasma membrane integrity and active mitochondrial function, respectively. The Trypan blue assay was performed after incubating cells for 3 h with the different doses of GTE or EGCG. Cells were incubated with Trypan blue (0.5%) for 20 min and the resulting percentage of blue cells, indicating a capture of the colourant due to plasma membrane rupture, were counted. Normal cell viability was considered to be 90%–95% colourless cells. The MTT assay is based on the reduction of a soluble pale yellow tetrazolium dye to water-insoluble purple formazan crystals in living cells 21. Cells (0.3 times 106 per mL) were incubated in polypropylene tubes with GTE (69.2 mug mL-1) or EGCG (10, 50 and 100 mug mL-1) for 2 h at 34°C under 95% O2 and 5% CO2. After this time, the cells were washed twice with M199 to remove the GTE/EGCG and then resuspended in 100 muL of fresh M199. Subsequently, 25 muL of MTT in PBS (5 mg mL-1) was added to the cell suspensions followed by 3 h of incubation at 37°C. This procedure avoids a direct reaction between the polyphenols and the MTT. It is known that polyphenols can reduce MTT in lieu of living cells, so results obtained may not reflect the true cell viability and instead mask the effects of polyphenols 22. After this incubation period, the cell suspensions were centrifuged at 400 times g for 5 min. The supernatant was removed and 100 muL DMSO was added to each tube to dissolve formazan crystals overnight. The dissolved pellet was transferred to microplates and the absorbance was measured at 630 nm in an ELISA reader. Appropriate controls were included (i.e., medium plus MTT without cells or cells treated with the toxic reagent saponin 0.1%). The animal care committee of the Federal University of Pernambuco (Recife, PE, Brazil) approved all treatments.
In vitro testosterone secretion
Cells (0.3 times 106 cells per 0.5 mL) were treated for 3 h with M199, GTE (6.92–692.0 mug mL-1), EGCG (5–200 mug mL-1) or EC (200 mug mL-1) in the absence or presence of hCG (1 mIU mL-1), dbcAMP (1 mmol L-1), LHRH (10-7 mol L-1), PDBu (200 nmol L-1) or androstenedione (1–100 mumol L-1) in a shaking water bath (60 Hz, 34°C) under an atmosphere of 95% O2 and 5% CO2. It is noteworthy that a cup of GTE (100 mL) usually contains 50–150 mg mL-1 of tea polyphenols 23. To study the reversibility of the inhibitory effect of GTE and EGCG, cells were preincubated for 15 min with M199 GTE (69.2 mug mL-1) or EGCG (100 mug mL-1), washed with fresh medium, allowed to recuperate for 60 min, and then incubated for 2 h with 0.5 mUI mL-1 hCG, 10-7 mol L-1 LHRH, 20 mumol L-1 22-hydroxycholesterol or 10 mumol L-1 androstenedione. This 15-min preincubation period was chosen because it was sufficient to detect an inhibitory effect of GTE or EGCG on testosterone production similar to that seen after 3 h incubation (data not shown). At the end of the second incubation, the cells were centrifuged and the supernatant was collected and stored at -20°C until used for testosterone measurement by radioimmunological analysis (RIA).
RIA and statistical analysis
Testosterone was measured directly (without extraction) in the incubation medium by a charcoal–dextran RIA method 24 that employs [3H]-testosterone as tracer and a primary antiserum raised in rabbits in our laboratory against testosterone-3-(0-carboxymethyl)oxime:BSA. Intra- and inter-assay coefficients of variation were 8.1% and 15.1%, respectively. The testosterone antibody showed < 0.1% cross-reactivity with androstenedione, dehydroepiandrosterone, androsterone, 17alpha-hydroxyprogesterone, beta-estradiol and estrone. None of the substances tested interfered with the assays. The data from the different analyses were reported as the mean plusminus SEM. of triplicate determinations and were representative of results obtained in at least two similar experiments. One-way ANOVA and Dunnet tests were used to examine differences among control and GTE/EGCG/EC-treated cells. P-values less than 0.05 were considered to be statistically significant.
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Results
GTE and EGCG inhibit basal and hCG-stimulated testosterone production
The modulatory effects of GTE and its pure constituents (EGCG and EC) were tested by incubating Leydig cell-enriched preparations with GTE or EGCG followed by determination of testosterone levels in the incubation medium. Cells were incubated with various concentrations (three different orders of magnitude) of GTE or EGCG. In Figures 1 and 2, Leydig cells were treated with and without GTE or the catechins EGCG and EC, respectively, in the absence or presence of maximum stimulation by hCG (indirect PKA activator). GTE and EGCG both produced an inhibitory effect on basal and stimulated testosterone production. At 6.92, 69.2 and 692 mug mL-1, GTE inhibited basal testosterone levels by 27.5% plusminus 4.8%, 60.5 plusminus 3.9% and 93.1% plusminus 0.2%, respectively, whereas EGCG at 5, 50 and 200 mug mL-1 produced 24.0% plusminus 6.7%, 37.9% plusminus 1.4% and 58.2% plusminus 1.4% inhibition, respectively. The inhibitory effects of 6.92, 69.2 and 692 mug mL-1 GTE on hCG-stimulated testosterone production were 11.2% plusminus 6.0%, 56.8% plusminus 1.5% and 99.4% plusminus 0.03%, respectively, whereas the inhibitory effect of 50 and 200 mug mL-1 EGCG on stimulation with hCG was 46.5% plusminus 7.8% and 98.8% plusminus 0.06%, respectively (the 5 mug mL-1 dose showed no significant inhibitory effect). Exposure to EC at 200 mug mL-1 did not induce a significant inhibitory effect on basal or stimulated testosterone production. To evaluate whether the effect of GTE or EGCG on testosterone production was due to a decrease in cell viability, the Trypan blue exclusion and MTT reduction assays were used. The Trypan blue exclusion test for cell viability showed that the inhibition was not due to GTE-induced toxicity at the concentrations of 6.92 mug mL-1 or 69.2 mug mL-1 or EGCG toxicity at any concentration used. This test showed that at the above-cited concentrations, the percentages of viable cells obtained after 3 h of incubation were comparable between the GTE- or EGCG-treated (91%) cells and the controls (95%). At the maximum concentration of GTE used (692 mug mL-1), cell viability was 80% of control. This difference (10%–15% fewer viable cells compared with the control group) could have contributed to the observed decrease in testosterone production. For this reason, this concentration was not used in further experiments in this study. When measured by MTT reduction, cell viability was not modified by any concentration of GTE or EGCG used. The two assays for cell viability showed that inhibition of testosterone production was not due to the toxicity of GTE (6.92 mug mL-1 or 69.2 mug mL-1) or EGCG (10–100 mug mL-1).
Figure 1.
Figure 1 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact or the author
Treatment of Leydig cells with different concentrations of green tea extract (GTE) affects basal and hCG-induced testosterone production. Leydig cells (0.3 times 106 per 0.5 mL) were incubated for 3 h with hCG (1 mIU mL-1) in the presence or absence of 6.92, 69.2 or 692 mug mL-1 GTE. Results are the mean plusminus SEM of three determinations repeated in two independent experiments. *P <0.0001, #P < 0.01, ##P < 0.0001, copared with the control.
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Figure 2.
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Treatment of Leydig cells with different concentrations of epigallocatechin-3-gallate (EGCG) affects basal and hCG-induced testosterone production. Leydig cells (0.3 times 106 per 0.5 mL) were incubated for 3 h with hCG (1 mIU mL-1) in the presence or absence of 5, 50 or 200 mug mL-1 EGCG or 200 mug mL-1 EC. Results are the mean plusminus SEM of three determinations repeated in two independent experiments. *P < 0.05, **P < 0.01, #P < 0.001, compared with the control.
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GTE and EGCG inhibit testosterone production elicited by activation of PKA and PKC
The action of GTE and EGCG was further investigated in the presence of a direct PKA activator, dbcAMP, and both an indirect and direct PKC activator, LHRH and PDBu, respectively. Table 1 shows testosterone production in the presence of each maximum stimulus alone or with GTE or EGCG. Testosterone production was reduced completely in all groups (to < 98%), regardless of the nature of the stimulus. These results indicate a probable interaction of GTE/EGCG with the signal-transduction process somewhere downstream of PKA and PKC.
Table 1 - Effect of GTE or EGCG added together with dbcAMP, LHRH or PDBu on testosterone production in rat Leydig cells preparation.
Table 1 - Effect of GTE or EGCG added together with dbcAMP, LHRH or PDBu on testosterone production in rat Leydig cells preparation. - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact or the authorFull table
EGCG inhibits the stimulatory effect of androstenedione on testosterone production
To further evaluate the effect of EGCG, the steroidogenic precursor androstenedione was used to support testosterone production (a measure of 17beta-hydroxysteroid dehydrogenase [17beta-HSD] activity). Androstenedione crosses the cell membrane and moves to the smooth endoplasmic reticulum of Leydig cells in which, after binding to 17beta-HSD, it is converted to testosterone. Several doses of androstenedione (1, 5, 10 and 20 mumol L-1) were used. These high concentrations were used to obviate the interference of any endogenous precursor. As shown in Figure 3, EGCG decreased testosterone production by approximately 50% at all concentrations of the precursor used. No significant inhibitory effect was observed in cells treated with EC.
Figure 3.
Figure 3 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact or the author
Effects of epigallocatechin-3-gallate (EGCG) on androstenedione-stimulated testosterone production. Leydig cells (0.3 times 106 per 0.5 mL) were incubated for 3 h with androstenedione (1–20 mumol L-1) in the absence (black square) or presence of 100 mug mL-1 EGCG (black triangle) or EC (white square). Results are the mean plusminus SEM of three determinations repeated in two independent experiments. *P < 0.001, **P < 0.0001, compared with respective androstenedione alone.
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