Inhibitory effects of gossypol on adrenal function Yan-Wan
Wu,
Constance L.
Chik, Barry D. Albertson, W.
Marston Linehan and Richard A. Knazek
Developmental Endocrinology Branch', NICHD, and Surgery Branch2, NCI, NIH, Bethesda, Maryland,
Abstract. Gossypol, an antifertility agent, has inhibitory actions on many membrane-associated enzymes, suggesting that this agent might have a generalized effect on cell membranes. This hypothesis was examined in the present membranes and dispersed cells prepared from human and rat adrenal glands. Four parameters were determined: microviscosity as measured by fluorescence polarization of human adrenal microsomal- and mitochondrial-enriched membranes, adrenal steroidogenic enzymes; and cAMP and cortisol responses to ACTH. It was found that gossypol increased the polarization constants of microsomes and mitochondria in a dose-dependent manner. Of the three adrenal enzymes tested, both 3\g=b\-hydroxysteroiddehydrogenase \g=D\5-\g=D\4isomerase and 11-hydroxylase were inhibited by gossypol, but not 21\x=req-\ hydroxylase. Using intact human adrenocortical cells, high doses of gossypol also inhibited the ACTH-stimulated cAMP and cortisol levels. The in vivo corticosterone response to ACTH in rats subjected to chronic gossypol treatment was also found to be reduced. These findings suggest that gossypol has multiple effects on adrenal function. Its effects on membrane microviscosity, adrenal steroidogenesis, cAMP and corticosterone responses to ACTH stimulation probably occur through a generalized membrane effect.
study using
In the 1930's and 40's outbreaks of unexplained were observed in several provinces of China. Entire villages went for a decade or more without a recorded birth. The agent causing the infertility was identified as gossypol, a binapthalenic component of cottonseed oil, used by citizens of these regions as cooking oil. Since that time, nu¬ merous studies have investigated the pharmaco¬ logy and toxicology of gossypol, especially as an
infertility
USA
antifertility agent in males ( 1 ). Although the mecha¬ nism of action of gossypol remains to be deter¬ mined, recent studies indicate that gossypol alters the activity of many membrane-associated enzymes including those involved in steroidogenesis (2-8). This raised the possibility that gossypol may affect adrenocortical function via a generalized action on membrane and steroidogenesis. Previous studies in our laboratory have demon¬ strated that a decrease in membrane microviscosity results in a concomitant increase in the detectable number of membrane-associated prolactin recep¬ tors (9-10). In contradistinction, an increase in membrane microviscosity induced by incorpora¬ tion of long-chain saturated fatty acids was accom¬ panied by a decrease in the responsiveness of human adrenal cells to ACTH stimulation in vitro, thus providing a possible explanation for the hypoadrenal state observed in adrenoleukodystrophy and adrenomyeloneuropathy (11). Other studies have also correlated changes in membrane micro¬ viscosity with alterations in the functionality of hor¬ mone receptors, antigenic determinants, and mem¬ brane-associated enzymes in a wide variety of cells (12). Since gossypol is known to affect many mem¬ brane-associated enzymes, a potential site of gossy¬ pol action is at the biological membrane. In this paper, it is demonstrated that exposure to gossypol results in an increase in membrane micro¬ viscosity, a decrease in steroidogenesis both in vivo and in vitro, and a decrease in responsiveness to ACTH. We propose that this is a mechanism by which gossypol can exert pathophysiological effects on the adrenal gland.
corticosterone (NEN Research products); [12T]cAMP (Bionetics Research Lab, Rockville, MD); cortisol anti¬ body (Western Chemical Research Corp, Fort Collins, CO); corticosterone antibody (ICN, Lisle, IL);
In vitro treatment with gossypol and ACTH Aliquots of 5 mmol/1 gossypol in ethanol stock solution were diluted with media to provide gossypol concentra¬ tions ranging from 0.05 to 50 (¿mol/1. The final concen¬ tration of ethanol in all incubation media including con¬ trols was 0.1% (v/v). Simultaneously, 250-liI aliquots of ACTH were added to quadruplicate wells to provide final concentrations ranging from 1 pmol/1 to 0.1 |imol/l in the culture wells. Basal production of cAMP and cortisol was measured after a 24-h culture of both control and gossypol-treated cells. The total volume in each well was 1 ml. Duplicate 50-|xl aliquots were removed from each well 24 h after ACTH addition and stored at 20°C prior to assay for cAMP and cortisol determination.
17-hydroxyprogesterone (50.0 mCi/mmol), [l,2-3H]4-an-
In vivo
Materials and Methods Materials The
following
were
purchased
from
suppliers
as
listed:
1,6 diphenylhexatriene (DPH), and tetrahydrofuran (THF)(Aldrich Chemical Co Inc, Milwaukee, WIS); ACTH(l-24) (Organon pharmaceuticals, West Orange,
NJ); silica gel LK6DF thin layer Chromatographie plates (Whatman Inc, Clifton, NJ); [3H]cortisol, and [3H]-
[4-MC]dehydroepiandrosterone (51.3 mCi/mmol), [4-l4C]
(48.5 Ci/mmol), [1,2-3H]11-deoxycortisol (58.2 Ci/mmol), and [4-14C]hydrocortisone (52.0 mCi/ mmol) (New England Nuclear, Boston, MA) ; gossypol acid
drostenedione
and gossypol (gifts from the National Research Institute for Family Planning, Beijing, China). All other drugs and chemicals were from commercial sources and were of the purest grades available.
Media preparation
Fetal calf serum was incubated three times with charcoal (0.75 g/300 ml) for 60 min at 37°C, centrifoged at 800 x g for 10 min, and filtered through a 0.2 ^m filter. The medium used in all experiments was Ham's F-12 contain¬ ing 10% charcoal treated fetal calf serum, 2 mmol/1 glutamine, 100 mg/1 streptomycin, and 100 000 U/l penicil¬ lin.
—
study on rat adrenal function with gossypol female Wistar rats (Charles River, Kingston, adult Eight NY) weighing 200-250 g were divided into two groups of four. The animals were housed at 24°C in a room having a 12 h light: 12 h dark cycle. A commercial rat diet and tap water were available ad libitum. The gossypol-treated an¬ imals received 25 mg gossypol acetic acid suspended in 0.25% carboxymethylcellulose/kg body weight per day via an oro-gastric tube. Control rats were fed an equal volume of the carrier. Body weights were measured weekly. At the end of 7 weeks, the animals were killed by decapitation and trunk blood was collected for determi¬ nation of corticosterone 30 min post ACTH stimulation (3 |xg ip). The adrenal glands were excised and stored at 70°C for subsequent measurement of membrane micro¬ viscosity. Samples of adrenal glands were fixed in Bouin's solution and histological sections prepared. —
Membrane Adrenocortical cell preparation
Human adult adrenal glands were obtained from 4 male patients with localized renal cell carcinoma, ages 35 to 45, who were otherwise healthy. At the time of nephrectomy, en bloc dissection of the primary neoplasm necessitated the excision of the adrenal gland. The adrenal cortex was dissected free from the medulla, minced into 1 to 2 mm pieces, and stirred gently for 1 h at 37°C in 20 ml of Ham's F-12 complete media containing collagenase (1 g/1); 2000 units of deoxyribonuclease were then added and the mix¬ ture was stirred for an additional 10 min. The resultant cell suspension was filtered through a fine mesh stainless steel screen, washed once, and resuspended in media. Cells were counted using a standard hemocytometer and placed into 24-well culture plates at a density of 30 000 cells/well. Cell viability as determined by trypan blue ex¬ clusion prior to plating was >95%. Cells were cultured at 37°C in a 5% C02/95% air environment. At the end of the treatment period, repeat determination of cell viabilitywas made after cells were dissociated from the culture wells. For all gossypol concentrations, viability remained
3=90%.
microviscosity measurement
Cultures of the human adrenal cells or freshly isolated rat adrenal cells were rinsed twice with Hanks balanced salt solution (HBSS, pH 7.2), scraped from the culture dishes and centrifuged at 150 x g for 10 min. Mitochondrialand microsomal-enriched fractions were isolated by dif¬ ferential centrifugation in 0.3 mol/1 sucrose at 15 000 x g for 20 min and 100 000 x g for 60 min after tissue homogenization in a ground glass dounce homogenizer. Using DPH as a probe, the microviscosities of these frac¬ tions were determined as described previously (13). Briefly, DPH was dissolved in THF at a concentration of 2 mmol/1 before being dispersed in HBSS, to provide a final concentration of 2 nmol/1. The membrane suspen¬ sions were diluted to concentrations of 33.3 mg protein/1 prior to loading with an equal volume of the HBSS-DPB solution at room temperature for 1 h. Each membrane sample was subjected to polarization analysis at 37°C using a Perkin-Elmer MPF-66 fluorescence spectrophotometer. The excitation wavelength was 366 nm, and the emission wavelength was 430 nm. Fluorescence polariza¬ tion (P) was calculated according to the equation: P=(IVG.IH)/(IV+G.IH), where Iv and IH are the relative fluo¬ rescence intensities measured at an angle of 90° to the
incident beam with the emission polarizer in the vertical and horisontal positions, respectively, and G=IV/IH mea¬ sured with the excitation polarizer in the horizontal po¬ sition.
Table 1. Dose responses of gossypol on fluorescence polarization of microsomal and mitochondrial membranes at 60 min.
Adrenal enzymes determination
Treatment
Fluorescence Normal human adrenal tissues were obtained from 2 ad¬ ditional male patients, ages 40 and 46, with renal cell
carcinoma undergoing unilateral nephrectomy. Micro¬ somal and mitochondrial fractions were prepared using differential centrifugation, resuspended in TRIS buffer and stored at 70°C until assayed for specific steroidogenic enzyme activities. Mitochondrial and microsomal proteins were estimated by the method of Lowry et al.
Control
Gossypol (0.05 nmol/1) Gossypol (0.5 (tmol/1) Gossypol (5 nmol/1)
polarization
Microsomes
Mitochondria
0.262±0.009 0.284±0.013 0.384±0.046*
0.259±0.012 0.280±0.018 0.329±0.027* 0.366±0.035*
0.544+0.101*
—
(14). The microsomal enzymes, 3ß-hydroxysteroid dehydrogenase A5-A4 isomerase (3ß-HSD) and 21-hydroxylase (21-OHase), and the mitochondrial enzyme, 11-hydroxylase (11-OHase), were measured by the incorporation of specific [ C]- or [H)-labelled substrates into products (dehydroepiandrosterone —» 4-androstenedione for 3ßHSD; 17-hydroxyprogesterone —> 11-deoxycortisol for 21-OHase; and 11-deoxycortisol —» cortisol for 11OHase) as described previously (15). Enzyme activities were computed as pmol of product produced per min of incubation per mg of microsomal or mitochondrial proteins, but are expressed collectively for each separate enzyme as percent of basal activity that was determined without addition of gossypol. Each adrenal was evaluated at two or more time points for two enzyme assays. All time points were run in duplicate.
Cortisol and corticosterone measurements
Radioimmunoassays for cortisol and corticosterone were performed using previously described charcoal adsorp¬ tion methods (16,17). Intra- and inter-assay coefficients of variation were
Wu,
Constance L.
Chik, Barry D. Albertson, W.
Marston Linehan and Richard A. Knazek
Developmental Endocrinology Branch', NICHD, and Surgery Branch2, NCI, NIH, Bethesda, Maryland,
Abstract. Gossypol, an antifertility agent, has inhibitory actions on many membrane-associated enzymes, suggesting that this agent might have a generalized effect on cell membranes. This hypothesis was examined in the present membranes and dispersed cells prepared from human and rat adrenal glands. Four parameters were determined: microviscosity as measured by fluorescence polarization of human adrenal microsomal- and mitochondrial-enriched membranes, adrenal steroidogenic enzymes; and cAMP and cortisol responses to ACTH. It was found that gossypol increased the polarization constants of microsomes and mitochondria in a dose-dependent manner. Of the three adrenal enzymes tested, both 3\g=b\-hydroxysteroiddehydrogenase \g=D\5-\g=D\4isomerase and 11-hydroxylase were inhibited by gossypol, but not 21\x=req-\ hydroxylase. Using intact human adrenocortical cells, high doses of gossypol also inhibited the ACTH-stimulated cAMP and cortisol levels. The in vivo corticosterone response to ACTH in rats subjected to chronic gossypol treatment was also found to be reduced. These findings suggest that gossypol has multiple effects on adrenal function. Its effects on membrane microviscosity, adrenal steroidogenesis, cAMP and corticosterone responses to ACTH stimulation probably occur through a generalized membrane effect.
study using
In the 1930's and 40's outbreaks of unexplained were observed in several provinces of China. Entire villages went for a decade or more without a recorded birth. The agent causing the infertility was identified as gossypol, a binapthalenic component of cottonseed oil, used by citizens of these regions as cooking oil. Since that time, nu¬ merous studies have investigated the pharmaco¬ logy and toxicology of gossypol, especially as an
infertility
USA
antifertility agent in males ( 1 ). Although the mecha¬ nism of action of gossypol remains to be deter¬ mined, recent studies indicate that gossypol alters the activity of many membrane-associated enzymes including those involved in steroidogenesis (2-8). This raised the possibility that gossypol may affect adrenocortical function via a generalized action on membrane and steroidogenesis. Previous studies in our laboratory have demon¬ strated that a decrease in membrane microviscosity results in a concomitant increase in the detectable number of membrane-associated prolactin recep¬ tors (9-10). In contradistinction, an increase in membrane microviscosity induced by incorpora¬ tion of long-chain saturated fatty acids was accom¬ panied by a decrease in the responsiveness of human adrenal cells to ACTH stimulation in vitro, thus providing a possible explanation for the hypoadrenal state observed in adrenoleukodystrophy and adrenomyeloneuropathy (11). Other studies have also correlated changes in membrane micro¬ viscosity with alterations in the functionality of hor¬ mone receptors, antigenic determinants, and mem¬ brane-associated enzymes in a wide variety of cells (12). Since gossypol is known to affect many mem¬ brane-associated enzymes, a potential site of gossy¬ pol action is at the biological membrane. In this paper, it is demonstrated that exposure to gossypol results in an increase in membrane micro¬ viscosity, a decrease in steroidogenesis both in vivo and in vitro, and a decrease in responsiveness to ACTH. We propose that this is a mechanism by which gossypol can exert pathophysiological effects on the adrenal gland.
corticosterone (NEN Research products); [12T]cAMP (Bionetics Research Lab, Rockville, MD); cortisol anti¬ body (Western Chemical Research Corp, Fort Collins, CO); corticosterone antibody (ICN, Lisle, IL);
In vitro treatment with gossypol and ACTH Aliquots of 5 mmol/1 gossypol in ethanol stock solution were diluted with media to provide gossypol concentra¬ tions ranging from 0.05 to 50 (¿mol/1. The final concen¬ tration of ethanol in all incubation media including con¬ trols was 0.1% (v/v). Simultaneously, 250-liI aliquots of ACTH were added to quadruplicate wells to provide final concentrations ranging from 1 pmol/1 to 0.1 |imol/l in the culture wells. Basal production of cAMP and cortisol was measured after a 24-h culture of both control and gossypol-treated cells. The total volume in each well was 1 ml. Duplicate 50-|xl aliquots were removed from each well 24 h after ACTH addition and stored at 20°C prior to assay for cAMP and cortisol determination.
17-hydroxyprogesterone (50.0 mCi/mmol), [l,2-3H]4-an-
In vivo
Materials and Methods Materials The
following
were
purchased
from
suppliers
as
listed:
1,6 diphenylhexatriene (DPH), and tetrahydrofuran (THF)(Aldrich Chemical Co Inc, Milwaukee, WIS); ACTH(l-24) (Organon pharmaceuticals, West Orange,
NJ); silica gel LK6DF thin layer Chromatographie plates (Whatman Inc, Clifton, NJ); [3H]cortisol, and [3H]-
[4-MC]dehydroepiandrosterone (51.3 mCi/mmol), [4-l4C]
(48.5 Ci/mmol), [1,2-3H]11-deoxycortisol (58.2 Ci/mmol), and [4-14C]hydrocortisone (52.0 mCi/ mmol) (New England Nuclear, Boston, MA) ; gossypol acid
drostenedione
and gossypol (gifts from the National Research Institute for Family Planning, Beijing, China). All other drugs and chemicals were from commercial sources and were of the purest grades available.
Media preparation
Fetal calf serum was incubated three times with charcoal (0.75 g/300 ml) for 60 min at 37°C, centrifoged at 800 x g for 10 min, and filtered through a 0.2 ^m filter. The medium used in all experiments was Ham's F-12 contain¬ ing 10% charcoal treated fetal calf serum, 2 mmol/1 glutamine, 100 mg/1 streptomycin, and 100 000 U/l penicil¬ lin.
—
study on rat adrenal function with gossypol female Wistar rats (Charles River, Kingston, adult Eight NY) weighing 200-250 g were divided into two groups of four. The animals were housed at 24°C in a room having a 12 h light: 12 h dark cycle. A commercial rat diet and tap water were available ad libitum. The gossypol-treated an¬ imals received 25 mg gossypol acetic acid suspended in 0.25% carboxymethylcellulose/kg body weight per day via an oro-gastric tube. Control rats were fed an equal volume of the carrier. Body weights were measured weekly. At the end of 7 weeks, the animals were killed by decapitation and trunk blood was collected for determi¬ nation of corticosterone 30 min post ACTH stimulation (3 |xg ip). The adrenal glands were excised and stored at 70°C for subsequent measurement of membrane micro¬ viscosity. Samples of adrenal glands were fixed in Bouin's solution and histological sections prepared. —
Membrane Adrenocortical cell preparation
Human adult adrenal glands were obtained from 4 male patients with localized renal cell carcinoma, ages 35 to 45, who were otherwise healthy. At the time of nephrectomy, en bloc dissection of the primary neoplasm necessitated the excision of the adrenal gland. The adrenal cortex was dissected free from the medulla, minced into 1 to 2 mm pieces, and stirred gently for 1 h at 37°C in 20 ml of Ham's F-12 complete media containing collagenase (1 g/1); 2000 units of deoxyribonuclease were then added and the mix¬ ture was stirred for an additional 10 min. The resultant cell suspension was filtered through a fine mesh stainless steel screen, washed once, and resuspended in media. Cells were counted using a standard hemocytometer and placed into 24-well culture plates at a density of 30 000 cells/well. Cell viability as determined by trypan blue ex¬ clusion prior to plating was >95%. Cells were cultured at 37°C in a 5% C02/95% air environment. At the end of the treatment period, repeat determination of cell viabilitywas made after cells were dissociated from the culture wells. For all gossypol concentrations, viability remained
3=90%.
microviscosity measurement
Cultures of the human adrenal cells or freshly isolated rat adrenal cells were rinsed twice with Hanks balanced salt solution (HBSS, pH 7.2), scraped from the culture dishes and centrifuged at 150 x g for 10 min. Mitochondrialand microsomal-enriched fractions were isolated by dif¬ ferential centrifugation in 0.3 mol/1 sucrose at 15 000 x g for 20 min and 100 000 x g for 60 min after tissue homogenization in a ground glass dounce homogenizer. Using DPH as a probe, the microviscosities of these frac¬ tions were determined as described previously (13). Briefly, DPH was dissolved in THF at a concentration of 2 mmol/1 before being dispersed in HBSS, to provide a final concentration of 2 nmol/1. The membrane suspen¬ sions were diluted to concentrations of 33.3 mg protein/1 prior to loading with an equal volume of the HBSS-DPB solution at room temperature for 1 h. Each membrane sample was subjected to polarization analysis at 37°C using a Perkin-Elmer MPF-66 fluorescence spectrophotometer. The excitation wavelength was 366 nm, and the emission wavelength was 430 nm. Fluorescence polariza¬ tion (P) was calculated according to the equation: P=(IVG.IH)/(IV+G.IH), where Iv and IH are the relative fluo¬ rescence intensities measured at an angle of 90° to the
incident beam with the emission polarizer in the vertical and horisontal positions, respectively, and G=IV/IH mea¬ sured with the excitation polarizer in the horizontal po¬ sition.
Table 1. Dose responses of gossypol on fluorescence polarization of microsomal and mitochondrial membranes at 60 min.
Adrenal enzymes determination
Treatment
Fluorescence Normal human adrenal tissues were obtained from 2 ad¬ ditional male patients, ages 40 and 46, with renal cell
carcinoma undergoing unilateral nephrectomy. Micro¬ somal and mitochondrial fractions were prepared using differential centrifugation, resuspended in TRIS buffer and stored at 70°C until assayed for specific steroidogenic enzyme activities. Mitochondrial and microsomal proteins were estimated by the method of Lowry et al.
Control
Gossypol (0.05 nmol/1) Gossypol (0.5 (tmol/1) Gossypol (5 nmol/1)
polarization
Microsomes
Mitochondria
0.262±0.009 0.284±0.013 0.384±0.046*
0.259±0.012 0.280±0.018 0.329±0.027* 0.366±0.035*
0.544+0.101*
—
(14). The microsomal enzymes, 3ß-hydroxysteroid dehydrogenase A5-A4 isomerase (3ß-HSD) and 21-hydroxylase (21-OHase), and the mitochondrial enzyme, 11-hydroxylase (11-OHase), were measured by the incorporation of specific [ C]- or [H)-labelled substrates into products (dehydroepiandrosterone —» 4-androstenedione for 3ßHSD; 17-hydroxyprogesterone —> 11-deoxycortisol for 21-OHase; and 11-deoxycortisol —» cortisol for 11OHase) as described previously (15). Enzyme activities were computed as pmol of product produced per min of incubation per mg of microsomal or mitochondrial proteins, but are expressed collectively for each separate enzyme as percent of basal activity that was determined without addition of gossypol. Each adrenal was evaluated at two or more time points for two enzyme assays. All time points were run in duplicate.
Cortisol and corticosterone measurements
Radioimmunoassays for cortisol and corticosterone were performed using previously described charcoal adsorp¬ tion methods (16,17). Intra- and inter-assay coefficients of variation were
