Inhibition of rabbit sperm acrosomal enzymes by gossypol

The effect of gossypol on the activities of 10 acrosomal enzymes of the rabbit sperm was evaluated. Acrosin, Azocoll proteinase, neuraminidase, and arylsulfatase were significantly inhibited or completely inactivated by 12–76 μM gossypol. Hyaluronidase, β-glucuronidase, and acid phosphatase were inhibited only at a higher concentration of gossypol (380 μM). Phospholipase C, alkaline phosphatase, and β-N-Acetyl glucosaminidase were not inhibited even at 380 μM gossypol. Gossypol was found to be a noncompetitive inhibitor of arylsulfatase with a Ki of 120 μM. The inhibition was reversible and dose-dependent. As the acrosomal enzymes were more sensitive to the inhibition by gossypol compared to sperm enzymes involved in glycolysis or energy production, these assays may serve as a more reliable indicator for monitoring the occurence of gossypol-induced sterility. © 1995 Wiley-Liss, Inc.     

Gossypol-induced free radical toxicity to isolated islet cells

1. The cytotoxicity of the polyphenolic potential male antifertility agent gossypol was investigated on isolated mouse islets cells. 2. Gossypol shared many properties with the diabetogenic agent alloxan. 3. Gossypol (0.1-1.0 mmol/l) induced a concentration-dependent increase of Trypan Blue uptake by the cells, indicating an increase of membrane permeability to the dye. 4. Trypan Blue uptake induced by 0.5 mmol/l gossypol was inhibited by concomitant incubation of the cells with enzymatic (200 mg/l superoxide dismutase, 200 mg/l catalase, 3 mmol/l cytochrome-c), or low-molecular weight (50 mmol/l D-mannitol) scavengers of oxygen radicals, and the metal chelator diethylenetriaminepentacetic acid (DTPA) (50 mumol/l). 5. The results support the hypothesis that gossypol is B-cytotoxic by generation of noxious free radicals and that when proposing gossypol as a male antifertility agent, studies to exclude gossypol as a diabetogenic agent should first be performed in vivo.     

Inhibition of DNA polymerase alpha by gossypol

Our earlier studies have shown that gossypol is a specific inhibitor of DNA synthesis in cultured cells at low doses. In an attempt to determine the mechanism for the inhibition of DNA synthesis by gossypol we observed that gossypol does not interact with DNA per se but may affect some of the enzymes involved in DNA replication. These studies indicated that gossypol inhibits both in vivo and in vitro the activity of DNA polymerase alpha (EC 2.7.7.7), a major enzyme involved in DNA replication, in a time- and dose-dependent manner. Kinetic analysis revealed that gossypol acts as a noncompetitive inhibitor of DNA polymerase alpha with respect to all four deoxynucleotide triphosphates and to the activated DNA template. Inhibition of DNA polymerase alpha does not appear to be due to either metal chelation or reduction of sulfhydryl groups on the enzyme. Gossypol also inhibited HeLa DNA polymerase beta in a dose-dependent manner, but had no effect on DNA polymerase gamma. These results suggest that inhibition of DNA polymerase alpha may account in part for the inhibition of DNA synthesis and the S-phase block caused by gossypol. The data also raise the possibility that gossypol may interfere with DNA repair processes as well.     

Inhibition of DNA polymerase α by gossypol

Our earlier studies have shown that gossypol is a specific inhibitor of DNA synthesis in cultured cells at low doses. In an attempt to determine the mechanism for the inhibition of DNA synthesis by gossypol we observed that gossypol does not interact with DNA per se but may affect some of the enzymes involved in DNA replication. These studies indicated that gossypol inhibits both in vivo and in vitro the activity of DNA polymerase α (EC 2.7.7.7), a major enzyme involved in DNA replication, in a time- and dose-dependent manner. Kinetic analysis revealed that gossypol acts as a noncompetitive inhibitor of DNA polymerase α with respect to all four deoxynucleotide triphosphates and to the activated DNA template. Inhibition of DNA polymerase α does not appear to be due to either metal chelation or reduction of sulfhydryl groups on the enzyme. Gossypol also inhibited HeLa DNA polymerase β in a dose-dependent manner, but had no effect on DNA polymerase γ. These results suggest that inhibition of DNA polymerase α may account in part for the inhibition of DNA synthesis and the S-phase block caused by gossypol. The data also raise the possibility that gossypol may interfere with DNA repair processes as well.     

The effect of gossypol on fast axonal transport and microtubule assembly

Gossypol at micromolar concentrations (2 microM) was found to inhibit axonal transport and a microsomal ATPase activity in the frog sciatic nerve, although axonal microtubules and the neuronal content of AMP, ADP and ATP were not affected. At slightly higher concentrations (30-40 microM), gossypol also inhibited microtubule assembly and neuronal energy metabolism. Gossypol accumulated in the nerve and the results indicate that gossypol may act as a potent neurotoxin.     

Effect of gossypol on few testicular enzymes in mature rats

Effect of oral administration of gossypol acetic acid (15 mg/kg/day) for 10 weeks, on certain enzymes, which may be taken as markers for the different stages of spermatogenesis, was studied in male albino rats. Gossypol produced a significant decrease in hyaluronidase and sorbitol dehydrogenase, while no change was observed in beta-glucuronidase and acid phosphatase. A significant increase in the total lactate dehydrogenase activity was observed in the testis. The possible significance of these findings is discussed.     

Effect of Gossypol on Some Oxidative Respiratory Enzymes

Gossypol was examined in relation to its effect on certain enzymes and enzyme complexes associated with the tricarboxylic acid cycle and the electron transport system. Succinic dehydrogenase and cytochrome oxidase activity from sweet potato was completely inhibited by gossypol at 7.5 x 10(-3)m and 2.0 x 10(-3)m, respectively. Succinoxidase activity of the same preparations was fully inhibited at a lower concentration, 2.5 x 10(-4)m. This concentration did not affect either succinic dehydrogenase or cytochrome oxidase, the primary and terminal enzymes of the succinoxidase complex. The nature of the intermediate step or steps inhibited at this concentration is not yet known. Gossypol was further shown to inhibit phosphorylation at concentrations having no appreciable effect on oxidation. Inhibition in general was not reduced by increased substrate concentrations in the enzyme systems examined, with the exception of cytochrome c for cytochrome oxidase. Bovine serum albumin was partially effective in reducing gossypol inhibition, provided that it was present before enzyme exposure to gossypol.     

Inhibitory effect of gossypol on basal and luteinization factor-stimulated progesterone synthesis in porcine granulosa cells.

Gossypol, a polyphenolic aldehyde, inhibits steroidogenesis and the reproductive system in both sexes. The present study was undertaken to investigate whether gossypol may affect progesterone biosynthesis in cultured porcine granulosa cells isolated from small (1-2 mm) follicles (SGC). SGC were cultured with gossypol, NO donor S-nitroso-N-acetylpenicillamine (S-NAP) or the specific NO-synthase inhibitor N-nitro-L-arginine methyl ester (L-NAME), in the presence or absence of follicular fluid isolated from large (5-8 mm) follicles (LFF) or conditioned media (CM) of granulosa cells isolated from large follicles (LGC). Gossypol enhanced the nitrite content in culture media of SGC and inhibited basal progesterone secretion by SGC. S-NAP (10(-3) M) inhibited progesterone secretion and enhanced the formation of cGMP by SGC. L-NAME had no effect on progesterone accumulation by SGC. The stimulatory effect of LFF or CM media on progesterone production by SGC in culture was also inhibited by S-NAP (10(-3)) and gossypol (10(-4) M). Moreover, gossypol inhibited forskolin-stimulated progesterone secretion, as well as substrate-enhanced conversion of 22-OH-cholesterol and pregnenolone to progesterone. These results suggest that the inhibitory effect of gossypol on progesterone secretion in culture of SGC may be mediated via NO generation.     

棉酚及其衍生物合成途径的研究进展

棉酚作为一种倍半萜烯类植物抗菌素广泛存在于棉花的根、种子等表皮组织的色素腺体里。该化合物已在医药、工业和农业等领域得到广泛应用。近来对棉酚及其衍生物合成途径的研究日趋深入,许多关键酶基因已得到克隆和分析。掌握棉酚及其衍生物的生物合成途径,并试图通过基因工程改造的方法来控制植物体棉酚的合成具有重要意义。概述了棉酚及其衍生物合成途径和关键酶的研究进展并展望其应用前景。     

Binding of gossypol to purified tubulin and inhibition of its assembly into microtubules

Gossypol is a polyphenolic pigment, which is employed as a male antifertility drug. It inhibits, among other reported effects, the growth of cultured mammalian cells, spermiogenesis, flagellar motility in Trypanosoma and sperm, dynein ATPase and the lactate dehydrogenase X (LDH-X) isozyme. We have characterized the non-covalent binding of gossypol to purified calf brain tubulin in 10 mM phosphate buffer, 0.1 mM GTP pH 7.0 at 25 degrees C. Equilibrium measurements were performed by difference spectroscopy. A peak at 435 nm was produced by the perturbation of gossypol light absorption upon binding to tubulin. The experimental isotherm was fitted by 1.96 +/- 0.06 gossypol binding sites per tubulin molecule, with identical apparent equilibrium binding constants of (7.5 +/- 1.1) X 10(4) M-1. The complex formed could be separated from free gossypol by gel chromatography. Binding of gossypol was independent of the presence of 0.1 mM GTP in the buffer. Gossypol did not affect the binding of ligands to the colchicine site. Gossypol interacted with vinblastine but apparently did not bind to the vinblastine sites of tubulin. Gossypol did not displace anilinonaphthalene sulphonate (ANS) bound to tubulin, but caused a strong (fivefold) quenching of its fluorescence. This indicated that gossypol probably binds in the vicinity of the ANS site of tubulin. Gossypol inhibited in vitro microtubule assembly at the same concentration range employed in the binding studies. An increase in the critical protein concentration required for polymerisation was observed, most simply interpreted by a stoichiometric mechanism. Gossypol did not induce any noticeable distortion of the microtubules observed under the electron microscope. This compound constitutes a new tubulin ligand and an inhibitor of microtubule assembly in vitro.     

Gossypol inhibits aromatase activity in cultured porcine granulosa cells

The present study investigated whether gossypol inhibited aromatase activity in cultured porcine granulosa cells. Aromatase activity was assayed by measuring (3)H-H(2)O released from [1beta-(3)H]-androstenedione. First, immature porcine granulosa cells were cultured with various doses of follicle stimulating hormone (FSH, 1 to 1000 ng/ml) for 1 to 5 d to determine optimal culture conditions for aromatase activity assay. Second, porcine granulosa cells were cultured with or without FSH in the presence or absence of gossypol. Gossypol, at 4 muM, significantly inhibited FSH-induced aromatase activity while showing no effect on basal aromatase activity. Gossypol did not inhibit cell proliferation during cell culture. These results suggest that gossypol inhibits aromatase activity by interfering with FSH induction of aromatase in cultured porcine granulosa cells.     

Effects of gossypol acetic acid on the absorptive and digestive functions of rat intestine

Oral administration of Gossypol acetic acid (10 mg/kg body wt./day, daily for 15 days), an experimental antifertility agent to male rats, caused significant reduction in the uptake of glucose, alanine, leucine and calcium in the small intestinal segments. Gossypol also caused significant decrease in the intestinal brush border membrane--associated enzymes, sucrase, lactase, maltase and alkaline phosphatase. Kinetic analysis indicated that Gossypol decreased the apparent velocity of the disaccharidases while the Km was not altered. It also caused a shift in the transition temperature in these enzymes and predictably changed the energy of activation both below and above the transition temperature, although the Arrhenius expressions of the temperature dependence still showed proximity and were parallel to the control group.     

Reversibility of the reproductive toxicity of gossypol in peripubertal bulls

Gossypol has been shown to impair sperm production in male ruminants. The purpose of this study was to determine if the adverse effects of gossypol on spermatogenesis in peripubertal bulls were reversible. Twenty-eight crossbred Angus bulls were allocated into treated and control groups at 11 months of age. For 8 weeks, treated bulls were fed a ration containing 8 mg of free gossypol per kilogram of body weight per day while control bulls were fed a soybean meal ration free of gossypol. At 28-days intervals, scrotal circumference was measured and semen collected to assess sperm motility and morphology. Seven control and seven treated animals were castrated 56 days after the start of the experiment and the testes were examined histologically. The remaining bulls were fed a gossypol-free diet for 210 days prior to castration. There were significant increases in primary and secondary sperm abnormalities in treated bulls 28 and 56 days after gossypol feeding. The number of sperm with proximal droplets was significantly higher in gossypol-treated bulls, suggesting testicular degeneration. There was no significant effect on the sperm motility, scrotal circumference, or histopathological characteristics of the testes. Four weeks after the end of gossypol feeding, primary and secondary abnormalities were still increased in gossypol-treated bulls, however in subsequent collection periods the percentage of abnormalities were similar between groups. At 210 days, there was no treatment effect on scrotal circumference, and histological characteristics of the testes were not different between groups. The deleterious effects of gossypol on the morphological characteristics of spermatozoa were reversible. Gossypol (8 mg/kg per day for 56 days) increased sperm abnormalities but the effects were reversible.     

Molecular mechanisms of gossypol action on sperm motility

1. Gossypol acetic acid inhibits collective motility of ejaculated ram spermatozoa. 2. Oxygen consumption was stimulated at low gossypol concentrations and inhibited as the concentrations are increased. 3. Gossypol inhibits respiration of permeabilized spermatozoa supported by durohydroquinome, which indicates a direct inhibition of mitochondrial electron transport chain. 4. The rapid reduction of mitochondrial dependent motility, high uncoupling effect and almost complete inhibition of mitochondrial calcium accumulation, indicate that gossypol inhibits motility in a mechanism by which mitochondrial uncoupling is involved.     

Cytotoxic effect of gossypol on colon carcinoma cells

Gossypol, a male contraceptive drug extracted from cottonseeds, has been found to have antiproliferative activity on tumour cells and is thought to be a potential anticancer drug. The aim of this study was to investigate the mechanisms of gossypol-induced cell death on two colon carcinoma cell lines, HT29 and LoVo. Firstly, we studied the effect of gossypol on the colony forming ability of these tumour cells, which is the main target of chemotherapeutic drugs. Using clonogenic assays, flow cytometry and DNA gel electrophoresis techniques, we have found that gossypol not only inhibited colony forming ability of these tumour cells, but we also observed cellular internucleosomal DNA fragmentation in the cells treated with 3 doses of gossypol and this was accompanied by the appearance of a sub-G1 apoptotic peak and morphological characteristics of apoptosis. Our results suggest that the gossypol induced cell death is via an apoptotic pathway and the effect of gossypol may not be cell cycle specific. Using Western blotting analysis, we found that the gossypol-induced apoptosis may not be involved in the regulation of p53 but possibly associated with the regulation of bcl-2 and Bax expression. Our evidence indicates that gossypol may provide a potential therapeutic benefit for the treatment of colon carcinoma and understanding the mechanisms of gossypol-induced cytotoxicity on tumour cells is essential for including this drug in clinical use.     

Similarities between a dipeptide hydrolase from brush-border and cytosol fractions of guinea-pig intestinal mucosa.

A dipeptide hydrolase from the brush border of guinea-pig intestinal mucosa was purified. The enzyme resembles another dipeptide hydrolase isolated from the cytosol fraction of intestinal mucosa. Studies on the binding of cytosol peptide hydrolase to brush-border membranes indicate that the enzyme found in the brush border may be a cytoplasmic contaminant.     

Isolation and characterization of a small intestinal surfactant-like particle containing alkaline phosphatase and other digestive enzymes

Digestive brush-border enzymes in particulate form have been reported in the intestinal lumen in vivo and in medium from organ explants in vitro. It has been suggested that these particles derive from membrane shedding of the apical brush border. This study describes the isolation and characterization of particles derived from the 105,000 x g supernatant fraction of intestinal luminal washings and from light scrapings of the mucosa itself after fat feeding of rats. These fractions were separated in a continuous NaBr gradient, producing a visible band of 1.07-1.08 g/liter density and resulting in a 15-fold enrichment of intestinal alkaline phosphatase in the band fraction. Other brush-border hydrolases were represented in the banded fraction, but at specific activities only 1/5th to 1/36th that of the brush border. The major phospholipid in the fraction was phosphatidylcholine (58 +/- 15%), containing 75% saturated fatty acids. In contrast, the major brush-border phospholipid was phosphatidyl-ethanolamine. These characteristics showed that the particles derived from the lumen and mucosal surface were not identical to fragments of the brush border. Electron microscopy of the banded fraction revealed partially coiled membrane fragments. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blots, some proteins (e.g. surfactant protein B, collagenous protein 4) were found in common between the intestinal particles and rat pulmonary surfactant. These data suggest the production of a particle secreted by rat intestine that differs from brush-border membranes and that shares some morphological and biochemical similarities with pulmonary surfactant.     

Catechin protects against ketoprofen-induced oxidative damage of the gastric mucosa by up-regulating Nrf2 in vitro and in vivo

Nonsteroidal anti-inflammatory drugs (NSAIDs), including ketoprofen, are widely used in clinical medicine. However, these drugs may damage the gastrointestinal mucosa. Some reports have suggested that intestinal diseases, such as ulcers, are associated with lipid peroxidation and oxidative damage in the mucosa. Phytochemicals, such as polyphenols, are common dietary antioxidants that possess many beneficial characteristics, such as antioxidant and anti-inflammatory capabilities. The objective of this study was to investigate the protective effects of polyphenols on ketoprofen-induced oxidative damage in the gastrointestinal mucosa. We evaluated the effects of catechin, theaflavin, malvidin, cyanidin and apigenin on the activity of antioxidant enzymes in human intestinal-407 (Int-407) cells and rat primary gastric cells treated with ketoprofen. The results indicated that catechin significantly (P<.05) decreased the levels of lipid peroxidation (40.5%) and reactive oxygen species (30.0%), and increased the activity of intracellular antioxidant enzymes glutathione peroxidase, glutathione reductase and total sulfhydryl groups. More importantly, the treatment of Sprague–Dawley rats with catechin (35 mg/kg/day) prior to the administration of ketoprofen (50 mg/kg/day) successfully inhibited oxidative damage and reversed the impairment of the antioxidant system in the intestinal mucosa. Western blot analysis revealed that catechin stimulated a time-dependent increase in both the nuclear factor erythroid 2-related factor 2 and total heme oxygenase-1 protein expression in Int-407 cells. These results suggest that catechin may have a protective effect on gastrointestinal ulcers.     

Enzyme activities of intestinal triacylglycerol and phosphatidylcholine biosynthesis in Atlantic salmon (Salmo salar L.)

The substitution of fish oil with plant-derived oil in diets for carnivorous fish, such as Atlantic salmon, has previously revealed the potentially deleterious supranuclear accumulation of lipid droplets in intestinal cells (enterocytes) which may compromise gut integrity, and consequently, fish health. This suggests that unfamiliar dietary lipid sources may have a significant impact on intestinal lipid metabolism, however, the mode of lipid resynthesis is largely unknown in teleost fish intestine. The present study aimed at characterising three key lipogenic enzymes involved in the biosynthesis of triacylglycerol (TAG) and phosphatidylcholine (PC) in Atlantic salmon enterocytes: monoacylglycerol acyltransferase (MGAT), diacylglycerol acyltransferase (DGAT), and diacylglycerol cholinephosphotransferase (CPT). Furthermore, to investigate the dietary effect of plant oils on these enzymes, two experimental groups of fish were fed a diet with either capelin (fish oil) or vegetable oil (rapeseed oil:palm oil:linseed oil, 55:30:15 w/w) as the lipid source. The monoacylglycerol (MAG) pathway was highly active in the intestinal mucosa of Atlantic salmon as demonstrated by MGAT activity (7 nmol [1-(14)C]palmitoyl-CoA incorporated min(-1) mg protein(-1)) and DGAT activity (4 nmol [1-(14)C]palmitoyl-CoA incorporated min(-1) mg protein(-1)), with MGAT appearing to also provide adequate production of sn-1,2-diacylglycerol for potential utilisation in PC synthesis via CPT activity (0.4 nmol CDP-[(14)C]choline incorporated min(-1) mg protein(-1)). Both DGAT and CPT specific activity values were comparable to reported mammalian equivalents, although MGAT activity was lower. Nevertheless, MGAT appeared not to be the rate-limiting step in salmon intestinal TAG synthesis. The homology between piscine and mammalian enzymes was established by similar stimulation and inhibition profiles by a variety of tested cofactors and isomeric substrates. The low dietary n-3/n-6 PUFA ratio presented in the vegetable oil diet did not significantly affect the activities of MGAT, DGAT, or CPT under optimised assay conditions, or in vivo intestinal mucosa lipid class composition, when compared to a standard fish oil diet.     

Gossypol: uncoupling of respiratory chain and oxidative phosphorylation

Gossypol produced adverse effects $\text{in vitro}$ on rat liver mitochondira. It stimulated mitochondrial respiration at low concentrations, inhibited it at high concentrations; abolished ADP/O and respiration control ratios; reversed inhibition caused by oligomycin; stimulated adenosine triphosphatase activity at low concentrations and inhibited it at high concentrations; and its effect on this enzyme was pH dependent. The possibility that gossypol may exert its toxic effect on poultry and animals by uncoupling respiratory chain-linked phosphorylation is discussed.