Gossypol isomers bind specifically to blood plasma proteins and spermatozoa of rainbow trout fed diets containing cottonseed meal

We investigated the role of gossypol isomers binding to blood plasma, seminal plasma and spermatozoa to elucidate gossypol anti-fertility action in the teleost fish, rainbow trout (Oncorhynchus mykiss). Growth and hematological indicators of males were depressed when fish meal protein in diets was completely replaced with cottonseed meal. The cottonseed meal contained equal proportions of (-) (47.8+/-1.6%) and (+) gossypol isomers. Concentrations of spermatozoa were decreased with increasing proportions of gossypol in diets (from 0.22% to 0.95%); however, sperm motility and fertilizing ability were not affected. In contrast to mammals, steroid hormone concentrations were not suppressed in fish given diets with gradual increase of gossypol level. Gossypol concentrations were 100-fold higher in blood plasma than in seminal plasma, confirming a barrier in gossypol transfer between the general circulation and the testis. Spermatozoa accumulated predominantly (+) enantiomer (65-75%) with decreasing proportions as dietary gossypol concentrations increased. Spermatozoa bound most of the gossypol contained in the semen; however, this did not result in impairment of the sperm motility apparatus. Teleost fish sperm rely on ATP stores that accumulate during maturation as a source of energy during activation. In addition, the duration of sperm movement is short in these fish. As such, we hypothesize that the major action of gossypol on mammalian sperm, which is uncoupling of oxidative phosphorylation, does not impair the energy supply required for flagellar beating in fish spermatozoa.     

In vitro inhibitory effect of gossypol from gossypol-acetic acid, and (+)- and (-)-isomers of gossypol on the growth of Edwardsiella ictaluri

AIM: This study was conducted to evaluate the toxic effect of gossypol from gossypol-acetic acid, and (+)- and (-)-isomers of gossypol on the growth of Edwardsiella ictaluri. METHODS AND RESULTS: Inhibitory effect of various concentrations of gossypol on the growth of E. ictaluri was determined. Bacterial recovery was performed by preincubation of bacteria in medium containing various concentrations of gossypol and subsequent activation of bacteria by inoculating on gossypol-free plates. Concentrations of racemic gossypol, (+)-gossypol and (-)-gossypol of 1.5 microg ml(-1) or higher significantly reduced the number of bacterial colonies compared with that of the control. The growth of E. ictaluri was completely inhibited on agar plates supplemented with 3 microg ml(-1), regardless of the forms of gossypol. The inhibitory effect of (+)-gossypol was higher than that of (-)-gossypol or gossypol-acetic acid. Recovery of E. ictaluri was <50% for all three forms of gossypol at concentrations of 5 microg ml(-1). Bacterial recovery remained relatively constant (6.5%) at gossypol concentrations from 10 to 100 microg ml(-1). Complete killing of E. ictaluri was not reached at gossypol levels up to 100 microg ml(-1). CONCLUSION: Gossypol-acetic acid, and (+)- and (-)-optical isomers have anti-bacterial effect against E. ictaluri. The results suggest the action is bacteriostatic rather than bactericidal. SIGNIFICANCE AND IMPACT OF THE STUDY: The therapeutic effect of gossypol against E. ictaluri may be useful in controlling enteric septicaemia of catfish.     

HPLC preparation of the chiral forms of 6-methoxy-gossypol and 6,6'-dimethoxy-gossypol

A concentrated mixture of gossypol, 6-methoxy-gossypol, and 6,6'-dimethoxy-gossypol was extracted from the root bark of St. Vincent Sea Island cotton with acetone. This extract was derivatized with R-(-)-2-amino-1-propanol to form diastereomeric gossypol Schiff's bases. Analytical-scale reverse-phase chromatography of these Schiff's bases produced six peaks, indicating separation of the enantiomeric forms of the three gossypol compounds. The elution order of the peaks was found to vary with the polarity of the mobile phase. The chromatography was scaled to a preparative level and was used to isolate each compound. After hydrolysis of the separated Schiff's bases, the original compounds were recovered by precipitation from solutions of diethyl ether, acetic acid, and water. Fifty injections yielded approximately 500 mg of each methoxy-gossypol enantiomer and 300 mg of each dimethoxy-gossypol enantiomer. Each compound was characterized for carbon and hydrogen content, optical rotation, UV-vis light absorption, and melting point. Standard curves were developed and were used to measure the concentration of each gossypol form in the root bark and dehulled seed of St. Vincent Sea Island cotton. In seed tissue, 48% of the gossypol compounds were methylated, and the (-)-optical form was found to be in a slight excess to the (+)-optical form (53-54%) for all three compounds. In root bark, 71% of the gossypol compounds were methylated, and the (+)-optical form was in excess to the (-)-optical form for all three compounds. However, in this tissue the extent of enantiomeric excess decreased with the degree of methylation, with 77% of the gossypol existing in the (+)-optical form and 59% of the 6,6'-dimethoxy-gossypol existing in the (+)-optical form.     

Differential effects of (+)- and (-)-gossypol enantiomers on LDH-C4 activity of hamster spermatogenic epithelium in vitro

Tubular fragments (spermatogenic epithelium) from immature hamsters were isolated and cultured with low doses of (+)- and (-)-gossypol enantiomers. The activity of lactate dehydrogenase isoenzyme LDH-C4 was estimated as a marker for spermatogenic cell development and alpha-ketoisovalerate was used as the substrate. In the absence of gossypol, the specific activity of LDH-C4 in the tubular fragments was increased 40% during incubation for 48 h. This developmental increase was suppressed by gossypol. The specific activity of LDH-C4 in the tubular fragments was lowered by gossypol, after 48 h of culture in the presence of low doses of racemic gossypol (1-4 microM) and 1% fetal calf serum. In this in-vitro system the (-)-enantiomer but not the (+)-enantiomer of gossypol affected the LDH-C4 activity. This is in agreement with other reports showing that only the (-)-enantiomer induces infertility. The observed action of gossypol on LDH-C4 activity in the tubular fragments may reflect gossypol-induced degeneration of spermatogenic cells. The present in-vitro system can be used to estimate the actions of gossypol derivatives, other investigational antifertility agents, and toxic agents on the spermatogenic epithelium.     

A circular dichroism study of (+) gossypol binding to proteins

The circular dichroism bands of (+) gossypol in the spectral region 300-400 nm have been shown to be sensitive to interactions with proteins. Using CD spectroscopy, gossypol has been shown to interact with lactate dehydrogenase, malate dehydrogenase, alkaline phosphatase, lysozyme, protamine and poly-L-lysine. Binding to proteins generally results in a pronounced red shift of the long wavelength CD band (approximately 380-430 nm) accompanied by a reduction in ellipticity. The changes in spectral parameters of the 1Lb binaphthyl transition may reflect a distortion from a nearly perpendicular gossypol conformation, on binding to proteins.     

Resolution of racemic gossypol and interaction of individual enantiomers with serum albumins and model peptides

Racemic gossypol has been resolved by HPLC separation of diastereomeric (-) norepinephrine adducts on a reverse-phase column. The binding constants for the interaction of the three gossypol forms (+, - and +/-) with human and bovine serum albumins have been determined by fluorescence quenching studies. The KD values demonstrate that all three forms bind equally effectively to the two proteins, suggesting an absence of chiral discrimination in albumin-gossypol interactions. Circular dichroism studies of (+)-gossypol binding to the model dibasic peptides, Boc-Lys-Pro-Aib-Lys-NHMe and gramicidin S, suggest that distortions of binaphthyl geometry may occur only for specific orientations of interacting residues at the receptor site.     

The peroxidative coupling of hemigossypol to (+)- and (-)-gossypol in cottonseed extracts

Peroxidase(s) present in embryo extracts of Gossypium hirsutum cv. Texas Marker 1 catalyzed a bimolecular coupling of [4-(3)H]-hemigossypol to [4,4'-(3)H(2)]-gossypol. The reaction was dependent on the addition of H(2)O(2) and was inhibited 71-94% by 1 and 10mM sodium azide. The phenolic coupling produced 53% (+)-gossypol and 47% (-)-gossypol in close agreement to the 49% (+)-gossypol and 51% (-)-gossypol found in the intact seed. The nearly racemic mixture of (+)-and (-)-gossypol produced in these embryo extracts can be accounted for by non-enzymatic random coupling of the free radicals of hemigossypol produced by the peroxidase. In contrast, peroxidase reaction mixtures containing crude embryo extracts of G. hirsutum var. marie-galante produced 73% (+)-gossypol and 27% (-)-gossypol. These data from the marie-galante extracts and the fact that these intact seed contain 95% (+)-gossypol suggest a regio-stereoselective bimolecular coupling of hemigossypol to gossypol. The development of the peroxidative coupling of hemigossypol to gossypol in maturing seed of G. hirsutum cv. Texas Marker 1 was correlated to the formation of gossypol and suggests that peroxidative coupling of hemigossypol contributes to gossypol biosynthesis.     

Inhibition of testicular LDH-X from laboratory animals and man by gossypol and its isomers

The inhibitory effect of (+)-, (-)-, (+/-)-gossypol and (+/-)-gossypol acetic acid upon testicular cytosolic LDH-X was measured in vitro. Gossypol acetic acid (0-100 mumol/l) inhibited LDH-X prepared from the testes of the mouse greater than rabbit greater than human greater than rat greater than hamster. There was no relationship between inhibition and in-vivo antifertility activity. LDH activity measured in vitro in serum of men and hamsters was unaffected by gossypol. Gossypol and its isomers were non-competitive inhibitors of human and hamster LDH-X with respect to the coenzyme NADH, competitive inhibitors of human LDH-X and noncompetitive-competitive inhibitors of hamster LDH-X with respect to the substrate alpha-ketobutyrate. Co-incubation with human serum albumin or poly-L-lysine but not lysine protected human and hamster LDH-X from gossypol.     

Different pathways of cell killing by gossypol enantiomers

Gossypol, a polyphenolic, aldehyde-containing constituent of cottonseed, produced partial responses (>50% reduction in tumor size) in some patients with advanced cancer and suppressed sperm as an antifertility agent for men. This action in vivo and its novel side effect profile suggest a specific mechanism of the action of gossypol. Using the random homozygous knockout approach of Li and Cohen (1), we developed a cell line resistant to killing by gossypol, but sensitive to methotrexate and doxorubicin. It showed stereospecific resistance to killing by (-) gossypol (ED(50) 4.9 microM) compared with wild type (ED(50) 2.0 microM). The resistant and wild-type cells were equally sensitive to (+) gossypol (ED(50) 8.8 and 8.4 microM, respectively), methotrexate, and doxyrubicin. We conclude that gossypol affects cells by a stereospecific pathway for (-) gossypol, possibly related to its selective effects, and a nonstereospecific pathway for (+) gossypol and higher concentrations of (-) gossypol. Further knowledge about the stereospecific pathway may lead to new therapeutic drugs.     

The effect of gossypol on the frequency of DNA-strand breaks in human leukocytes in vitro

Gossypol, a human antifertility agent isolated from the cotton plant, was found to induce a dose-dependent increase in the frequency of DNA-strand breaks in human leukocytes exposed to 2-40 micrograms/ml of the drug for 1 h in serum-free medium in vitro. DNA-strand breaks were studied by alkaline elution or alkaline unwinding of DNA followed by hydroxylapatite-chromatography. No decrease of gossypol-induced DNA-strand breaks was observed after post-treatment incubation times up to 24 h, whereas X-ray-induced DNA breaks disappeared within 2 h under the same incubation conditions. Cells exposed to gossypol in the presence of 10% fetal calf serum showed no or little increase of DNA breaks, suggesting that serum proteins inhibit the DNA-damaging activity of the drug. Both optical isomers of gossypol induced DNA-strand breaks. However, the effect of (-)-gossypol was only about half of that of (+)-gossypol and the racemic form. The induction and persistence of DNA-strand breaks by gossypol, as well as the reduction of this effect in the presence of serum should be considered in the evaluation of the potential in vivo genotoxicity of the drug.     

Identification of the major antioxidative metabolites in biological fluids of the rat with ingested (+)-catechin and (-)-epicatechin.

(+)-Catechin and (-)-epicatechin are known to be biologically effective antioxidants present in the human diet, particularly in wine and tea. We studied the metabolism of these compounds to elucidate the truly active structures in biological fluids by their oral administration to rats. Without any treatment with beta-glucuronidase and sulfatase, a pair of metabolites were detected at much higher concentrations in the plasma, bile, and urine than the originally ingested compounds. Each major metabolite found in the plasma at the highest concentration was excreted in both the bile and urine, and was purified from urine. Their chemical structures were established to be (+)-catechin 5-O-beta-glucuronide and (-)-epicatechin 5-O-beta-glucuronide by MS and NMR analyses. These glucuronide conjugates exhibited high antioxidative activities as superoxide anion radical scavengers like their parent compounds. It is concluded that (+)-catechin 5-O-beta-glucuronide and (-)-epicatechin 5-O-beta-glucuronide are the biologically active in vivo structures of the ingested polyphenolic antioxidants.     

Probenecid-induced changes in the clearance of pranoprofen enantiomers

Probenecid is known to inhibit the elimination of several acidic drugs. Its influence on the pharmacokinetics of pranoprofen was investigated in rabbit after a single intravenous injection of racemic mixture (5 mg/kg). Levels of (-)-(R)- and (+)-(S)-pranoprofen and their glucuronide (after hydrolysis with sodium hydroxide) were determined in plasma, urine, and several tissues. The plasma concentration of the (+)-(S)-isomer was higher than that of the (-)-(R)-form. Oral coadministered probenecid (100 mg/kg) resulted in an increased plasma concentration of both enantiomers. Probenecid reduced the apparent total clearance and excretion of pranoprofen enantiomers in urine. It had a slight effect on the tissue distribution of pranoprofen at the dose used, but significantly reduced the formation of glucuronide for both enantiomers to the same extent in kidney microsomes. The differences caused by probenecid were significant with respect to its ability to inhibit glucuronidation in the kidney and subsequent excretion into urine, but enantioselective effects were negligible.     

Optically active gossypol as a circular dichroism probe of interactions with serum albumins

The (+)-enantiomer of the polyphenolic binaphthyl gossypol, has been shown to be a useful CD probe of interactions with human and bovine serum albumin. (+)-Gossypol binds to albumin with same affinity as recemic (±)-gossypol, as shown by fluorescence quenching, and also displaces bilirubin from its albumin binding site. The CD characteristics of bound gossypol are different in the case of the two proteins.     

Stereoselective disposition and chiral inversion of KE-298, a new antirheumatic drug,in rats

The present study was an attempt to elucidate the relationship between stereoselective pharmacokinetics and protein binding of KE-298 and its active metabolites, deacetyl-KE-298 (M-1) and S-methyl-KE-298 (M-2). Metabolic chiral inversion was also investigated. The levels of unchanged KE-298 in plasma after oral administration of (+)-(S)-KE-298 to rats were lower than those of (−)-(R)-KE-298, whereas the levels of M-1 and M-2 after administration of (+)-(S)-KE-298 were higher than after (−)-(R)-KE-298. In vitro, rat plasma protein binding of (+)-(S)-KE-298 was lower than that of (−)-(R)-KE-298. In contrast, the binding of (+)-(S)-M-1 and (+)-(S)-M-2 was higher than that of (−)-(R)-M-1 and (−)-(R)-M-2. Displacement studies revealed that the (+)-(S) and (−)-(R)-enantiomers of KE-298 and their metabolites bound to the warfarin binding site on rat serum albumin. These results suggest that the stereoselective plasma levels in KE-298 and its metabolites were closely related to enantiomeric differences in protein binding, attributed to quantitative differences in binding to albumin rather than to the different binding sites. Unidirectional chiral inversion was detected after oral administration of either (−)-(R)-KE-298 or (−)-(R)-M-2 to rats both yielding (+)-(S)-M-2. Chirality 9:22–28, 1997 © 1997 Wiley-Liss, Inc.     

Inhibitory effects of gossypol on corticosteroid 11-beta-hydroxysteroid dehydrogenase from guinea pig kidney: a possible mechanism for causing hypokalemia

Inhibition of 11-beta-hydroxysteroid dehydrogenase (11-beta-OHSD) in the kidney can cause excess mineralocorticoid effect and hypokalemia. To find out if gossypol, a potential oral contraceptive for men that has been associated with cases of hypokalemia, inhibits this enzyme, its effect on guinea pig kidney was studied. Working with microsomes from the kidney cortex, and using corticosterone as the substrate, racemic gossypol was found to be a competitive inhibitor of 11-beta-OHSD with a Ki of 67 +/- 5 microM. The (+) enantiomer was a little more potent than the (-) enantiomer. Microsomes from the kidneys of animals given gossypol for 2 weeks had lower enzyme activities than saline-treated animals. Microsomes from a strain of hairless guinea pigs had lower intrinsic enzyme activity than the normal animals. We conclude that there is genetic variation in the activity of this enzyme and that it can be inhibited by gossypol.     

Fiber-based liquid-phase micro-extraction of mebeverine enantiomers followed by chiral high-performance liquid chromatography analysis and its application to pharmacokinetics study in rat plasma

The applicability of two-phase liquid-phase micro-extraction (LPME) in porous hollow polypropylene fiber for the sample preparation and the stereoselective pharmacokinetics of mebeverine (MEB) enantiomers (an antispasmodic drug) in rat after intramuscular administration were studied. Plasma was assayed for MEB enantiomer concentrations using stereospecific high-performance liquid chromatography with ultraviolet detection after a simple, inexpensive, and efficient preconcentration and clean-up hollow fiber-based LPME. Under optimized micro-extraction conditions, MEB enantiomers were extracted with 25 μl of 1-octanol within a lumen of a hollow fiber from 0.5 ml of plasma previously diluted with 4.5 ml alkalized water (pH 10). The chromatographic analysis was carried out through chiral liquid chromatography using a DELTA S column and hexane-isopropyl alcohol (85:15 v/v) containing 0.2% triethylamine as mobile phase. The mean recoveries of (+)-MEB and (-)-MEB were 75.5% and 71.0%, respectively. The limit of detection (LOD) was 3.0 ng/ml with linear response over the concentration range of 10-2500 ng/ml with correlation coefficient higher than 0.993 for both enantiomers. The pharmacokinetic studies showed that the mean plasma levels of (+)-MEB were higher than those of (-)-MEB at almost all time points. Also, (+)-MEB exhibited greater t(max) (peak time in concentration-time profile), C(max) (peak concentration in concentration-time profile), t(1/2) (elimination half-life), and AUC(0-240 min) (area under the curve for concentration versus time) and smaller CL (clearance) and V(d) (apparent distribution volume) than its antipode. The obtained results implied that the absorption, distribution, and elimination of (-)-MEB were more rapid than those of (+)-MEB and there were stereoselective differences in pharmacokinetics.     

Plasma gossypol dynamics in white-tailed deer: Implications for whole cottonseed as a supplemental feed

Whole cottonseed (WCS) is a potential supplemental feed for white-tailed deer (Odocoileus virginianus) in rangeland conditions because of its high digestible energy and protein content, moderate fiber content, and resistance to degradation in moist conditions. WCS also contains the polyphenolic secondary metabolite gossypol, which reduces palatability to non-target monogastric species but may be of concern for deer nutrition. Plasma gossypol stabilization when fed a constant dry matter intake, plasma gossypol depletion after WCS was removed from the diet, and the relationship between WCS consumption and plasma gossypol concentration was studied in 10 mature male (N = 5) and female (N = 5) captive white-tailed deer. Consumption of WCS by 73 free-ranging white-tailed deer (59 males, 14 females) was estimated using results of the captive study. Plasma gossypol concentrations declined exponentially and averaged 0.74 μg/mL 35 days after WCS was removed from the diet. Plasma gossypol concentration was linearly related to WCS consumption (P < 0.001), with females having 0.35 μg/mL greater (P = 0.04) plasma gossypol than males for a given rate of dry matter consumption. All female and 93% of male white-tailed deer captured in WCS supplemented pastures had detectable plasma gossypol. Female averaged 1.88 μg/mL of plasma gossypol and males averaged 4.84 μg/mL of plasma gossypol. Based on the captive deer data, these plasma values suggest an average WCS consumption of ∼2.6 g/kg BW/day for female free-ranging deer and ∼5.6 g/kg BW/day for male free-ranging deer. Inferentially, a large proportion of free-ranging white-tailed deer in rangeland conditions will consume WCS, with females consuming 125 g WCS/day and males consuming 428 g WCS/day. That plasma gossypol levels decrease rapidly after cottonseed is removed from the diet suggests that the long withdrawal periods often used prior to breeding season may not be needed. However, although 93% of gossypol was eliminated from the animals after a five-week withdrawal period, a small amount of gossypol can still be detected. While our preliminary data on these animals suggests that these levels are not detriment to animal health or reproduction, ranch managers may want to take a conservative approach to the feeding of WCS until these questions are answered.     

Effect of extracellular volume expansion on erythrocyte sodium transport in rats.

The effects of extracellular volume expansion (EVE) on the major sodium transport systems and sodium and potassium contents in rat erythrocytes have been examined in the present study. Study has been performed in anesthetized Wistar rat weighing about 300 g. Acute extracellular volume expansion (EVE) was induced by a constant intravenous saline infusion (3% body wt, 3 hours). Rats anaesthetized and catheterized but not expanded were used as controls. Arterial blood samples from control and expanded rats were obtained at the same time, and assayed immediately. Intracellular sodium and potassium concentration and ouabain sensitive (Na(+)-K(+)-pump) and bumetanide sensitive (Na(+)-K(+)-cotransport system) outward Na+ fluxes in erythrocytes were measured. The effect of plasma on erythrocyte transport was also analyzed by measuring 86Rb uptake. Neither of two plasma cations (Na+ and K+) were modified by the EVE. Also intracellular Na+ and K+ levels remained unvariable. Total Na+ efflux was not modified by EVE, but pump-mediated Na+ efflux was smaller after than before EVE. The ouabain-inhibible Na+ efflux rate constant decreased after EVE (from 687 +/- 81 to 525 +/- 29 h-1 x 10(-3); P less than 0.05). Both Na(+)-K(+)cotransport-mediated Na+ efflux and passive permeability increased significantly after EVE. The incubation with plasma from saline-infused animals induced a significant decrease in Rb uptake rate constant, that was not observed after incubation with plasma from non-expanded rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stereoselective distribution of hydroxychloroquine in the rabbit following single and multiple oral doses of the racemate and the separate enantiomers

Hydroxychloroquine (HCQ) stereoselective distribution was investigated in rabbits after 20 mg/kg po of racemic-HCQ (rac-HCQ) and 20 mg/kg po of each enantiomer, 97% pure (?)-(R)-HCQ and 99% pure (+)-(S)-HCQ. Concentrations were 4 to 6 times higher in whole blood than in plasma. Melanin did not affect plasma and whole blood levels since concentrations did not differ between pigmented and nonpigmented animals. After single and multiple doses of the separate enantiomers, only 5–10% of the antipode could be measured, in blood or plasma. Therefore, there was no significant interconversion from one enantiomer into the other. Following rac-HCQ, plasma (+)-(S)-levels always surpassed (?)-(R)-ones while in whole blood, (?)-(R)-HCQ concentrations were always the highest. When the enantiomers were administered separately, blood concentrations achieved after (?)-(R)-HCQ were higher, especially after multiple doses. These observations suggest that (?)-(R)-HCQ is preferentially concentrated by cellular components of blood. This enantioselective distribution of HCQ could be secondary to a stereoselective protein binding to plasma proteins, although a more specific binding of (?)-(R)-HCQ to blood cells cannot be ruled out. Since in whole blood (?)-(R)-HCQ is retained in cellular components, metabolism would favour the more available (+)-(S)-enantiomer. © 1994 Wiley-Liss, Inc.     

Renal transport of gossypol in the rabbit

The present work was carried out to investigate the transport characteristics of gossypol, a toxic weak organic acid (pK = 7.2) contained in cottonseed, into the rabbit renal cortical slice. The uptake of gossypol increased linearly during a 2-hr incubation after which it leveled off with the average slice-to-medium concentration ratio (S/M) slightly above 20. In the presence of metabolic inhibitors, the S/M gossypol leveled off at about 9, suggesting an extensive binding of gossypol to tissue proteins. The uptake of gossypol was significantly inhibited by p-aminohippurate (PAH), probenecid, ouabain, and DIDS, all of which are known inhibitors of renal organic anion transport. However, the gossypol uptake was not affected by tetraethylammonium (TEA), a prototypical organic cation. Kinetic studies indicated that the apparent Km for gossypol transport is 0.28 mM, and also that probenecid inhibits gossypol transport in a competitive manner. It is concluded that gossypol is transported by the renal tubule through the classic organic anion system.