Anita P. Hoffer

Antiproliferative effect of gossypol and its optical isomers on human reproductive cancer cell lines

The antiproliferative effect of gossypol and its optical isomers on various human cell lines of reproductive and nonreproductive tissue origin was studied. Various reproductive cancer cell lines of ovarian, gestational, and testicular origin were highly sensitive (IC50 values of 0.86-1.98) to gossypol. The antiproliferative action of gossypol was not restricted to reproductive cancers, as non-reproductive cancer cell lines were also equally sensitive (IC50 values of 0.69-3.55). In addition, actively proliferating untransformed cells such as fibroblasts and PHA-activated lymphocytes were also sensitive (IC50 values of 0.87-2.51). (-)-Gossypol was 3.6-12.4 times more potent than (+)-gossypol and 1.48-2.65 times more potent than (+/-)-gossypol. The most sensitive indicator of gossypol action was a decrease in DNA synthesis followed by inhibition of protein synthesis and uptake of rhodamine-123 by mitochondria as tested in an ovarian cancer cell line (OVCA 433) and a fibroblast line (Hs27). These results indicate that gossypol possesses a general nonselective antiproliferative action toward human cells in vitro. Further, the pharmacologic activity of gossypol as an antiproliferative agent is primarily attributable to its (-) isomer, which is also the active isomer as a contraceptive.

Ultrastructural, Biochemical, and Endocrine Studies on the Effects of Gossypol and its Isomeric Derivatives on The Male Reproductive Tract

Chinese scientists have reported that in clinical trials involving more than 8000 men tested over a four-year period gossypol was found to be a 99.89% effective, safe, and reversible contraceptive [1]. Treatment for eight weeks with 20 mg of gossypol daily decreases sperm counts to well below 4 x 106/ml; a maintenance dose of 40 mg of gossypol/wk thereafter sustains infertility. Gossypol does not appear to affect plasma testosterone levels in human subjects [2, 3] or in monkeys [4, 5], and the drug is without adverse effects on sexual function. A few minor side effects occur but they are transient and disappear spontaneously within the first few weeks of treatment. Hypokalemic paralysis, a potentially serious side effect reported by Chinese investigators in 66/8806 cases (0.75%) [6], was not observed in Brazilian men treated with gossypol [7] and it has since been suggested that the incidence of hypokalemia in China may be related more to regional differences in dietary intake than to effects of gossypol on serum potassium [8]. More recently, double-blind studies in gossypol-treated men in China have failed to confirm a causal relationship between gossypol and hypokalemia [9]. Interestingly, there is a high incidence of spontaneously occurring periodic hypokalemic paralysis in Oriental men [10].

ANTI-FERTILITY AND OTHER ACTIONS OF GOSSYPOL ANALOGUES

From a series of gossypol derivatives studied, we conclude that the carbonyl groups of gossypol are needed for inhibition of erythrocyte anion transport and the hydroxy groups affect but are not essential to that inhibition. In an in vitro mouse erythroleukemia cytocidal assay, the most active compounds were gossypol and apogossypol. The latter was not active in the inhibition of erythrocyte anion transport or in a spermicidal assay. Of the more simple structures related to gossypol, those that were active in the cytocidal and spermicidal assays were bi-aromatic, linked by a 1- and not a 4-carbon chain and had free phenolic hydroxyl groups. These results are included in a discussion of the specificity and mechanism of action of gossypol.

Quantitative analysis of germ cells and Leydig cells in rat made infertile with gossypol.

This study utilized improved methods of fixation and plastic embedding to quantitatively evaluate the effects of gossypol on germ cells and Leydig cells in testes of rats made infertile with gossypol. Rats were fed by gavage with 10, 20 or 30 mg/kg per day of gossypol for 9 weeks; control animals received the vehicle alone. Numbers of A spermatogonia, preleptotene and pachytene spermatocytes, and step 7 or 8 spermatids per Sertoli cell were counted in stages VII-VIII of the cycle of the seminiferous epithelium. Although high doses (30 mg/kg) of gossypol produced a significant decrease in the relative number of germ cells compared with vehicle-treated controls, no significant deviation in the relative number of germ cells was noted between controls and rats made infertile with 10 or 20 mg/kg/day of gossypol. Stereologic techniques were used to assess the changes in the Leydig cells. No significant deviation in the Leydig cell morphology, cell number, or cell volume was noted as a result of gossypol treatment at the dose levels employed. It appears that germ cell depletion, such as that caused by high doses of gossypol, is not mediated by a change in Leydig cell function. The present report emphasizes the importance of studies to determine the minimal effective doses for gossypols antifertility activity in animal models as well as in man.

Glycogen accumulations in differentiating mesonephric ducts and tubuli in male human embryos

SummaryHuman mesonephric duct epithelial cells contained empty appearing regions in the infranuclear cytoplasm when prepared for transmission electron microscopy using glutaraldehyde and osmium fixation. The same regions stained positively with PAS in Epon sections for light microscopy suggesting that glycogen was present. Incubation with saliva abolished the reaction. For electron microscopy the glycogen stained very intensely if a mixture of osmium tetroxide and potassium ferrocyanide was used instead of osmium alone. Glycogen accumulations were present between the ages of 5 to 10 weeks and absent at the age of 15 weeks. Reports by others indicate that glycogen may be present in different reactive forms in relation to its staining behaviour after various fixatives. The present results, and similar studies in other tissues, indicate that osmium tetroxide-potassium ferrocyanide fixative should be used routinely for preservation of embryos and fetuses and where indicated, for ultrastructural identification of glycogen and cytoplasmic filaments in clinical specimens.