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Clastogenicity of a male contraceptive, gossypol, in mammalian cell cultures with and without the metabolic activation by S9 mix
ENVIRONMENTAL
RESEARCH
36, 138-143 (1985)
Clastogenicity of a Male Contraceptive, Gossypol, in Mammalian Cell Cultures with and without the Metabolic Activation by S9 Mix’ JAN C. LIANG* Department
of Cell Biology,
The University at Houston,
AND WEI-SAN
of Texas Houston,
YES
M.D. Anderson Texas 77030
Hospital
and Tumor
Institute
Accepted March 5. 1983 The clastogenicity of a potential male contraceptive, gossypol, was examined in cultured Chinese hamster cells with and without the presence of a metabolic activation system (rat liver S9 mix). Gossypol at concentrations of 1, 5, and 10 kg/ml did not induce chromosome breakage either in the presence or absence of the S9 mix. The ability of this compound to induce chromosome breakage and polyploidy was further examined in human lymphocyte cultures. Neither increased frequencies of chromosome breakage nor polyploidy was found in lymphocyte cultures from two healthy donors. The present study indicates that gossypol does not cause genetic damage at the chromosomal level. This is consistent with previously reported findings that it does not produce mutations in the Ames test. Gossypol has been under clinical trial in China for years and shown to be effective in 99.9% of over 10,000 men tested with no or mild side effects. If this compound can be further proven to be “safe” and approved for world-wide use as a male contraceptive, it would be for the benefit of all mankind. Q 1985 Academic Press, Inc.
INTRODUCTION
Gossypol is a polyphenolic compound found in leaves, stems, roots, and seeds of certain species of cotton plants (Berardi and Goldblatt, 1980). Since the discovery of its antifertility effect on human males, gossypol has been proposed for use as a male contraceptive (National Coordinating Group for Male Contraception, 1978). One of the major concerns about using gossypol as a male contraceptive is that genetic toxicity of this compound has not been fully investigated. In a study by investigators from the Batelle Institute (Geneva), gossypol was found to have tumor-initiating and -promoting activity from skin tests in mice (Haroz and Thomasson, 1980). But since gossypol is an impure product, it is not clear whether or not the same component in gossypol induces both infertility and tumors. Another observation which suggested potential genetic hazzards of gossypol was that malformed spermatozoa were found in gossypol-fed animals as well as in human male volunteers (Zatuchni and Osborn, 1981; National Coordinating Group for Male Contraception, 1978). Since a wide variety of mutagens, t Supported in part by research Grant R-808582 from the U.S. Environmental Protection Agency, Washington, D.C. * To whom correspondence should be addressed. 3 On leave from Department of Histology and Embryology, Capital Medical College of China, Beijing, China. 0013-9351/85 $3.00 Copyright 0 1985 by Academic Press, Inc. All rights of reproduction in any form reserved
138
CLASTOGENICITY
OF GOSSYPOL
139
clastogens, and carcinogens have been shown to induce increased numbers of abnormal spermatozoa (Wyrobek and Bruce, 1978), it is important to know if gossypol causes any genetic damage. Studies on gossypol-induced genetic damage have been few thus far. Ye et al. (1983) reported that gossypol inhibited the synthesis of DNA, RNA, and protein in mammalian cell cultures. Colman et al. (1979) and de Peyster and Wang (1979) found that gossypol did not induce gene mutations in the Ames Salmonella/ mammalian microsome test. The effects of gossypol on chromosomes of human lymphocytes were investigated by Cai et al. (1981) and Tsui et al. (1983). Both studies indicated that gossypol did not induce chromosome breakage. However, in neither study was any metabolic activation system incorporated. In the study by Cai et al. (1981), a significantly increased frequency of polyploids was found in lymphocyte cultures treated with 9 kg/ml of gossypol as compared to the control. But in a preliminary study by Ye et al. (1983), the incidence of polyploids was not found to be different between the gossypol-treated and the control cultures. Because a large number of carcinogens require metabolic activation to cause chromosome breakage, and further, agents that induce polyploidy represent a class of carcinogens which interfere with cell division processes, the purposes of the present study are (1) to elucidate potential clastogenic (chromosome breaking) actions of gossypol in mammalian cell cultures with the activation system incorporated and (2) to confirm or disprove the finding that gossypol induces polyploidy. MATERIALS
AND METHODS
Chinese hamster fibroblast cultures. Diploid Chinese hamster line Don was maintained in McCoy’s 5a medium supplemented with 10% fetal bovine serum (GIBCO, Grand Island, N.Y.) and 0.1 pg/ml of neomycin (Elkins-Sinn, Inc., N.J.). Exponentially growing cells (60-70% confluent) in T25 flasks (Corning) were treated with 1, 5, and 10 Fg/ml of gossypol acetate (Sigma Chemical Co., St. Louis, MO.) for 5-48 hr with and without the presence of S9 mix (0.5 ml in 10 ml of culture medium). Gossypol was dissolved in dimethyl sulfoxide (DMSO) to a final concentration of 0.2% in the medium. DMSO alone, with and without the S9 mix, served as untreated controls. As a positive control, cyclophosphamide (a clastogen which requires metabolic activation) was tested simultaneously at 100 and 200 FM with and without the S9 mix. Duplicate cultures were used for each experimental condition and two separate experiments were performed. At the end of the incubation, cells were harvested with I-hr colcemid (0.05 kg/ml) pretreatment and air-dried slide preparations were made. Slides were then coded and the mitotic index (MI) and chromosome breakage frequency were determined. MI was determined by counting a minimum of 1000 cells and chromosome breakage frequency was estimated from reading 50 metaphases for each culture under each treatment condition in each experiment. Therefore, at each treatment condition, a total of 4000 cells were counted for MI determination and 200 cells were analyzed for chromosome aberrations. Preparation ofthe S9 mix. Rat liver homogenate was obtained from Dr. Thomas
140
LIANG
AND YE
Connor at The University of Texas Health Science Center at Houston, School of Public Health. The procedure for preparing the S9 mix is as follows: Young male Long-Evans hooded rats (Blue Spruce Farms, Altament, N.Y.), 200-2508, were injected intraperitoneally with 500 mg/kg of Aroclor 1254 dissolved in corn oil. The animals were sacrificed 5 days later and the liver homogenate was prepared according to the procedure of Ames er al. (1975). The S9 homogenate was mixed with cofactors before each experiment and the mixture was referred to as the S9 mix. One ml of S9 mix contained 0.3 ml of S9 homogenate and 0.7 ml of cofactors (11.5 mM MgCl,, 47 mM KCl, 7.1 mM glucose-6-phosphate, 5.7 mM NADP, and 140 mM K,HPO,KH,PO, buffer at pH 7.4). Human lymphocytes. Human lymphocyte cultures were initiated with peripheral blood from two healthy donors in RPM1 1640 medium supplemented with 15% fetal bovine serum and phytohemagglutinin (Burroughs-Wellcome, Greenville, N.C.). Gossypol at concentrations of 1, 5, and 10 pg/ml was added to the cultures at the time of culture initiation as described above, and cells were harvested after 48 and 72 hr incubation with 1-hr colcemid (0.05 pg/ml) pretreatment. Cells treated with the solvent DMSO (0.2%) alone served as controls. Conventional air-dried preparations were made and slides were coded before they were subjected to cytogenetic analyses. The frequency of polyploid cells was estimated from 800 metaphases and the frequency of chromosome aberrations was determined by analyzing 200 metaphases for each culture condition. RESULTS
Gossypol was found to be toxic to both Chinese hamster fibroblasts and human lymphocytes. Severe mitotic inhibition and cell death were noted in Chinese hamster fibroblasts at a concentration of 25 kg/ml and in human lymphocytes at 50 kg/ml. The concentrations of gossypol used in the present study were chosen to be at and under 10 kg/ml to ensure the availability of a suitable number of metaphases for chromosome analysis. At the three concentrations (1, 5, 10 p.g/ ml) tested, gossypol alone was not found to affect the mitotic index in Chinese hamster fibroblasts (Table 1). In the presence of the S9 mix, gossypol caused a slight decrease in the MI. Since the S9 mix alone also caused a slight decrease TABLE MITOTIC
INDEX AND CHINESE
1
CHROMOSOME ABERRATION FREQUENCY HAMSTER (DON) FIBROBLASTS
IN
Ml Agents Gossypol
Cyclophosphamide
breaks/cell
Concentration
-s9
+s9
-s9
+s9
0 1 pg/ml 5 iAm1 10 p,g/ml
0.03 0.02 0.03 0.02
0.02 0.01 0.01 0.01 0.04 0.03
0.08 0.06 0.07 0.08 0.09 0.08
0.09 0.05 0.07 0.08 0.18 0.40
100
PM
0.05
200
FM
0.06
Note. Fibroblasts were exposed to gossypol for 5 hr with or without the presence of rat liver microsomes (S9 mix)
CLASTOGENICITY
OF GOSSYPOL
141
of the MI, the declined MI in gossypol-treated cells in the presence of the S9 mix was likely due to the toxicity of the S9 mix. The frequencies of chromosome breakage induced by gossypol in the presence or absence of the S9 mix for 5 hr are shown in Table 1. Since only chromatid gaps and breaks were observed, data were simply expressed as breaks/cell to include gaps. While the frequencies of cyclophosphamide-induced chromosome breakage followed a dose-related increase in the presence of the S9 mix, the frequencies of chromosome breakage in gossypol-treated cells remained at the control level with or without S9 mix. Longer exposure period (24 and 48 hr) also did not result in increased chromosome breakage (data not shown). The frequencies of chromosome aberrations and polyploids in cultured human lymphocytes treated with 1, 5, and 10 pg/ml of gossypol for 72 hr are shown in Table 2. Neither of the two cultures from separate donors showed significantly elevated numbers of chromosome aberrations nor did they exhibit increased frequencies of polyploids. When the exposure period was reduced to 48 hr to allow only the first- and second-division mitoses for analysis, similar results were obtained (data not shown). DISCUSSION
Gossypol is considered by the World Health Organization (WHO) to be the only agent, thus far investigated, that has the promise to be used as a male contraceptive worldwide in the foreseeable future (Prasad and Diczfalusy, 1982). The results of a clinical trial conducted in China indicated that the toxicity of gossypol in humans was generally mild. The primary side effect was hypokalemia which might be mitigated by the addition of potassium in the diet of gossypol users. Various studies on the genetic toxicity of gossypol also indicated that this agent was rather “safe.” Using the Ames Safmonellulmammalian microsome test, several investigators showed that gossypol was nonmutagenic (Colman et al., 1979; de Peyster and Wang, 1979). In human lymphocyte cultures, gossypol was TABLE 2 FREQUENCIESOFCHROMOSOMEABERRATIONS
Gossypol dose b&d) 0
Donor
Chrtd gaps
Chrtd breaks
ANDPOLYPLOIDS
Isochrtd gaps
IN
lsochrtd breaks
I
HUMAN LYMPHOCYTES
Lesions”
Polyploidsb
0 2
2 1
2
I
1
1
1 2
1 1
1
2 I
4 I
5
1 2
1
1 1
2 1
3 0
10
1 2
2 I
5 1
2 0
1
I
Note. Lymphocytes were treated with gossypol for 72 hr. il Number of lesions observed per 200 metaphases. b Number of polyploids observed per 800 metaphases.
I
142
LIANG
AND YE
not found to cause chromosome breakage (Cai et al., 1981; Tsui et al., 1983), sister chromatid exchanges (Tsui et al., 1983), or micronucleus induction (Tsui et al., 1983). Gossypol, after metabolic activation by liver microsomes (S9), was shown in the present study to be nonclastogenic. Therefore, it appears that gossypol does not damage DNA. Our finding that gossypol did not induce polyploidy in human lymphocyte cultures was contrary to that of Cai et al. (1981). Because more blood samples and cells were analyzed in the present study than in that of Cai et al., we believe that data strongly indicate that gossypol does not cause polyploidy. The mechanisms by which gossypol causes infertility are not yet fully understood. One of the likely possibilities is that this compound specifically affects the metabolism of spermiogenic cells. This concept is supported by the finding of Lee et al. (1982) that sperm-specific lactate dehydrogenase X isozyme is selectively inhibited by gossypol and by the finding of Poso et al. (1980) that fructose utilization in human spermatozoa is profoundly inhibited by this drug. Cytological observations that gossypol inhibits the motility of human spermatozoa in vitro (Waller et al., 1980; Kalla and Vasudar, 1981) and rat spermatozoa in vivo (unpublished data) also support this view. Perhaps the most convincing evidence which indicates a direct action of gossypol on mature sperm is the finding that gossypol causes degeneration of the mitochondrial sheath of the middle piece in the sperm of rats and men, resulting in bulging, fraying, bending of the tail, and decapitation (Oko and Hrudka, 1982; Hadley et al., 1981; Nadakavukaven et al., 1979; National Coordinating Group for Male Contraceptives, 1978). Conceivably, the antifertility effect of gossypol can be achieved by the failure of these damaged sperms to perform the fertilization function. The safety of gossypol as a male contraceptive drug is further characterized by the findings of the present communication that it is not clastogenic in somatic mitotic cells. Preliminary observations in our laboratory also indicate that it does not cause chromosome aberrations in either spermatogonial mitoses or diakinesis, metaphase I, and metaphase II cells in rats. Observations of Majumdar et al. (1982) that gossypol does not cause sperm head abnormalities in mice also suggest that gossypol does not pose a genetic hazard. Further research to determine the mechanisms of toxic effects of gossypol and to minimize the toxicity of gossypol by modifying the chemical structure of this compound will greatly facilitate the approval of this compound as a male contraceptive by the drug regulatory agencies. ACKNOWLEDGMENTS The authors thank Dr. T. C. Hsu for his interest and support of this investigation, Dr. T. H. Connor for the S9 preparations, and Mrs. Ruby Kirkpatrick for secretarial assistance.
REFERENCES Ames, B. N., McCann, J., and Yamasaki E. (1975). Methods for detecting carcinogens and mutagens with the SaZmoneZZalmammalian microsome mutagenecity test. Mutat. Res. 31, 347-364. Berardi, L. S., and Goldblatt, L. A. (1980). Gossypol. In “Toxic Constitutents of Plant Foodstuffs” (I. E. Liener, Ed.), pp. 183-237. Academic Press, New York. Cai, Y., Liu, Y., Xu, G., Li, S., and Shieh, S. (1981). Effect of gossypol on the frequency of chro-
CLASTOGENICITY
OF GOSSYPOL
143
mosomal aberrations and sister chromatid exchanges in human peripheral lymphocyte in vitro. Chieh Pou Hsueh Pao (Acta Anat. Sinica) 12, 293-297. Colman, N., Gardner, A., and Herbert, V. (1979). Non-mutagenicity of gossypol in the Salmonella/ mammalian-microsome plate assay. Environ. Mutagen. 1, 315-320. de Peyster, A., Wang, Y. Y. (1979). Gossypol-proposed contraceptive for men passes the Ames test. N. Et@. J. Med. 30, 275-276. Hadley, M. A., Lin, Y. C., and Dym. M. (1981). Effects of gossypol on the reproductive system of male rats. J. Androl. 2, 190-199. Haroz, R. K., and Thomasson. J. (1980). Tumor initiating and promoting activity of gossypol. Toxicol. Lett. Suppl. 6, 72. Kalla, N. R., and Vasudar, M. (1981). Studies on the male antifertility agent-gossypol acetic acid. II. Effect of gossypol acetic acid on the motility and ATPase activity of human spermatozoa. Andrologia 13, 95-98. Lee, C. Y. G.. Moon, Y. S., Yuan, J. H., and Chen, A. F. (1982). Enzyme inactivation and inhibition by gossypol. Mol. Cell. Biochetn. 47, 65-70. Majumdar. S. K., Ingraham, H. J., and Prymowicz. D. A. (1982). Gossypol-an effective male contraceptive was not mutagenic in sperm head abnormality assay in mice. Canad. J. Genet. Cytol. 24 (6). 777-780. Nadakavukaren, M. J., Sorensen, R. H., and Tone, J. N. (1979). Effect of gossypol on the ultrastructure of rat spermatozoa, Cell Tissue Res. 204, 293-296. National Coordinating Group for Male Contraceptives (1978). A new antifertility agent for males Chin. Med. J. (Peking English Ed.) 6, 417-428. Oko, R., and Hrudka. F. (1982). Segmental aplasia of the mitochondrial sheath and sequelae induced by gossypol in rat spermatozoa. Biol. Reprod. 26, 183-195. Poso, H., Wichmann, K., Janne, J., and Luukkainin, T. (1980). Gossypol, a powerful inhibitor or human spermatozoa1 metabolism. Lancer 1 (8173), 885-886. Prasad, M. R. N., and Diczfalusy, E. (1982). Gossypol. Int. J. Androl., Suppl. 5, 53-70. Tsui, Y-C., Creasy, M. R., and Hulten. M. A. (1983). The effect of the male contraceptive agent gossypol on human lymphocytes in vitro: Traditional chromosome breakage, micronuclei, sister chromatid exchange, and cell kinetics. J. Med. Genet. 20, 81-85. Wailer, D. P., Zaneveld, L. J. D., and Fong, H. H. S. (1980). In vitro spermicidal activity of gossypol. Contraception 22, 183-187. Wyrobek, A. J., and Bruce, W. R. (1978). The induction of sperm-shape abnormalities in mice and humans. In “Chemical Mutagens, Principles and Methods for their Detection” (A. Hollaender and F. J. de Serres, Eds.), pp. 257-285. Plenum Press, New York. Ye, W., Liang, J. C.. and Hsu, T. C. (1983). Toxicity of a male contraceptive, gossypol, in mammalian cell cultures, In Vitro 19, 53-57. Zatuchni, G. I., and Osbom. C. K. (1981). Gossypol: A possible male antifertility agent, report of a workshop. Res. Front. Fertil. Regul. 1, I-15.
RESEARCH
36, 138-143 (1985)
Clastogenicity of a Male Contraceptive, Gossypol, in Mammalian Cell Cultures with and without the Metabolic Activation by S9 Mix’ JAN C. LIANG* Department
of Cell Biology,
The University at Houston,
AND WEI-SAN
of Texas Houston,
YES
M.D. Anderson Texas 77030
Hospital
and Tumor
Institute
Accepted March 5. 1983 The clastogenicity of a potential male contraceptive, gossypol, was examined in cultured Chinese hamster cells with and without the presence of a metabolic activation system (rat liver S9 mix). Gossypol at concentrations of 1, 5, and 10 kg/ml did not induce chromosome breakage either in the presence or absence of the S9 mix. The ability of this compound to induce chromosome breakage and polyploidy was further examined in human lymphocyte cultures. Neither increased frequencies of chromosome breakage nor polyploidy was found in lymphocyte cultures from two healthy donors. The present study indicates that gossypol does not cause genetic damage at the chromosomal level. This is consistent with previously reported findings that it does not produce mutations in the Ames test. Gossypol has been under clinical trial in China for years and shown to be effective in 99.9% of over 10,000 men tested with no or mild side effects. If this compound can be further proven to be “safe” and approved for world-wide use as a male contraceptive, it would be for the benefit of all mankind. Q 1985 Academic Press, Inc.
INTRODUCTION
Gossypol is a polyphenolic compound found in leaves, stems, roots, and seeds of certain species of cotton plants (Berardi and Goldblatt, 1980). Since the discovery of its antifertility effect on human males, gossypol has been proposed for use as a male contraceptive (National Coordinating Group for Male Contraception, 1978). One of the major concerns about using gossypol as a male contraceptive is that genetic toxicity of this compound has not been fully investigated. In a study by investigators from the Batelle Institute (Geneva), gossypol was found to have tumor-initiating and -promoting activity from skin tests in mice (Haroz and Thomasson, 1980). But since gossypol is an impure product, it is not clear whether or not the same component in gossypol induces both infertility and tumors. Another observation which suggested potential genetic hazzards of gossypol was that malformed spermatozoa were found in gossypol-fed animals as well as in human male volunteers (Zatuchni and Osborn, 1981; National Coordinating Group for Male Contraception, 1978). Since a wide variety of mutagens, t Supported in part by research Grant R-808582 from the U.S. Environmental Protection Agency, Washington, D.C. * To whom correspondence should be addressed. 3 On leave from Department of Histology and Embryology, Capital Medical College of China, Beijing, China. 0013-9351/85 $3.00 Copyright 0 1985 by Academic Press, Inc. All rights of reproduction in any form reserved
138
CLASTOGENICITY
OF GOSSYPOL
139
clastogens, and carcinogens have been shown to induce increased numbers of abnormal spermatozoa (Wyrobek and Bruce, 1978), it is important to know if gossypol causes any genetic damage. Studies on gossypol-induced genetic damage have been few thus far. Ye et al. (1983) reported that gossypol inhibited the synthesis of DNA, RNA, and protein in mammalian cell cultures. Colman et al. (1979) and de Peyster and Wang (1979) found that gossypol did not induce gene mutations in the Ames Salmonella/ mammalian microsome test. The effects of gossypol on chromosomes of human lymphocytes were investigated by Cai et al. (1981) and Tsui et al. (1983). Both studies indicated that gossypol did not induce chromosome breakage. However, in neither study was any metabolic activation system incorporated. In the study by Cai et al. (1981), a significantly increased frequency of polyploids was found in lymphocyte cultures treated with 9 kg/ml of gossypol as compared to the control. But in a preliminary study by Ye et al. (1983), the incidence of polyploids was not found to be different between the gossypol-treated and the control cultures. Because a large number of carcinogens require metabolic activation to cause chromosome breakage, and further, agents that induce polyploidy represent a class of carcinogens which interfere with cell division processes, the purposes of the present study are (1) to elucidate potential clastogenic (chromosome breaking) actions of gossypol in mammalian cell cultures with the activation system incorporated and (2) to confirm or disprove the finding that gossypol induces polyploidy. MATERIALS
AND METHODS
Chinese hamster fibroblast cultures. Diploid Chinese hamster line Don was maintained in McCoy’s 5a medium supplemented with 10% fetal bovine serum (GIBCO, Grand Island, N.Y.) and 0.1 pg/ml of neomycin (Elkins-Sinn, Inc., N.J.). Exponentially growing cells (60-70% confluent) in T25 flasks (Corning) were treated with 1, 5, and 10 Fg/ml of gossypol acetate (Sigma Chemical Co., St. Louis, MO.) for 5-48 hr with and without the presence of S9 mix (0.5 ml in 10 ml of culture medium). Gossypol was dissolved in dimethyl sulfoxide (DMSO) to a final concentration of 0.2% in the medium. DMSO alone, with and without the S9 mix, served as untreated controls. As a positive control, cyclophosphamide (a clastogen which requires metabolic activation) was tested simultaneously at 100 and 200 FM with and without the S9 mix. Duplicate cultures were used for each experimental condition and two separate experiments were performed. At the end of the incubation, cells were harvested with I-hr colcemid (0.05 kg/ml) pretreatment and air-dried slide preparations were made. Slides were then coded and the mitotic index (MI) and chromosome breakage frequency were determined. MI was determined by counting a minimum of 1000 cells and chromosome breakage frequency was estimated from reading 50 metaphases for each culture under each treatment condition in each experiment. Therefore, at each treatment condition, a total of 4000 cells were counted for MI determination and 200 cells were analyzed for chromosome aberrations. Preparation ofthe S9 mix. Rat liver homogenate was obtained from Dr. Thomas
140
LIANG
AND YE
Connor at The University of Texas Health Science Center at Houston, School of Public Health. The procedure for preparing the S9 mix is as follows: Young male Long-Evans hooded rats (Blue Spruce Farms, Altament, N.Y.), 200-2508, were injected intraperitoneally with 500 mg/kg of Aroclor 1254 dissolved in corn oil. The animals were sacrificed 5 days later and the liver homogenate was prepared according to the procedure of Ames er al. (1975). The S9 homogenate was mixed with cofactors before each experiment and the mixture was referred to as the S9 mix. One ml of S9 mix contained 0.3 ml of S9 homogenate and 0.7 ml of cofactors (11.5 mM MgCl,, 47 mM KCl, 7.1 mM glucose-6-phosphate, 5.7 mM NADP, and 140 mM K,HPO,KH,PO, buffer at pH 7.4). Human lymphocytes. Human lymphocyte cultures were initiated with peripheral blood from two healthy donors in RPM1 1640 medium supplemented with 15% fetal bovine serum and phytohemagglutinin (Burroughs-Wellcome, Greenville, N.C.). Gossypol at concentrations of 1, 5, and 10 pg/ml was added to the cultures at the time of culture initiation as described above, and cells were harvested after 48 and 72 hr incubation with 1-hr colcemid (0.05 pg/ml) pretreatment. Cells treated with the solvent DMSO (0.2%) alone served as controls. Conventional air-dried preparations were made and slides were coded before they were subjected to cytogenetic analyses. The frequency of polyploid cells was estimated from 800 metaphases and the frequency of chromosome aberrations was determined by analyzing 200 metaphases for each culture condition. RESULTS
Gossypol was found to be toxic to both Chinese hamster fibroblasts and human lymphocytes. Severe mitotic inhibition and cell death were noted in Chinese hamster fibroblasts at a concentration of 25 kg/ml and in human lymphocytes at 50 kg/ml. The concentrations of gossypol used in the present study were chosen to be at and under 10 kg/ml to ensure the availability of a suitable number of metaphases for chromosome analysis. At the three concentrations (1, 5, 10 p.g/ ml) tested, gossypol alone was not found to affect the mitotic index in Chinese hamster fibroblasts (Table 1). In the presence of the S9 mix, gossypol caused a slight decrease in the MI. Since the S9 mix alone also caused a slight decrease TABLE MITOTIC
INDEX AND CHINESE
1
CHROMOSOME ABERRATION FREQUENCY HAMSTER (DON) FIBROBLASTS
IN
Ml Agents Gossypol
Cyclophosphamide
breaks/cell
Concentration
-s9
+s9
-s9
+s9
0 1 pg/ml 5 iAm1 10 p,g/ml
0.03 0.02 0.03 0.02
0.02 0.01 0.01 0.01 0.04 0.03
0.08 0.06 0.07 0.08 0.09 0.08
0.09 0.05 0.07 0.08 0.18 0.40
100
PM
0.05
200
FM
0.06
Note. Fibroblasts were exposed to gossypol for 5 hr with or without the presence of rat liver microsomes (S9 mix)
CLASTOGENICITY
OF GOSSYPOL
141
of the MI, the declined MI in gossypol-treated cells in the presence of the S9 mix was likely due to the toxicity of the S9 mix. The frequencies of chromosome breakage induced by gossypol in the presence or absence of the S9 mix for 5 hr are shown in Table 1. Since only chromatid gaps and breaks were observed, data were simply expressed as breaks/cell to include gaps. While the frequencies of cyclophosphamide-induced chromosome breakage followed a dose-related increase in the presence of the S9 mix, the frequencies of chromosome breakage in gossypol-treated cells remained at the control level with or without S9 mix. Longer exposure period (24 and 48 hr) also did not result in increased chromosome breakage (data not shown). The frequencies of chromosome aberrations and polyploids in cultured human lymphocytes treated with 1, 5, and 10 pg/ml of gossypol for 72 hr are shown in Table 2. Neither of the two cultures from separate donors showed significantly elevated numbers of chromosome aberrations nor did they exhibit increased frequencies of polyploids. When the exposure period was reduced to 48 hr to allow only the first- and second-division mitoses for analysis, similar results were obtained (data not shown). DISCUSSION
Gossypol is considered by the World Health Organization (WHO) to be the only agent, thus far investigated, that has the promise to be used as a male contraceptive worldwide in the foreseeable future (Prasad and Diczfalusy, 1982). The results of a clinical trial conducted in China indicated that the toxicity of gossypol in humans was generally mild. The primary side effect was hypokalemia which might be mitigated by the addition of potassium in the diet of gossypol users. Various studies on the genetic toxicity of gossypol also indicated that this agent was rather “safe.” Using the Ames Safmonellulmammalian microsome test, several investigators showed that gossypol was nonmutagenic (Colman et al., 1979; de Peyster and Wang, 1979). In human lymphocyte cultures, gossypol was TABLE 2 FREQUENCIESOFCHROMOSOMEABERRATIONS
Gossypol dose b&d) 0
Donor
Chrtd gaps
Chrtd breaks
ANDPOLYPLOIDS
Isochrtd gaps
IN
lsochrtd breaks
I
HUMAN LYMPHOCYTES
Lesions”
Polyploidsb
0 2
2 1
2
I
1
1
1 2
1 1
1
2 I
4 I
5
1 2
1
1 1
2 1
3 0
10
1 2
2 I
5 1
2 0
1
I
Note. Lymphocytes were treated with gossypol for 72 hr. il Number of lesions observed per 200 metaphases. b Number of polyploids observed per 800 metaphases.
I
142
LIANG
AND YE
not found to cause chromosome breakage (Cai et al., 1981; Tsui et al., 1983), sister chromatid exchanges (Tsui et al., 1983), or micronucleus induction (Tsui et al., 1983). Gossypol, after metabolic activation by liver microsomes (S9), was shown in the present study to be nonclastogenic. Therefore, it appears that gossypol does not damage DNA. Our finding that gossypol did not induce polyploidy in human lymphocyte cultures was contrary to that of Cai et al. (1981). Because more blood samples and cells were analyzed in the present study than in that of Cai et al., we believe that data strongly indicate that gossypol does not cause polyploidy. The mechanisms by which gossypol causes infertility are not yet fully understood. One of the likely possibilities is that this compound specifically affects the metabolism of spermiogenic cells. This concept is supported by the finding of Lee et al. (1982) that sperm-specific lactate dehydrogenase X isozyme is selectively inhibited by gossypol and by the finding of Poso et al. (1980) that fructose utilization in human spermatozoa is profoundly inhibited by this drug. Cytological observations that gossypol inhibits the motility of human spermatozoa in vitro (Waller et al., 1980; Kalla and Vasudar, 1981) and rat spermatozoa in vivo (unpublished data) also support this view. Perhaps the most convincing evidence which indicates a direct action of gossypol on mature sperm is the finding that gossypol causes degeneration of the mitochondrial sheath of the middle piece in the sperm of rats and men, resulting in bulging, fraying, bending of the tail, and decapitation (Oko and Hrudka, 1982; Hadley et al., 1981; Nadakavukaven et al., 1979; National Coordinating Group for Male Contraceptives, 1978). Conceivably, the antifertility effect of gossypol can be achieved by the failure of these damaged sperms to perform the fertilization function. The safety of gossypol as a male contraceptive drug is further characterized by the findings of the present communication that it is not clastogenic in somatic mitotic cells. Preliminary observations in our laboratory also indicate that it does not cause chromosome aberrations in either spermatogonial mitoses or diakinesis, metaphase I, and metaphase II cells in rats. Observations of Majumdar et al. (1982) that gossypol does not cause sperm head abnormalities in mice also suggest that gossypol does not pose a genetic hazard. Further research to determine the mechanisms of toxic effects of gossypol and to minimize the toxicity of gossypol by modifying the chemical structure of this compound will greatly facilitate the approval of this compound as a male contraceptive by the drug regulatory agencies. ACKNOWLEDGMENTS The authors thank Dr. T. C. Hsu for his interest and support of this investigation, Dr. T. H. Connor for the S9 preparations, and Mrs. Ruby Kirkpatrick for secretarial assistance.
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