Mutagenicity of gossypol analyzed by induction of meiotic micronuclei in vitro

Mutation Research, 208 (1988) 69-72

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Elsevier MTRI_ 0109

Mutagenicity of gossypol analyzed by induction of meiotic micronuclei in vitro Liu D e - y u a, J a a n a Lfihdetie b a n d M a r t t i P a r v i n e n c "National Research lnstitutej~r Family Planning, No. 12 Da Hui Zhi Lu, Haidian District, Beijing (People's Republic' ~?['China). Departments ~)[~'Anatomy and 'Medical Genetics, Institute of Biomedicine, University of Turku, SF-20520 Turku (Finland)

(Accepted 25 January 1988)

Kevwo~tl~': Rat; Spermatogenesis;Meioticdivision; Spermatids; Gossypol; Micronuclei

Summary C h r o m o s o m e breakage caused by mutagens in male germ cells can be analyzed by micronucleus induction during meiotic division. This can be followed in vitro by culturing seminiferous tubular segments from stages of the epithelial cycle that contain late pachytene and diakinetic primary spermatocytes. We studied the mutagenic potential of a male contraceptive, gossypol, in this test system using adriamycin (10 ng/ml) as a reference mutagen. A small but significant increase in the frequency of micronuclei was induced with concentrations of 10 and 20 #g/ml of gossypol, while cytotoxic effects appeared at concentration of 20 ~g/ml and were evident at 50 tzg/ml. Analysis of meiotic micronucleus induction in vitro seems to be a sensitive ~est system of male germ-cell mutagenesis, but further studies on the possible mutagenic effects of gossypol are needed.

Gossypol, a phenolic compound isolated from cottonseed oil, has been widely used in China as an antifertility agent for men (for references, see Prasad and Diczfalusy, 1982). Its effects" on germ cells have been extensively investigated. Uncoupling of oxidative phosphorylation, morphological damage to mitochondria and specific inhibition of L D H - X enzyme have been the most c o m m o n findings (for references, see Baccetti et al., 1986). The toxicity, mutagenicity and carcinogenicity of

Correspondence: Martti Parvinen, Institute of Biomedicine, Department of Anatomy, University of Turku, Kiinamyllynkatu 10, SF-20520 Turku (Finland).

gossypol have not been studied systematically. Most mutagenicity studies carried out thus far have yielded negative results. We decided to study the possible genotoxicity of gossypol by the in vitro meiotic micronucleus method that has been shown to be a very sensitive male germ-cell mutagenicity assay for S-independent agents (Toppari et al., 1986).

Materials and methods Separation and culture o f d e f i n e d seminiferous tubule segments

Segments of seminiferous tubules were isolated from young adult (3-5 months) Sprague-Dawley

0165-7992/88/$ 03.50 (~3 1988 Elsevier Science Publishers B.V. (Biomedical Division)

70 Weak Strong spot spot I L _ -v

Dark vii-viii

Pale zone Weak spot Ix-xN x -x v

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Fig. 1. A schematic tracing of the transiltuminated, freshly isolated rat seminiferous tubule with characteristic light absorption zones. Squash preparations are isolated from stages XII and XIII (narrow areas between horizontal lines) to make sure that no cells at meiotic division are present in the tubular segment at the onset of culture. The segment about 2 mm in length marked with an asterisk is selected for culture at the onset of the weak spot zone.

rats by transillumination-assisted microdissection (Parvinen and Vanha-Perttula, 1972), according to a scheme depicted in Fig. 1. Stages X I I - X I I I of the epithelial cycle at the border of pale and weak spot transillumination zones were identified by phasecontrast microscopy (Toppari et al., 1986). Segments of 2 m m in length were separated under sterile conditions in H a m ' s F 12/Dulbecco's minimum essential medium (MEM) 1:1 (Flow Laboratories, Irvine, Scotland), supplemented with Hepes (15 raM, Sigma, St. Louis, MO), gentamycin (10 ~g/ml, Neofarma, Sein~ijoki, Finland) and sodium bicarbonate (1.2 g/l), pH 7.4. They were incubated in 100 ~1 of the medium in 96-well culture plates (Nunclon, A / S Nunc, Roskilde, Denmark) in a humidified atmosphere of 5% CO2 in air at 32°C for 3-5 h, before addition of gossypol acetic acid (National Research Institute for Family Planning, Beijing, People's Republic of China) or adriamycin (Farmitalia Carlo Erba, Barcelona, Spain). Gossypol was dissolved in ethanol and adjusted to 1, 10, 20 and 50 gg/ml in 0.1% final concentration of ethanol in the tissue culture medium. Adriamycin was dissolved in 100 /zl of the same medium to a final concentration of 10 ng/ml. The culture was continued for 4 days, the time required for development of late primary spermatocytes from stages X I I - X I I I of the epithelial cycle to spermatids through meiotic divisions (Parvinen et al., 1983). For morphological analysis, the seminiferous tubule segments were fixed in Bouin's fluid for 1 h, embedded in paraffin, sectioned at 5 /~m and stained with periodic acid-Schiff-bematoxylin.

Assessment of micronuclei The micronuclei were scored from squash preparations as described by L~,hdetie and Parvinen (1981). Briefly, the slides were frozen in liquid nitrogen and the coverslips removed, fixed in acetic acid-ethanol (1:3) and stained with Hoecbst 33258 fluorescent dye. The scoring was performed at 1000-fold magnification in a fluorescence microscope. The number of micronuclei in 500 early spermatids was counted in each slide in a blind fashion.

Statistical analyses Calculations of statistical significance were based on the Poisson distribution (Lfibdetie and Parvinen, 1981). Since there was no significant difference between the 2 control groups (with and without 0.1% ethanol), these groups were pooled.

Results

Completion of meiotic divisions was evident in the tubules cultured in the presence of 0-20 ~g/ml of gossypol or 10 ng/ml of adriamycin, while 20 /~g/ml of gossypol arrested and killed some of the dividing meiotic cells, and the concentration of 50 ~ g / m l was clearly toxic to the cultured seminiferous tubules. Adriamycin (10 ng/ml) or gossypol in a concentration of 1 /~g/ml did not cause alterations in histological appearance compared with the controls. In tubules cultured in the medium only or in a medium containing 0.1°70 ethanol, the frequencies of micronuclei were 0.29% and 0.35%, respectively. This difference is not statistically significant (Fig. 2). Adriamycin (10 ng/ml), the positive control, induced micronuclei during meiotic divisions in a frequency of 4.10%, which is compatible with earlier observations (4.42°70, Toppari et al., 1986). At the concentration of 1 /zg/ml, gossypol did not increase the micronucleus frequency (0.34%) while a slight but significant ( p < 0 . 0 1 ) increase was found with 10 and 20 ~g/ml of gossypol (0.79 and 0.84%, respec:ively, Fig. 2).

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0 L.

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2

0mr c

CE

A

GI

GI0

G20

Fig. 2. Frequencies of micronuclei (%) after cultures of stage XII-XIII seminiferous tubule segments in control (C, 21 experiments, 10 500 spermatids scored), 0.1% ethanol control media (CE, 20 experiments, 10 000 spermatids), and in the presence of 10 n g / m l of adriamycin (A, 20 experiments, 10 011 spermatids), or 1 (GI, 16 experiments, 8005 spermatids), 10 (G10, 14 experiments, 7000 spermatids) and 20 (G20, 5 experiments, 2500 spermatids) txg/ml of gossypol in the tissue culture medium. The SD is indicated at the top of the bars. Significant differences were found between adriamycin and all other groups (/7<0.01) and between 10 and 20 #g/ml gossypol groups compared with controls (p<0.01).

Discussion

Meiotic micronuclei are signs of chromosome breakage during meiosis. Lagging acentric chromosome fragments obtain an own nuclear envelope during telophase and become visible as micronuclei in early spermatids. Adriamycin, which intercalates with DNA and causes chromosome breakage by this mechanism (for review, see Vig, 1977), has been used as a reference mutagen for meiotic micronucleus methods both in vivo and in vitro (L~ihdetie, 1983; Toppari et al., 1986). Unlike many other chemicals, e.g., alkylating agents, the action is S-independent and similar to that of X-rays (Hsu et al., 1982). The in vitro analysis of meiotic micronucleus induction measures the mutagenic insults to the late prophase of meiosis, i.e., cells in the late Gz phase of the meiotic cell cycle, but does not reveal the mutagenic potential of agents that only affect DNA synthesis. During rat meiosis, DNA replica-

tion takes place in the preleptotene stage primary spermatocytes about 3 weeks before meiotic division. At present, it is not possible to cultivate rat seminiferous tubules from the onset of meiosis (preleptotene in stages VII and VIII) to its completion (division in stage XIV). The daily dose of gossypol used in male contraception is 20 mg (Peyster and Wang, 1979). Since its half-life is long and an accumulation in the tissues is possible (Prasad and Diczfalusy, 1982), it is difficult to estimate the actual contraceptive concentration of gossypol in the human testis. This is the first report about a weak mutagenic effect of gossypol in mammalian germ cells. Gossypol has not been found to be mutagenic in Ames' SalmoneIla/microsome assay nor in the mouse sperm-head abnormality assay (Majumdar et al., 1982b, a). However, species differences exist and it has been reported that gossypol has no infertility effect nor testicular toxicity in the mouse (Hahn et al., 1981). Synaptonemal complex formation and function were not affected by gossypol treatment of rats (Bhagirath and Kundu, 1985). However, these results do not exclude the possibility of germ cell mutagenicity at later stages of spermatogenesis. Such effects can be studied by the present micronucleus method. A germ cell-specific toxic action of gossypol may also imply differences in the mutagenic response of germ cells compared with somatic cells. Studies with Chinese hamster ovary cells or cultured human lymphocytes did not reveal chromosome breakage, micronuclei or increase of sister-chromatid exchange, although the mitotic index was shown to be reduced (Tsui et al., 1983; Ye et al., 1983). Recent observations of Rosenberg and Adlakha (1986) suggest that gossypol interferes with DNA synthesis by covalently modifying DNA polymerase and possibly altering the DNA template. Furthermore, gossypol-induced DNA-strand breaks have been observed in human fibroblasts and leukocytes in vitro (Nordenskj61d and Lambert, 1984; Yu et al., 1986). These observations together with the present results and with the data suggesting a tumorinducing or -promoting activity of gossypol acetic

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acid (Haroz and Thomasson, 1980) make further evaluations of the mutagenic potential of gossypol important.

Acknowledgements We thank Ms. Leena Simola and Mr. Henrik Wikgren for skillful technical assistance, and the Academy of Finland (Project no. 200 at the Medical Research Council) for financial support. Ms. Liu De-yu was a research and training fellow supported by the World Health Organization during this work.

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mutagenic in sperm head abnormality assay in mice, Can. J. Genet. Cytoh, 24, 777-780. Majumdar, S.K., J.D. Thatcher, E.H. Dennis, A. Cutrone, M. Stockage and M. Hammond (1982b) Mutagenic evaluation of two male contraceptives: 5-thio-D-glucose and gossypol acetic acid, J. Hered., 73, 76-77. Nordenskj61d, M., and B. Lambert (1984) Gossypol induces DNA strand breaks in human fibroblasts and sister chromatid exchanges in human lymphocytes in vitro, J. Med. Genet., 21, 129-132. Parvinen, M., and T. Vanha-Perttula (1972) Identification and enzyme quantitation of the stages of the seminiferous epithelial wave in the rat, Anat. Rec., 174, 435-450. Parvinen, M., W.W. Wright, D.M. Phillips, J.P. Mather, N.A. Musto and C.W. Bardin (1983) Spermatogenesis in vitro: completion of meiosis and early spermiogenesis, Endocrinology, 112, 1150-1152. Peyster, A. De, and Y.Y. Wang (1979) Gossypol - proposed contraceptive for men passes the Ames test, New Engt. J. Med., 301, 275-276. Prasad, M.R.N., and E. Diczfalusy (1982) Gossypol, Int. J. Androl., Suppl. 5, 53-70. Rosenberg, L.J., and R.C. Adlakha (1986) Mechanism of inhibition of DNA synthesis by gossypol, J. Cell Biol., 103, 172a. Toppari, J., J, L~ihdetie, P. Hfirk6nen, E. Eerola and M. Parvinen (1986) Mutagen effects on cultured seminiferous tubules: induction of meiotic micronuclei by adriamycin, Mutation Res., 171, 149-156. Tsui, Y.-C., M.R. Creasy and M.A. Hulten (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. Vig, B.K. (1977) Genetic toxicology of mitomycin C, actinomycins, daunomycin and adriamycin, Mutation Res., 39, 189-238. Ye, W.-S., J.C. Liang and T.C. Hsu (1983) Toxicity of a male contraceptive, gossypol, in mammalian cell cultures, In Vitro, 19, 53-57. Yu, C., M. Sten, M. Nordenskj61d, B. Lambert, S.A. Marlin and R.H. Zhou (1986) The effect of gossypol on the frequency of DNA-strand breaks in human leukocytes in vitro, Mutation Res., 164, 71-78. Communicated by B. Lambert