Chromosomal aberrations caused by sesquiterpene lactones in chinese hamster ovary cells

Chromosomal aberrations caused by sesquiterpene lactones in chinese hamster ovary cells

BiochemicelSvsfmr,,.tic=amdEcology,VoL 13. No. 3, pp. 365-369, 1985. Primed in Greet Britain. 0305-1978/85 $3.00+0.00 PecgamonPre~ Ltd. Chromosomal ...

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BiochemicelSvsfmr,,.tic=amdEcology,VoL 13. No. 3, pp. 365-369, 1985. Primed in Greet Britain.

0305-1978/85 $3.00+0.00 PecgamonPre~ Ltd.

Chromosomal Aberrations Caused By Sesquiterpene Lactones in Chinese Hamster Ovary Cells B. F. ABEYSEKERA, Z. ABRAMOWSKI and G. H. N. TOWERS Department of Botany, University of British Columbia, Vancouver, B. C., V6T 2B1, Canada Key Word Index--Sesquiterpene lactones; cytotoxicity; chromosomal aberrations; Chinese hamster ovary cells. Abstract--The cytotoxic behaviour of 20 sasquiterpene lactonas toward Chinese hamster ovary cells was examined. The structural pre-recluisite for cytotoxicity was the o-methylene y-lactone moiety. Certain sesquiterpene lactonas caused chromosomal aberrations suggesting that DNA was the cellular target. The cellular target for most of these compounds, however, is probably not the nucleus and the cytotoxicity may be accounted for by Michael-type additions with sulphydryl groups of enzymes and other proteins.

Introduction Sesquiterpene lactones, a class of natural products that form the 'bitter principles' of many species of Compositae, cause allergic contact dermatitis in humans [1], poison livestock [2, 3], and are insect feeding deterrents [4]. Some also display anti-tumour and cytotoxic activity [5-9] as well as antibiotic behaviour [10]. One of the main requisites for biological activity is the presence of a - C H = C - C = O moiety as pert of an ester or ketone or lactone [11,12]. The mechanism of cytotoxicity is thought to be a Michael-type addition reaction between this moiety and the sulphydryl groups of enzymes and other proteins [13]. However, an alternative hypothesis is that these compounds alkylate DNA, causesingle strand breaks and interfere with the DNA template [14-16]. Our results indicate that certain sesquiterpene lactones cause chromosomal aberrations in Chinese hamster ovary (CHO) cells and, therefore, that the cell nucleus is the target of these toxic compounds. Results and Discussion Of the sescluiterpene lactones tested, helenalin (1) proved to be the most toxic (Table 1 ). The closely related and more lipophilic balduilin (2) was surprisingly less toxic as increasing (Received 6 March 1984) 365

lipophilicity was expected to confer greater cytotoxicity. The result might be due to the lower solubility of balduilin in the test medium or to the different stereochernistries of the hydroxyl group of helenalin and the acetoxy group of balduinino In keeping with the observations of Kupchan et al. [11] and Lee et al. [12], all compounds containing an a-methylene Flactone moiety showed cytotoxic behaviour. The three compounds tested that did not have this structural feature, namely isotenulin (3), usantonin (12) and desacetoxymatricarin (20) were essentially non-toxic. It is remarkable that isotenulin is non-toxic since it has a /9unsubstituted cyclopentenone moiety which was shown to be responsible for the antimicrobial effect observed with helenalin and 11,13-dihydrohelenalin [17]. This might be because of the different biological models used to test these compounds or because of slight stereochemical differences between the molecules. It is noteworthy that desacetoxymatricarin (20) showed a small percentage of chromosome aberrations even though its cyclopentenone system was B-substituted. The presence of a cyclopentenone moiety, in addition to the a-methylene y-lactone, is known to enhance the activity of certain sesquiterpene lactones and, as expected, perthenin (4) was about 10 times more toxic than coronopilin (5) or damsin (6). In the series, isoalantolactone

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(7), ivalin (8) and ivasperin (9), the cytotoxicity increased with increasing lipophilicity. Similarly, the more lipophilic acetate, cumambrin A (17), was about twice as toxic as the free alcohol, cumambrin B (18). Chromosomal aberrations were clearly demonstrable with helenalin, parthenin, coronopilin, damsin and ivasperin and, to a lesser extent, with isoalantolactone, pinnatifidin, pyrethrosin, cumambrin B, grossheimin and desacetoxymatricarin. This is an important result since, prior to this, proof of chromosomal aberrations had only been furnished for parthenin. This also provides evidence that certain sesquiterpene lactones may attack DNA directly. In all cases there was an abrupt change from toxicity to normality. This is in marked contrast to results obtained with other compounds tested in this laboratory using this method on CHO cells [18, 19]. Thus, with phototoxic furanoqu~nolines or furanocoumarins there is a much wider range of mitotic inhibition (MI) following the least toxic concentration and the percentage of metaphase plates with at least one chromosomal aberration (B) decreased gradually from 100% to 0% over a wide range of concentrations. It is interesting that with furanocoumarins, adducts with DNA have been isolated [20] and the nucleus established as the target of these compounds. It appears that sesquiterpene lactones are cytotoxic for a number of reasons; genuine genotoxicity is exhibited by only a few. Helenalin has been shown to inhibit, m vivo and in vitro, both DNA and protein synthesis in Ehrlich ascites carcinoma [21,22] and P-388 lymphocyte leukemia cells [13]. DNA polymerase and thymidylate synthetase activities are inhibited [21 ] as well as inosine phosphate dehydrogenase activities. The activities of these enzymes are suppressed at concentrations consistent with in vivo anti-neoplastic activity and could be the result of Michael additions of lactones to sulphydryl functions of those enzymes. The inhibition of those enzymes, in turn, lead to inhibition of DNA synthesis. More recently, helenalin and a synthetic derivative have been shown to inhibit protein synthesis primarily at the level of initiation in P-388 lymphocytic leukemia cells [23]. The formation of the 48S initiation complex was inhibited. How this affects mitosis, leading to the

B.F. ABIEYSEKERAETAL

production of chromosomal aberrations, remains to be explained.

Experimental Materia/s. Se~luiterpene lactones were isolated by publishad procedures from various plant sources [24]. The identities and purity of all compounds were confirmed by TLC, m.p. and spectroscopic methods. Chinese hamster ovary cells were kindly provided by Professor H. Stich, Cancer Research Centre, Vancouver. Cytotoxicity tests. CHO cells were cultured in Eagle's minimum essential medium (MEM) supplemented with 10% foetal calf serum (FCS) and streptomycin sulphate (29.6 pg/ml), penicillin G, N F. sodium (125 pg/ml), kanarnycin (lO0pg/ml), fungizone (2.5/~g/ml) and 7.5% NaHCO= (1 rng/ml). Calls of stock cultures, grown in 240 ml plastic culture flasks (Falcon) at 37" in a CO= incubator at 100% humidity, were dispersed, centrifuged and resuependad in fresh medium. For seeding, the suspension was diluted to an approximate density of 7 x 10 = cells/mL A quantity (2 ml) of this suspension were seeded on a 22 mm = coverslip in a 35 x 10 mm Falcon plastic dish and kept in MEM with 1 0% FCS at 37" for 2 days to achieve a 60-80% confluency of cells. The compounds to be tested were dissolved in 95% EtOH or sterile HaO and diluted in MEM containing 2.5% FCS. The EtOH concentration in the first dilution did not exceed 1%. Subsequent two-fold dilutions were made and 1 ml of each was added to the Petri dishes after removing the tissue culture medium. Cultures were exposed to the chemicals for 3 h at 37". After treatment, the test solutions were removed, the coverslips washed twice with MEM and fresh medium containing 10% FCS added to the Petri dishes. Samples were incubated for 16 h. At 4 h prior to harvesting, 0.2 ml colchicine (0.01% in 2.5% MEM) was added. Calls were then treated with 1% sodium citrate for 20min, followed by fixation in Carnoy's solution for 20min. Air-dried coverslips were stained with 2% acid orcein, mounted and 100 metaphasa plates analysed in each case for chromosome breakages and exchanges. The results in Table 1 represent the mean of at least three replicates.

A c k n o w l e d g e m e n t = t ~ - W e thank the Natural Sciences and Engineering Research Council of Canada for supporting this research and Professor H. Stich and Dr. M. P. Rosin for help with the genotoxicity tests. We also thank Dr. A. Romo de Vivar for a small sample of helenalin and Dr. E Rodriguez for a gift of damsin and isotenulin.

References 1. Mitchell, J. C., Dupuis, G. and Towers, G. H. N. (1972) Br. J. Derm. 87, 235. 2. Ivie, G. W., Witzel, D. A., Herz. W., Kannan, R.. Norman, J. O., Rushing, D. D., Johnson, J. H., Rowe, L. D. and Veech, J. A. (1975) J. Agric. Food Chem. 23, 841. 3. Towers, G. H N., Mitchell, J. C., Rodriguez, E., Bennett, F. D. and Rao, P. V. S. (1977) J. Sci. Ind. Res. 36, 672.

CHROMOSOMAL ABERRATIONS IN CHINESE HAMSTER

4. Burnett, W. C., Jones, S. B., Mabcy, T. J. and Padolinea, W. (1974) Biochem. Syst. Ecol. 2, 25. 5. Hartwell, J. L and Abbott, B.J. (1969) Adv. Pharrr~col. Chemothet. 7, 117. 6. IJe, K. H., Huang, E. S., Piantadosi, C., Pagano, J. S. and Geiuman, T. A. (1971) CancerRes. 31, 1649. 7. Lee, IL H., Meck, R., Piantadosi, C. and Huang, E. S. (1973) J. Med. Chem. 16, 299. 8. Woynerow~i, J. W. and Konopa, J. (1981) Molec. Pham'mcol. 19, 97. 9. Mew, D., Baiza, F., Towers, G. H. N. and Levy, J. G. (1982) Pbnta Med. 46, 23, 10. Rodriguez, E., Towers, G. H. N. and Mitchell, J. C. (1976) Phytochem/stry 18, 1573. 11. Kupchan, S. M., F.akin, M. A. and Thomas, A. M. (1971 ) J. Med. Chem. 14, 1147. 12. Lee, K. H., Fumikawa, H. and Huang, E. S. (1972) J. Meal. Chem. 15, 609. 13. Hall, I. H., lee, K. H., Mar, E. C., Statues, C. O. and Waddell, T. G. (1977) J. Meal. Chem. 20, 333. 14. Vaidya, V. G., KuIkarni, I. and Nagesampagi, B. A. (1978) Ind. J. Exp. Biol. 18, 1117. 15. Woynarowski, J. W., Bearrnan, T. A. and Konopa, J, (1981) Biochem. Pharmaco/. 30, 3005.

[email protected]@

16. Jones, D. H., Kim, H. L and Donne, y, K, C. (1981) Res. Commun. Chem. Path. Phannacol. 34, 161. 17. Lee, K. H., Ikuba, T., Wu, R. Y. and Geissman, T. A. (1977) Phytochemistry 18, 1177. 18. Towers, G. H. N. and Ab¢amowski, 7- (1983) J. Nat. Prod. [email protected], 576. 19. Abeysekers, B. F., Abramowski, 7- and Towers, G. H. N (1983) Photochem. Photobiol. 38, 311 20. Kanne, D., Straub, K., Rapoport, H. and Hearst, J. E. (1982) Biochemistry 21,861. 21. Hall, I. H., Lee, K. H., Statues, C. O., EIGaby, S. A., Ibuka, T., Wu, Y. S., Kimura, T. and Haruna, M. (1978) J. Phann. Sci. 67, 1235. 22. Hall, I. H., Lee, K. H., Okano, M., Sims, D., Ibuka, D., Liou, Y. F. and Imakura, Y. (1981) J. Pharm. Sci. 70, 1147. 23. Liou, Y. F., Hall, I. F., Lee, K. H., Williams, W. L and Chaney, S. G:(1983) Biochim. Biophys. Acta [email protected], 190. 24. Fischer, N. H., Olivier, E. J. and Fischer, H. D. (1979) Progress in the Chemistry of Organic Natural Products Vol. 38, p. 47. Swinger, New York.

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