Environmental Health Perspectives 103, Supplement 7, October 1995
Effects of Estrogens on Microtubule Polymerization in Vitro: Correlation with Estrogenicity
Manfred Metzler and Erika Pfeiffer
Department of Chemistry, University of Kaiserslautern, Kaiserslautern, Germany
Abstract
Several estrogens of diverse classes, namely stilbene estrogens, steroidal estrogens, phytoestrogens, mycoestrogens, lignans, and bisphenol A, were studied for their ability to interfere with the assembly of microtubules under cell-free conditions. Inhibition of microtubules in intact cells may lead to the induction of micronuclei and aneuploidy and thereby contribute to estrogen-mediated carcinogenesis. Diethylstilbestrol and various other stilbene estrogens, as well as bisphenol A, were good inhibitors of microtubule polymerization whereas the other tested compounds were devoid of this activity. Therefore, the ability of estrogenic substances to interact with microtubules does not correlate with the hormonal activity. --
Environ Health Perspect 103(Suppl 7):21-22 (1995)
Key words: microtubules, stilbene estrogens, steroidal estrogens, phytoestrogens, mycoestrogens, lignans, bisphenol A
This paper was presented at the Symposium on Estrogens in the Environment, III; Global Health Implications held 9-11 January 1994 in Washington, DC. Manuscript received: March 15, 1995; manuscript accepted: April 4, 1995.
Work in our laboratory has been supported by the Deutsche Forschungsgemeinschaft (grant Me 574/9-1).
Address correspondence to Dr. M. Metzler, Department of Chemistry, University of Kaiserslautern, P.O. Box 3049, D-67653 Kaiserslautern, Germany. Telephone: 49 6312053217. Fax: 49 6312053200.
Abbreviations used: E2, 17ß-estradiol; DES, diethylstilbestrol; SHE, Syrian hamster embryo; MT, microtubule.
Introduction
Both natural estrogens, e.g., 17ß-estradiol (E2), and synthetic estrogens, e.g., diethylstilbestrol (DES), are associated with human neoplasia (1). In experimental animals E2 and DES as well as several of their analogs induce tumors in various tissues, e.g., the kidney of the male Syrian golden hamster (1,2). Moreover, primary cells such as Syrian hamster embryo (SHE) fibroblasts are morphologically and neoplastically transformed by E2 and DES (3-5). In short-term assays, carcinogenic estrogens fail to induce gene mutations and DNA damage but lead to numerical and structural chromosomal aberrations (6,7). In particular, nonrandom aneuploidies were observed in DES-transformed SHE cells (8). Several laboratories have reported that DES has colchicinelike effects, causing inhibition of microtubule (MT) polymerization under cell-free conditions as well as disruption of the mitotic spindle and the cytoplasmic MT complex in mammalian cells (9-16). To clarify the question whether interaction with MTs is a property shared by all estrogens, we have studied several estrogens of diverse classes, namely stilbene estrogens, steroidal estrogens, phytoestrogens, mycoestrogens, and lignans, for their ability to interfere with MT assembly. Bisphenol A, used for the manufacture of polycarbonate plastics and recently identified as an environmental estrogen, has also been included in this study.
Methods
The MT polymerization assay used in our laboratory has been described before (17). Briefly, the test compound dissolved in ethanol or dimethyl sulfoxide was added to 0.5 ml (final volume) of 10 µM MT proteins from bovine brain in assembly buffer; concentrations of the estrogens varied between 10 and 100 µM; the concentration of the organic solvent was 2%. Following an incubation period of 20 min at 37°C, MT assembly was started by adding 0.5 mM guanosine triphosphate, and the increase in turbidity was measured at 350 nm for 30 min. The control incubation containing all components except the test compound was used as reference (100% assembly). Depolymerization at 4°C was carried out to confirm MT formation and detect aggregation.
Results
The result of our study is summarized in Table 1. All stilbene estrogens tested at 50-µM concentrations caused a 10 to 40% inhibition of MT assembly. The data are published in detail elsewhere (17). The plastic estrogen bisphenol A proved to be a powerful inhibitor, exhibiting about half the activity of DES (18). In contrast, none of the tested steroidal estrogens showed a detectable inhibitory effect on MT polymerization even at 100-µM concentrations. The same lack of effect was observed for the phytoestrogens, mycoestrogens, and lignans listed in Table 1. However, several of these compounds could not be tested at 100-µM concentrations due to limitations in solubility. The highest concentrations tested without precipitation of the estrogens are given in Table 1.
Discussion
When the inhibitory effects of the various compounds for MT assembly are compared with their known estrogen receptor binding affinities, it becomes obvious that no correlation between these two properties exists. Stilbene estrogens with very low estrogen receptor binding, such as Z,Z-dienestrol, indanestrol, or threo-hexestrol (19), inhibit MT polymerization as well as the strong estrogens DES, E,E-dienestrol, or erythro-hexestrol. On the other hand, powerful steroidal estrogens such as E2, 17
-ethinyl-E2, and 11ß-methoxy-17-ethinyl-E2 (moxestrol) fail to inhibit MT assembly, as do the moderately strong phytoestrogens and mycoestrogens coumestrol, genistein, and zearalenone.
Recent studies on the mechanism of the DES-mediated inhibition of MT polymerization have shown that DES has two binding sites on the tubulin heterodimer (20), which is the major constituent of MT proteins. One of the binding sites of DES is related to that of colchicine, as both compounds compete for this site. Binding to this site probably causes the inhibition of MT assembly. The other DES binding site seems to have no relevance for MT polymerization, as it also binds E2, which displays no inhibitory effect.
It should be pointed out that the effect of a compound on MT assembly observed under cell-free conditons may be different in intact cells where metabolism of the estrogen can either diminish or enhance the propensity for tubulin binding. An increased inhibition of MT assembly might be expected for all compounds capable of quinone formation, e.g., DES, indenestrol A, the catechol estrogens 2- and 4-hydroxy-E1 and E2, and possibly coumestrol, as these reactive intermediates should covalently bind to thiol groups of tubulin essential for MT polymerization (21). Furthermore, mechanisms other than inhibition of MT formation may be employed by certain estrogens to cause chromosomal nondisjunction or chromosomal breakage. Examples are E2 and coumestrol (22): E2 appears to damage the kinetochore and thereby impair the attachment of the mitotic spindle to the chromatids, whereas coumestrol seemingly has clastogenic effects in mammalian cells. Therefore, the noncovalent interaction with tubulin and subsequent inhibition of MT assembly is only one among several mechanisms whereby estrogens can cause chromosomal damage. This study has shown that the ability of an estrogenic substance to bind tubulin does not correlate with the hormonal activity.
Reference
1. IARC. Sex Hormones (II). IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans. Vol 21. Lyon:International Agency for Research on Cancer, 1979.
2. Li JJ, Li SA, Klicka JK, Parsons JA, Lam LTK. Relative carcinogenic activity of various synthetic and natural estrogens in the Syrian hamster kidney. Cancer Res 43:5200-5204 (1983).
3. McLachlan JA, Wong A, Degen GH, Barrett JC. Morphological and neoplastic transformation of Syrian hamster embryo fibroblasts by diethylstilbestrol and its analogs. Cancer Res 42:3040-3045 (1982).
4. Tsutsui T, Maizumi H, McLachlan JA, Barrett JC. Aneuploidy induction and cell transformation by diethylstilbestrol: a possible chromosomal mechanism in carcinogenesis. Cancer Res 43:3814-3821 (1983).
5. Tsutsui T, Suzuki N, Fukuda S, Sato M, Maizumi H, McLachlan JA, Barrett JC. 17ß-Estradiol-induced cell transformation and aneuploidy of Syrian hamster embryo cells in culture. Carcinogenesis 8:1715-1719 (1987).
6. Barrett JC, Wong A, McLachlan JA. Diethylstilbestrol induces neoplastic transformation without measurable gene mutation at two loci. Science 212:1402-1404 (1981).
7. Degen GH, Metzler M. Sex hormones and neoplasia: genotoxic effects in short term assays. Arch Toxicol Suppl 10:264-278 (1987).
8. Ozawa N, Oshimura M, McLachlan JA, Barrett JC. Nonrandom karyotypic changes in immortal and tumorigenic Syrian hamster cells induced by diethylstilbestrol. Cancer Genet Cytogenet 38:271-282 (1989).
9. Sawada M, Ishidate M Jr. Colchicine-like effect of diethylstilbestrol (DES) on mammalian cells in vitro. Mutat Res 57:175-182 (1978).
10. Parry EM, Danford N, Parry JM. Differential staining of chromosomes and spindle and its use as an assay for determining the effect of diethylstilbestrol on cultured mammalian cells. Mutat Res 105:243-252 (1982).
11. Sato Y, Murai T, Tsumuraya M, Saito H, Kodama M. Disruptive effect of diethylstilbestrol on microtubules. Gann 75:1046-1048 (1984).
12. Sharp DC, Parry JM. Diethylstilboestrol: the binding and effects of diethylstilboestrol upon the polymerisation and depolymerisation of purified microtubule protein in vitro. Carcinogenesis 6:865-871 (1985).
13. Hartley-Asp B, Deinum J, Wallin M. Diethylstilbestrol induces metaphase arrest and inhibits microtubule assembly. Mutat Res 143:231-235 (1985).
14. Tucker RW, Barrett JC. Decreased numbers of spindle and cytoplasmic microtubules in hamster embryo cells treated with a carcinogen, diethylstilbestrol. Cancer Res 46:2088-2095 (1986).
15. Wheeler WJ, Cherry LM, Downs T, Hsu TC. Mitotic inhibition and aneuploidy induction by naturally occurring and synthetic estrogens in Chinese hamster cells in vitro. Mutat Res 171:31-41 (1986).
16. Sakakibara Y, Saito I, Ichinoseki K, Oda T, Kaneko M, Saito H, Kodama M, Sato Y. Effects of diethylstilbestrol and its methyl ethers on aneuploidy induction and microtubule distribution in Chinese hamster V79 cells. Mutat Res 263:269-276 (1991).
17. Pfeiffer E, Metzler M. Effects of steroidal and stilbene estrogens and their peroxidative metabolites on microtubular proteins. In: Hormonal Carcinogenesis (Li JJ, Nandi S, Li SA, eds). New York:Springer Publishing, 1992;313-317.
18. Pfeiffer E, Metzler M. Bisphenol A inhibits microtubule polymerization in vitro and disrupts the cytoplasmic microtubule complex in cultured V79 cells. In: Hormonal Carcinogenesis, Vol 2 (Li JJ, Li SA, Nandi S, Gustafsson JA, Sekely LI, eds). New York:Springer Publishing, in press.
19. Korach KS, Metzler M, McLachlan JA. Estrogenic activity in vivo and in vitro of some diethylstilbestrol metabolites and analogs. Proc Natl Acad Sci USA 75:468-471 (1978).
20. Pfeiffer E, Neuwirth B, Metzler M. Interaction of diethylstilbestrol and estradiol with tubulin: evidence for different sites of noncovalent binding. Toxicologist 14:135 (1994).
21. Epe B, Harttig U, Stopper H, Metzler M. Covalent binding of reactive estrogen metabolites to microtubular protein as a possible mechanism of aneuploidy induction and neoplastic cell transformation. Environ Health Perspect 88:123-127 (1990).
22. Schuler M, Huber K, Zankl H, Metzler M. Induction of micronucleation, spindle disturbances and mitotic arrest in human chorionic villi cells by 17ß-estradiol, diethylstilbestrol and coumestrol. In: Hormonal Carcinogenesis, Vol 2 (Li JJ, Li SA, Nandi S, Gustafsson JA, Sekely LI eds). New York:Springer Publishing, in press.
Last Update: September 18, 1998
