Induction of mammalian cell transformation and genotoxicity by 2 ...

Carcinogenesis vol.21 no.4 pp.735–740, 2000

Induction of mammalian cell transformation and genotoxicity by 2-methoxyestradiol, an endogenous metabolite of estrogen

Takeki Tsutsui, Yukiko Tamura, Makoto Hagiwara, Takashi Miyachi, Hirohito Hikiba, Chikahiro Kubo and J.Carl Barrett1,2 Department of Pharmacology, The Nippon Dental University, School of Dentistry at Tokyo, Tokyo 102-8159, Japan and 1Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Science, PO Box 12233, Research Triangle Park, NC 27709, USA 2To

whom correspondence should be addressed Email: [email protected]

2-Methoxyestradiol (2-MeOE2) is an endogenous metabolite of 17β-estradiol and a proposed inhibitor of tumor growth and angiogenesis. However, 2-MeOE2 is also an inhibitor of microtubule assembly and other microtubule inhibitors, e.g. colcemid and diethylstilbestrol, induce aneuploidy and cell transformation in cultured mammalian cells. To assess the in vitro carcinogenicity and related activity of 2MeOE2, the abilities of this metabolite to induce cell transformation and genetic effects were studied simultaneously using Syrian hamster embryo (SHE) fibroblasts. Growth of these cells was reduced by treatment with 2MeOE2 at 0.1–1.0 µg/ml in a concentration-dependent manner. Treatment of SHE cells with 2-MeOE2 at 0.3 or 1.0 µg/ml for 2–48 h also resulted in a concentration- and treatment time-related increase in the mitotic index and the percentage of multinucleated cells. Treatment with 2-MeOE2 at 0.1–1.0 µg/ml for 48 h induced a statistically significant increase in the frequencies of morphological transformation of SHE cells in a concentration-dependent manner. A statistically significant increase in the frequencies of somatic mutations at the Na⍣/K⍣ ATPase or hprt locus was also observed in cells treated with 2-MeOE2 for 48 h at 0.1 or 0.3 µg/ml, respectively. Treatment of SHE cells with 2-MeOE2 at 0.3 or 1.0 µg/ml for 24 h induced chromosome aberrations, mainly breaks, exchanges and chromosome pulverization. The incidence of chromosome aberrations was not affected by co-treatment with αnaphthoflavone, an inhibitor of 2-hydroxylase that inhibits oxidative conversion of 2-MeOE2 to 2-hydroxyestradiol, but the incidence was slightly increased by co-treatment with L-ascorbic acid. Numerical chromosomal changes in the near diploid range and in the tetraploid and near tetraploid ranges were also detected in 2-MeOE2-treated cells. These findings indicate that 2-MeOE2 has cell transforming and genotoxic activities in cultured mammalian cells and potential carcinogenic activity. Introduction 2-Methoxyestradiol (2-MeOE2), an endogenous metabolite of 17β-estradiol (E2), inhibits cell proliferation (1,2) and Abbreviations: B[a]P, benzo[a]pyrene; CFE, colony-forming efficiency; DES, diethylstilbestrol; DMSO, dimethylsulfoxide; E2, 17β-estradiol; 2-OHE2, 2-hydroxyestradiol; 2-MeOE2, 2-methoxyestradiol; Oua, ouabain; SHE, Syrian hamster embryo; TG, 6-thioguanine. Published by Oxford University Press

angiogenesis in vitro (2) and suppresses the growth of tumors in vivo (2). 2-MeOE2 also blocks mitosis of human cancer cells that lack estrogen receptors in metaphase (1). A proposed mechanism by which 2-MeOE2 blocks mitosis is via inhibition of microtubule assembly (1,3) and interference with mitotic spindle dynamics (4). Microtubule-disrupting agents, e.g. colcemid and diethylstilbestrol (DES), a synthetic estrogen, exhibit transforming and genotoxic activities in cultured mammalian cells (5–8). DES and colcemid disrupt polymerization of microtubules and induce a specific type of genetic change, i.e. aneuploidy, in hamster (7–10) and human cells (11). Aneuploidy induction correlates with the ability to induce morphological transformation of Syrian hamster embryo (SHE) cells with parallel dose– response curves (7,8). Treatment of synchronized cultures with DES results in a cell cycle-dependent induction of aneuploid cells that parallels the induction of cell transformation, with the greatest level observed following treatment during the mitotic phase. Parallel dose–response curves for cell transformation and aneuploidy induction by DES are observed when the synchronized cultures are treated during the mitotic phase of the cell cycle (7). A non-random chromosome gain accompanies DES-induced immortalization and tumorigenic conversion of SHE cells (12). In addition, a good correlation has been demonstrated in Chinese hamster cells between aneuploidy induction and cell transformation (13). These suggest that conformational or functional changes in microtubule organization lead to aneuploidy induction and cell transformation. Therefore, the present study was carried out to determine the ability of 2-MeOE2 to induce cell transformation and genetic effects in SHE cells. The results obtained indicate that 2-MeOE2 has cell transforming and genotoxic activities in cultured mammalian cells and potential carcinogenic activity. Materials and methods Cells and chemicals SHE fibroblast cultures were established from 13 day gestation hamster fetuses and grown as previously described (7). 2-MeOE2 and E2 were purchased from Sigma (St Louis, MO) and dissolved in dimethylsulfoxide (DMSO) at 3 and 10 mg/ml, respectively. α-Naphthoflavone was purchased from Wako Pure Chemical (Osaka, Japan) and dissolved in DMSO at 10 mM. DMSO was added to control cultures at a final concentration of ⬍0.33%. L-Ascorbic acid phosphate magnesium salt n-hydrate was obtained from Wako Pure Chemical. 6-Thioguanine (TG), ouabain (Oua) and benzo[a]pyrene (B[a]P) were obtained from Sigma. Growth curve Cells (3⫻104) in logarithmic growth phase were plated on 35 mm dishes (Falcon, Oxnard, CA). After overnight incubation, the cells were treated with 2-MeOE2 at various concentrations for 24–72 h. The number of cells per 35 mm dish was determined after trypsinization. Cell counts are presented as means ⫾ SD from four dishes per counting point (24, 48 and 72 h after start of treatment). Mitotic inhibition and multinucleation Cells were plated on 60 mm dishes at a density of 1–2⫻105 cells/dish and, after overnight incubation, were treated with 2-MeOE2 or E2 at the designated concentrations for 2–48 h. After trypsinization, the cells were treated with

735

T.Tsutsui et al. 0.9% sodium citrate at room temperature for 13 min, fixed with Carnoy’s solution (methanol:acetic acid, 3:1), and stored at room temperature for 4–7 days. The suspension of cells in fixative was dropped onto glass slides wetted with 100% ethanol and then air dried, as previously described (14). This procedure allowed nuclei to remain intact in the cell cytoplasm. The slides were stained with Giemsa and ⬎1000 cells were scored for each experimental group. Cellular transformation and somatic mutations Cells (2.5⫻105) were plated into 75 cm2 flasks (Falcon), incubated overnight and treated with 2-MeOE2 for 48 h. After trypsinization, a part of the cell suspension was assayed for morphological transformation and the remaining cells were subcultured at a density of 4.0⫻105 cells/75 cm2 flask for mutation experiments. For morphological transformation, 2000 cells were replated on 100 mm dishes (20 dishes for each group) and incubated for 7 days to form colonies. The cells were fixed with absolute methanol and stained with a 10% aqueous Giemsa solution. The number of surviving colonies with ⬎50 cells and morphologically transformed colonies were scored by previously established criteria (7). For mutation experiments, the cells were grown for an expression time of 4 days and then 105 cells were plated on 100 mm dishes (10 dishes/group) with medium containing 18 µM TG or 1.1 mM Oua and incubated for 7 days for colony formation (15). The mutation frequency was calculated as described previously (15). Chromosome aberrations and chromosome number SHE cells were plated into 75 cm2 flasks at 2.5⫻105 cells/flask for the 48 h treatment group and 5.0⫻105 cells/flask for the 24 h treatment group. After overnight incubation, the cells were treated with 2-MeOE2 at 0.1–1.0 µg/ml for 24 or 48 h. In some experiments, cells were simultaneously treated with 2-MeOE2 (1.0 µg/ml) and α-naphthoflavone (30 µM) for 24 h. Cells were also co-treated with 2-MeOE2 (1.0 µg/ml) and L-ascorbic acid (0.5 mM) for 24 h. Three hours before the end of the treatment time, colcemid (Gibco, Grand Island, NY) was administered at 0.2 µg/ml and metaphase chromosomes were prepared as described previously (7). After trypsinization, cells were treated with 0.9% sodium citrate at room temperature for 13 min, fixed in Carnoy’s solution (methanol:acetic acid, 3:1) and spread on glass slides by the air drying method. Specimens were stained with a 3% Giemsa solution in 0.07 M phosphate buffer (pH 6.8) for 7 min. For determination of both chromosome aberrations and chromosome number, 100 metaphases per experimental group were scored. Achromatic lesions greater than the width of the chromatid were scored as gaps unless there was displacement of the broken piece of chromatid. If there was displacement, these were recorded as breaks.

treatment up to 8 h after treatment and then decreased. Although the inducibility of mitotic inhibition by E2 was lower than that by 2-MeOE2, the inducibility of multinucleation by E2 was comparable with that by 2-MeOE2 (Figure 3). The colony-forming efficiency (CFE) of SHE cells following treatment with 2-MeOE2 at 0.03–1.0 µg/ml for 48 h decreased in a concentration-dependent manner (Table I).

Fig. 1. Growth of SHE cells treated with 2-MeOE2. SHE cells were plated in triplicate on 35 mm dishes at a density of 3⫻104 cells/dish. After overnight incubation, cells were treated with 2-MeOE2 at concentrations of 0 (d), 0.1 (j), 0.3 (m) and 1.0 (.) µg/ml for 24, 48 and 72 h. Bars denote SD. When not indicated, the SD is within the symbol.

Results When SHE cells were treated with 2-MeOE2 at 0.1 µg/ml for 24–72 h, cellular growth was not significantly decreased, compared with that of control cells. At higher concentrations (0.3–1.0 µg/ml), growth of SHE cells was decreased by treatment with 2-MeOE2 in a concentration-dependent manner. In particular, growth was markedly decreased by treatment with 2-MeOE2 at 1.0 µg/ml (Figure 1). As 2-MeOE2 is a microtubule-disrupting agent (1,3), we examined the effects of 2-MeOE2 on mitotic inhibition and multinucleation in SHE cells. Although no significant increase in either the mitotic index or the percentage of multinucleated cells was observed in cells treated with 2MeOE2 at 0.1 µg/ml, treatment of SHE cells at 0.3 or 1.0 µg/ml for 2–48 h induced a concentration- and treatment time-related increase in the mitotic index and the percentage of multinucleated cells (Figure 2). The mitotic index began to increase 2 h after treatment and reached a maximum 8 h after treatment. When the treatment time was extended to 48 h, the mitotic index decreased and the percentage of multinucleated cells increased. The percentage of multinucleated cells reached a maximum 48 h after treatment (Figure 2). The effects of 2-MeOE2 on mitotic inhibition and multinucleation were compared with E2. SHE cells were treated with E2 for 2–48 h at 20 µg/ml, which induces inhibition of SHE cell growth comparable with that induced by treatment with 2-MeOE2 at 1.0 µg/ml (14). The mitotic index increased with 736

Fig. 2. Time dependence of mitotic index and multinucleation of SHE cells induced by 2-MeOE2. Cells (1–2⫻105) were plated on 60 mm dishes and after overnight incubation treated with 2-MeOE2 at the indicated concentrations for 2–48 h. After trypsinization, the cells were spread on glass slides by the air drying method and analyzed for mitotic index (d) and multinucleation (m) as described in Materials and methods. The mitotic index and the percentage of multinucleated cells of control SHE cells were 2.44 and 2.25%, respectively.

Cell transformation and genotoxicity by 2-MeOE2

Little or a slight decrease in CFE was observed in cells treated with 2-MeOE2 at 0.03 or 0.1 µg/ml. When SHE cells were treated with 2-MeOE2 at 0.3 µg/ml, the CFE was decreased to 20% of controls and 1.0 µg/ml 2-MeOE2 reduced the CFE to 0.3%. Treatment with 2-MeOE2 at 0.03–1.0 µg/ml for 48 h induced morphological transformation of SHE cells in a concentrationdependent manner (Table I). A statistically significant increase in the frequency of morphological transformation was elicited by treatment with 2-MeOE2 at 0.1, 0.3 or 1.0 µg/ml. Treatment with 2-MeOE2 for 48 h at 0.1 or 0.3 µg/ml also induced a statistically significant increase in the frequencies of somatic mutations at the Na⫹/K⫹ ATPase locus and the hprt locus, respectively. The mutation frequencies induced were lower than those induced by treatment with 1 µg/ml B[a]P, used as a positive control (Table I). Few chromosome aberrations were detected in cells treated with 2-MeOE2 at 0.1 µg/ml for 24 h, but treatment with 2-MeOE2 at 0.3 or 1.0 µg/ml resulted in a statistically significant level of chromosome aberrations in SHE cells, mainly breaks, exchanges and chromosome pulverization (Table II). The percentages of cells with either breaks or chromosome pulverization were significantly increased, when compared with those of the control cultures. The percentage of cells with exchanges induced by 2-MeOE2 was low, but

Fig. 3. Time dependence of mitotic index and multinucleation of SHE cells induced by E2. Cells (1–2⫻105) were plated on 60 mm dishes and after overnight incubation treated with E2 at the indicated concentration for 2–48 h. After trypsinization, the cells were spread on glass slides by the air drying method and analyzed for mitotic index (d) and multinucleation (m) as described in Materials and methods.

this type of chromosomal aberration was not observed in ⬎1500 metaphases scored in the control cultures. The incidence of chromosomal aberration was not affected by co-treatment with α-naphthoflavone but was slightly increased by cotreatment with L-ascorbic acid (Table II). In addition, treatment of SHE cells with 2-MeOE2 at 0.3 or 1.0 µg/ml for 48 h induced numerical chromosome changes in the near diploid range and/or in the tetraploid range (Table III). Heteroploid cells with the tetraploid and near tetraploid number of chromosomes were predominantly increased by the treatment. Discussion 2-MeOE2 induced morphological transformation in SHE cells at concentrations of 艌0.1 µg/ml. Treatment of SHE cells with transforming concentrations of 2-MeOE2 induced gene mutations and chromosomal abnormalities, including structural and numerical chromosome changes in the treated cells. SHE cell growth was inhibited by 2-MeOE2. Concentrations that inhibited cell growth resulted in increased mitotic cells in a concentration- and time-dependent manner, suggesting that inhibition of growth appears to result from mitotic arrest by 2-MeOE2. Multinucleation of SHE cells was also induced by 2-MeOE2 at concentrations that inhibited cell growth. In addition, chromosome losses and gains in SHE cells, which are consistent with non-disjunctional mechanisms, were elicited by the treatment. 2-MeOE2 binds to the colchicine site of tubulin and inhibits microtubule assembly (3). Fragmented centrosomes, multiple polar formations and abnormal spindle formations are observed when dividing MCF-7 and HeLa cells are treated with a high concentration of 2-MeOE2 (3). The abnormal dynamics of microtubule formation in the cytoskeleton and in the spindle figure may be a causal mechanism for induction of multinucleation and aneuploidy observed in SHE cells. Treatment of SHE cells with 2-MeOE2 at 1 µg/ml induced growth inhibition and multinucleation of cells comparable with treatment with E2 at 20 µg/ml. In addition, frequencies of both cell transformation and aneuploidy induced by 2-MeOE2 were more than 20-fold higher than those induced by E2 (16). These observations indicate that 2-MeOE2 is more active in inducing mitotic inhibition, multinucleation, cell transformation and aneuploidy in SHE cells than E2. Somatic mutations at the Na⫹/K⫹ ATPase and the hprt loci

Table I. Induction of morphological transformation and somatic mutations in SHE cells by 2-MeOE2 Chemical

2-MeOE2

B[a]P

Concentration (µg/ml)

0 0.03 0.1 0.3 1.0 1.0

Relative survival (%)

100b 97.7 81.9 19.9 0.3 60.0

No. of morphologically transformed colonies/ no. of colonies scored 1/8940c 3/8734 10/7321 4/1777 1/29 12/5372

Transformation (%)

0.01 0.03 0.14d 0.23e 3.45e 0.22e

No. of mutant colonies/ no. of cells assayed

Specific locus mutation frequencya

Ouar

TGr

Ouar

TGr

0/7⫻106 0/4⫻106 10/7⫻106 1/7⫻106 ND 12/2⫻106

0/7⫻106 0/4⫻106 0/7⫻106 8/7⫻106 ND 10/2⫻106

⬍7.0⫻10–6 ⬍4.0⫻10–6 8.0⫻10–6e 8.0⫻10–7

⬍7.0⫻10–6 ⬍4.0⫻10–6 ⬍7.0⫻10–6 5.7⫻10–6d

3.4⫻10–5e

2.5⫻10–5e

aCalculated

as described previously (15). was 11.89 ⫾ 0.3% (SD). independent experiments. dStatistically different from control (P ⬍ 0.05, χ2 test). eStatistically different from control (P ⬍ 0.01, χ2 test). bActual plating efficiency cData compiled from two

737

T.Tsutsui et al.

Table II. Chromosome aberrations in SHE cells treated with 2-MeOE2 alone or in combination with either α-naphthoflavone or L-ascorbic acid for 24 ha Type of aberrationb (%)

Chemical (concentration)

2-MeOE2 (0 µg/ml) 2-MeOE2 (0.1 µg/ml) 2-MeOE2 (0.3 µg/ml) 2-MeOE2 (1.0 µg/ml) α-Naphthoflavone (30 µM) α-Naphthoflavone (30 µM) ⫹ 2-MeOE2 (1.0 µg/ml) L-Ascorbic acid (0.5 mM) L-Ascorbic acid (0.5 mM) ⫹ 2-MeOE2 (1.0 µg/ml)

Aberrant metaphases (%)

G

B

E

D

O

CP

1.0 1.0 0 0 1.0 3.0 0 0

0 0 3.0 6.0 0 2.0 0 5.0

0 0 1.0 2.0 0 3.0 0 1.0

0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0

0 1.0 9.0 26.0 0 22.0 0 43.0

1.0 2.0 10.0c 32.0d 1.0 29.0d 0 48.0d

aFor each bG, gaps;

group, 100 metaphases were scored. B, breaks; E, exchanges; D, dicentric chromosomes; O, ring chromosomes; CP, chromosome pulverization. different from control (P ⬍ 0.05, χ2 test). dStatistically different from control (P ⬍ 0.01, χ2 test). cStatistically

Table III. Distribution of chromosome number per metaphase of SHE cells treated with 2-MeOE2a Concentration (µg/ml)

No. of chromosomes 42

0 0.1 0.3 1.0

43 1

44

45

95 88 19 1

1 6

46

2

47

48

1

⬎80 5 10 72 99

Diploid cells (%) 95.0 88.0 19.0 1.0

Heteroploid cells (%) Near diploidb

Tetraploid and near tetraploid

0 2.0 9.0c 0

5.0 10.0 72.0c 99.0c

aFor each group, bAneuploid cells

100 metaphases were scored. with a chromosome number within 1–4 of the diploid chromosome number (2N ⫽ 44). cStatistically different from control (P ⬍0.01, χ2 test).

were induced in SHE cells treated with 2-MeOE2. However, a concentration-dependent increase in the mutation frequencies was not detected, which might be due to the sensitivity of the assay system and/or a weak mutagenic activity of 2MeOE2. 2-MeOE2 is not mutagenic in the Ames test with Salmonella typhimurium TA100 in the presence of a rat liver S9 mixture (17). 2-MeOE2 can be converted to 2hydroxyestradiol (2-OHE2) by in vivo demethylation (18,19), suggesting that the mutagenic activity of 2-MeOE2 in SHE cells could be elicited by an oxidative metabolite of 2-MeOE2, 2-OHE2. However, we have observed that the mutagenic activity of 2-OHE2 in SHE cells is positive only in cells treated at a concentration of 1.0 µg/ml (20), which is higher than the concentrations of 2-MeOE2 (0.1 and 0.3 µg/ml) that induced SHE cell mutations (Table I). This indicates that 2-MeOE2 may be inherently mutagenic to SHE cells. Chromosome pulverization was observed in SHE cells treated with 2-MeOE2 at high frequencies, compared with other types of chromosome aberrations, e.g. breaks and exchanges. Chromosome pulverization occurs in interphase nuclei in multinucleated cells when the nuclei in a common cytoplasm undergo asynchronous entry into mitosis (21). Multi- or micronucleated cell formation resulting from cell fusion, failure of cytokinesis following normal nuclear division, cell division with lagging chromosomes or chromosome fragments or random aggregation of mitotic chromosomes is considered to lead to chromosome pulverization (21). The microtubule-disrupting activity and/or chromosome-damaging activity of 2-MeOE2 may be involved in chromosome pulverization induced by 2-MeOE2. Chromosome aberrations in SHE cells induced by 2-MeOE2 can result from the oxidative conversion of 2-MeOE2 738

to 2-OHE2, which has the ability to induce chromosome aberrations in SHE cells (20). Similar levels of chromosome aberrations were, however, induced in both cultures treated with 2-MeOE2 alone and cultures co-treated with 2-MeOE2 and α-naphthoflavone. Mobley et al. (22) proposed another metabolic pathway of 2-MeOE2. Their potentiometric studies showed that 2-MeOE2 can undergo oxidation of the phenolic moiety followed by oxidative demethylation, which results in E2-2,3-quinone (22). Cavalieri et al. (23) demonstrated that E2-2,3-quinone is capable of DNA adduct formation. Because SHE cells have endogenous metabolizing enzymes that exhibit oxidative and peroxidative activities (24,25), it is plausible that the oxidative conversion of 2-MeOE2 to its quinone metabolite participates in the cytological and genetic effects on SHE cells. However, co-treatment of SHE cells with 2-MeOE2 and L-ascorbic acid showed a slight increase in the incidence of chromosome aberrations when compared with that induced by 2-MeOE2 alone, suggesting that the genetic effects on SHE cells observed in the present study are induced by 2-MeOE2 rather than its quinone derivatives. The results from multiple end-points measured in the present experiments are qualitatively summarized in Table IV. Morphological transformation in SHE cells was induced by 2-MeOE2 at 0.1 µg/ml, which was the efficacious concentration for inducing mutations but not for inducing either mitotic inhibition or chromosomal abnormalities, including structural and numerical changes. Mitotic inhibition and chromosomal abnormalities were induced by 2-MeOE2 at 艌0.3 µg/ml, corresponding to the enhancement of transformation activity. As 2-MeOE2 induced genetic effects at the concentration that reduced cell survival to 0.3%, morphological transformation

Cell transformation and genotoxicity by 2-MeOE2

Table IV. Comparative qualitative findings and multiple end-point measurements of SHE cells exposed to 2-MeOE2

indicate that 2-MeOE2 has cell-transforming and genotoxic activities in cultured mammalian cells and potential carcinogenic activity.

Concentration Morphological Somatic (µg/ml) transformation mutations

References

0.03 0.1 0.3 1.0

⫺a ⫹ ⫹⫹ ⫹⫹⫹

aConcentration-associated bNot

⫺ ⫹ ⫹ NDb

Mitotic Chromosomal inhibition abnormalities

⫺ ⫺ ⫹ ⫹⫹

Structural

Numerical

⫺ ⫺ ⫹ ⫹⫹

⫺ ⫺ ⫹ ⫹⫹

increase in measured SHE cell end-points.

determined.

in SHE cells treated with 2-MeOE2 could be pertinent to the genetic effects of 2-MeOE2 rather than to cytotoxicity. The results in the present studies suggest the involvement of multiple genetic effects, including somatic mutations and chromosomal abnormalities, in the SHE cell transforming activity of 2-MeOE2. Multiple effects of 2-MeOE2 can act together to cause different genetic alterations leading to cell transformation. The concentrations of 2-MeOE2 used in the present experiments are much higher than human serum levels of 2-methoxyestrogens, ranging from ⬍10 pg/ml (adult males) to 3700 pg/ml (pregnant females) (26). However, levels of 2-MeOE2 inside cells are unknown and specific cell types in which it accumulates may exist. The effects of 2-MeOE2 are concentration-dependent and effects at physiological concentrations may exist but would be rare. However, if 2-MeOE2 is used as a pharmaceutical agent, higher concentrations may cause significant mutational effects and represent a potential hazard. Endogenous estrogens have been implicated as a possible etiological factor in the causation of certain types of human cancers, such as breast, endometrium, ovary, prostate and brain cancers (reviewed in ref. 27). The carcinogenic activity of estrogens and their metabolites is well demonstrated using the Syrian hamster kidney tumor model (17,28). In the hamster kidney model, E2 exhibits carcinogenic activity, whereas 2-MeOE2 does not (17). This is in contrast to our findings that 2-MeOE2 has cell-transforming and mutagenic activities in vitro. The differences between activity in vitro and in vivo can be explained by many mechanisms. (i) The lack of carcinogenic activity may be due to the anti-angiogenic activity of 2-MeOE2 (2). (ii) Because mitotic spindle poisons induce aneuploidy only in dividing cells, the carcinogenicity of 2-MeOE2 may be observed only in rapidly dividing cells. (iii) Although the serum concentration of 2-methoxyestrogens is very high in pregnant women (26), 2-MeOE2 has a high binding affinity for the plasma protein sex hormone-binding globulin (29), which may block its transforming activity in vivo. (iv) The relative binding affinity of 2-MeOE2 for the cytosol estrogen receptor of rat uterus is ⬍0.1% of E2 (29) and 2-MeOE2 has low hormonal activity (30). Each of these properties might contribute to masking of the potential carcinogenic activity of 2-MeOE2 in vivo. Further studies of this chemical in vivo may yield new insights into the mechanisms of carcinogenicity of estrogens. In the present study, we have demonstrated that 2-MeOE2 induces morphological transformation, somatic mutations and/ or chromosomal abnormalities in SHE cells. The results

1. Seegers,J.C., Aveling,M-L., von Aswegen,C.H., Cross,M., Koch,F. and Joubert,W.S. (1989) The cytotoxic effects of estradiol-17β, catecholestradiols and methoxyestradiols on dividing MCF-7 and HeLa cells. J. Steroid Biochem., 32, 797–809. 2. Fotsis,T., Zhang,Y., Pepper,M,S., Adlercreutz,H., Montesano,R., Nawroth,P.P. and Schweigerer,L. (1994) The endogenous oestrogen metabolite 2-methoxyestradiol inhibits angiogenesis and suppresses tumor growth. Nature, 368, 237–239. 3. D’Amato,R.J., Lin,C.M., Flynn,E., Folkman,J. and Hamel,E. (1994) 2Methoxyestradiol, an endogenous mammalian metabolite, inhibits tubulin polymerization by interacting at the colchicine site. Proc. Natl Acad. Sci. USA, 91, 3964–3968. 4. Attalla,H., Ma¨ kela¨ ,T.P., Adlercreutz,H. and Andersson,L.C. (1996) 2Methoxyestradiol arrests cells in mitosis without depolymerizing tubulin. Biochem. Biophys. Res. Commun., 228, 467–473. 5. Rao,P.N. and Engleberg,J. (1967) Structural specificity of estrogens in the induction of mitotic chromatid non-disjunction in HeLa cells. Exp. Cell Res., 48, 71–81. 6. Danford,N. and Parry,J.M. (1981) Abnormal cell division in cultured human fibroblasts after exposure to diethylstilbestrol. Mutat. Res., 103, 379–383. 7. Tsutsui,T., Maizumi,H., McLachlan,J.A. and Barrett,J.C. (1983) Aneuploidy induction and cell transformation by diethylstilbestrol: a possible chromosomal mechanism in carcinogenesis. Cancer Res., 43, 3814–3821. 8. Tsutsui,T., Maizumi,H. and Barrett,J.C. (1984) Colcemid-induced neoplastic transformation and aneuploidy in Syrian hamster embryo cells. Carcinogenesis, 5, 89–93. 9. Cox,D.M. and Puck,T.T. (1969) Chromosomal nondisjunction: the action of colcemid on Chinese hamster cells in vitro. Cytogenetics, 8, 158–169. 10. Tucker,R.W. and Barrett,J.C. (1986) Decreased numbers of spindle and cytoplasmic microtubules in hamster embryo cells treated with a carcinogen, diethylstilbestrol. Cancer Res., 46, 2088–2095. 11. Tsutsui,T., Suzuki,N., Maizumi,H. and Barrett,J.C. (1990) Aneuploidy induction in human fibroblasts: comparison with results in Syrian hamster fibroblasts. Mutat. Res., 240, 241–249. 12. Ozawa,N., Oshimura,M., McLachlan,J.A. and Barrett,J.C. (1989) Nonrandom karyotypic changes in immortal and tumorigenic Syrian hamster cells induced by diethylstilbestrol. Cancer Genet. Cytogenet., 38, 271–282. 13. Li,R., Yerganian,G., Duesberg,P., Kraemer,A., Willer,A., Rausch,C. and Hehlmann,R. (1997) Aneuploidy correlated 100% with chemical transformation of Chinese hamster cells. Proc. Natl Acad. Sci. USA, 94, 14506–14511. 14. Tsutsui,T., Suzuki,N., Maizumi,H. and Barrett,J.C. (1990) Comparison of human versus Syrian hamster cells in culture for induction of mitotic inhibition, binucleation and multinucleation, following treatment with four aneuploidogens. Toxicol. In Vitro, 4, 75–84. 15. Barrett,J.C., Bias,N.E. and Ts’o,P.O.P. (1978) A mammalian cellular system for the concomitant study of neoplastic transformation and somatic mutation. Mutat. Res., 50, 121–136. 16. Tsutsui,T., Suzuki,N., Fukuda,S., Sato,M., Maizumi,H., McLachlan,J.A. and Barrett,J.C. (1987) 17β-Estradiol-induced cell transformation and aneuploidy of Syrian hamster embryo cells in culture. Carcinogenesis, 8, 1715–1719. 17. Liehr,J.G., Fang,W.F., Sirbasku,D.A. and Ulubelen,A.A. (1986) Carcinogenicity of catechol estrogens in Syrian hamsters. J. Steroid Biochem., 24, 353–356. 18. Theron,C.N., Russel,V.A. and Taljaard,J.J.F. (1986) Substrate dependency of specific and non-specific estrogen-2/4-hydroxylase activities measured by the radio-enzymatic method in rat brain microsomes. J. Steroid Biochem., 25, 585–591. 19. Martucci,C.P. and Fishman,J. (1993) P450 enzymes of estrogen metabolism. Pharmacol. Ther., 57, 237–257. 20. Tsutsui,T., Tamura,Y., Yagi,E. and Barrett,J.C. (2000) Involvement of genotoxic effects in the initiation of estrogen-induced cellular transformation: studies using Syrian hamster embryo cells treated with 17β-estradiol and eight of its metabolites. Int. J. Cancer, in press. 21. Ikeuchi,T. and Matsui,S. (1974) The phenomenon of ‘chromosome pulverization’. Symp. Cell Biol., 26, 67–76.

739

T.Tsutsui et al. 22. Mobley,J.A., Bhat,A.S. and Brueggemeier,R.W. (1999) Measurement of oxidative DNA damage by catechol estrogens and analogues in vitro. Chem. Res. Toxicol., 12, 270–277. 23. Cavalieri,E.L., Stack,D.E., Devanesan,P.D., Todorovic,R., Dwivedy,I., Higginbotham,S., Johansson,S.L., Patil,K.D., Gross,M.L., Gooden,J.K., Ramanathan,R., Cerny,R.L. and Rogan,E.G. (1997) Molecular origin of cancer: catechol estrogen-3,4-quinones as endogenous tumor initiators. Proc. Natl Acad. Sci. USA, 94, 10937–10942. 24. Pienta,R.J. (1980) Transformation of Syrian hamster embryo cells by diverse chemicals and correlation with their reported carcinogenic and mutagenic activities. In deSerres,F.J. and Hollaender,A. (eds) Chemical Mutagens, Principles and Methods for Their Detection. Plenum Press, New York, NY, Vol. 6, pp. 175–202. 25. Degen,G.H., Wong,A., Eling,T.E., Barrett,J.C. and McLachlan,J.A. (1983) Involvement of prostaglandin synthetase in the peroxidative metabolism of fibroblast cell cultures. Cancer Res., 43, 992–996.

740

26. Berg,D., Sonsalla,R. and Kuss,E. (1983) Concentrations of 2methoxyestrogens in human serum measured by a heterologous immunoassay with an 125I-labelled ligand. Acta Endocrinol., 103, 282–288. 27. Zhu,B.T. and Conney,A.H. (1998) Functional role of estrogen metabolism in target cells: review and perspectives. Carcinogenesis, 19, 1–27. 28. Li,J.J. and Li,S.A. (1987) Estrogen carcinogenesis in Syrian hamster tissues: role of metabolism. Fed. Proc., 46, 1858–1863. 29. Ball,P. and Knuppen,R. (1990) Formation, metabolism, and physiologic importance of catecholestrogens. Am. J. Obstet. Gynecol., 163, 2163–2170. 30. Martucci,C.P. and Fishman,J. (1979) Impact of continuously administered catechol estrogens on uterine growth and luteinizing hormone secretion. Endocrinology, 105, 1288–1292. Received August 6, 1999; revised November 10, 1999; accepted November 22, 1999