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Studies on the genotoxicity of the mammalian lignans enterolactone and enterodiol and their metabolic precursors at various endpoints in vitro

Studies on the genotoxicity of the mammalian lignans enterolactone and enterodiol and their metabolic precursors at various endpoints in vitro

Mutation Research 416 Ž1998. 115–124 Studies on the genotoxicity of the mammalian lignans enterolactone and enterodiol and their metabolic precursors...

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Mutation Research 416 Ž1998. 115–124

Studies on the genotoxicity of the mammalian lignans enterolactone and enterodiol and their metabolic precursors at various endpoints in vitro Sabine E. Kulling, Eric Jacobs, Erika Pfeiffer, Manfred Metzler

)

Institute of Food Chemistry, Department of Chemistry, UniÕersity of Karlsruhe, PO Box 6980, D-76128 Karlsruhe, Germany Received 7 April 1998; revised 26 May 1998; accepted 29 May 1998

Abstract The mammalian lignans enterolactone ŽENL. and enterodiol ŽEND. are formed by intestinal bacteria from the plant lignans matairesinol ŽMAT. and secoisolariciresinol ŽSEC., respectively, which are ingested with different types of food. ENL and END are weak estrogens. According to epidemiological and biochemical studies, lignans may act as anticarcinogens, but little is known about their genotoxic potential. We have therefore investigated the effects of ENL, END, MAT and SEC on cell-free microtubule assembly and at the following genetic endpoints in cultured male Chinese hamster V79 cells: disruption of the cytoplasmic microtubule complex, induction of mitotic arrest, induction of micronuclei and their characterization by CREST staining, and mutagenicity at the HPRT gene locus. The lignans were tested at concentrations of 200 mM in the cell-free system and 100 mM in cultured cells, which represents the limit of solubility in each assay. The established aneuploidogen diethylstilbestrol and the clastogen 4-nitroquinoline-N-oxide were used as positive reference compounds. As none of the four lignans had any activity at the endpoints studied, we conclude that ENL, END, MAT and SEC are devoid of aneuploidogenic and clastogenic potential under the experimental conditions used in this study. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Mammalian lignans; Plant lignans; Enterolactone; Enterodiol; Matairesinol; Secoisolariciresinol; Genotoxicity

Abbreviations: BSA, Bovine serum albumin; CREST, Calcinosis, Raynaud’s phenomenon, oesophagal motility abnormalities, scleroX X dactyly and telangiectasia; COL, Colchicine; DAPI, 4 ,6 -Diamidino-2-phenylindole; DES, Diethylstilbestrol, E-3,4-bisŽ p-hydroxyphenyl.hex-3-ene; DMEM, Dulbecco’s modified Eagle’s medium; DMSO, Dimethylsulfoxide; E 2 , 17b-Estradiol; EDTA, Ethylenediaminetetraacetate, sodium salt; END, Enterodiol, 2,3-bisŽ3-hydroxybenzyl.butane-1,4-diol; ENL, Enterolactone, trans-2,3-bisŽ3-hydroxybenzyl.-gbutyrolactone; FCS, Fetal calf serum; FITC, Fluorescein isothiocyanate; GCrMS, Gas chromatographyrmass spectrometry; GTP, Guanosine triphosphate; HPRT, Hypoxanthine phosphoribosyltransferase; MAT, Matairesinol, trans-2,3-bisŽ3-methoxy-4-hydroxybenzyl.g-butyrolactone; MN, Micronucleus; MT, MicrotubuleŽs.; MTP, Microtubule proteins; NQO, 4-Nitroquinoline-N-oxide; PBS, Phosphatebuffered saline; PBS-CMF, Phosphate-buffered saline free of calcium and magnesium; SEC, Secoisolariciresinol, 2,3-bisŽ3-methoxy-4-hydroxybenzyl.butane-1,4-diol; SRB, Sulforhodamine B; 6-TG, 6-thioguanine ) Corresponding author. Fax: q49-721-608-7255; E-mail: [email protected] 1383-5718r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 3 - 5 7 1 8 Ž 9 8 . 0 0 0 8 2 - 5

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1. Introduction Enterolactone ŽENL, Fig. 1. and enterodiol ŽEND. were the first lignans identified in humans and animals in 1980 w1x. These mammalian lignans exhibit weak estrogenic activity w2–4x and are formed from the plant lignans matairesinol ŽMAT. and secoisolariciresinol ŽSEC., respectively, by bacteria in the intestinal tract w5,6x. MAT and SEC are ingested with different types of food, especially flaxseed and whole grain products but also vegetables and fruits w7x. Plasma levels of ENL and END can be as high as 500 ngrml w8,9x, and urinary excretion can exceed 30 mmolrday w9x. Thus, the plasma concentration of mammalian lignans can be more than 1000 times higher than the normal circulating level of the endogenous steroidal estrogen 17b-estradiol ŽE 2 . w3x and may affect the hormonal system.

There is considerable evidence from epidemiological studies correlating high concentrations of lignans in body fluids with a low incidence of hormone-dependent tumors, in particular breast cancer w10–12x. These findings suggest an anticarcinogenic potential for ENL and END which is supported by several in vitro and in vivo investigations. For example, it was shown that flaxseed inhibits mammary and colon carcinogenesis in rats w13,14x. Furthermore, ENL and END significantly inhibit the growth of human colon tumor cells w15x, and the E 2-induced proliferation of MCF-7 breast cancer cells is inhibited by ENL w3x. The protective effects of mammalian lignans may be due to their ability to compete with E 2 for the type II estrogen receptor w3,9,16x, to induce sex hormone binding globulin w9,16,17x, to inhibit placental aromatase w9,17–19x, to enhance b-glucuronidase activity w14x and to act as antioxidants w20x. In contrast to the beneficial effects, little attention has thus far been paid to possible adverse health effects of mammalian lignans. The structural similarity to the synthetic estrogen diethylstilbestrol ŽDES, Fig. 1., an established human and animal carcinogen, prompted us to study the genotoxic potential of the mammalian lignans ENL and END and their plant precursors MAT and SEC in different in vitro systems.

2. Materials and methods 2.1. Chemicals, microtubule proteins, and cell culture

Fig. 1. Chemical structures of mammalian and plant lignans and of diethylstilbestrol ŽDES..

Ž".-ENL and Ž".-END were synthesized according to a modification of the method of Pelter et al. w21x. Žy.-MAT was kindly provided by Dr. Kasper ŽUniversity of Berlin. and Ž".-SEC by Matthias ŽUniversity of Bayreuth.. DES was purSchottner ¨ chased from Sigma. The purity of the compounds was ) 99% according to GCrMS after trimethylsilylation with N,O-bisŽtrimethylsilyl.acetamide. All other chemicals were obtained from either Sigma, Fluka or Serva and were of the highest purity available. Microtubule proteins ŽMTP. were prepared from bovine brain by two cycles of assembly and disassembly according to the method of Shelanski w22x.

S.E. Kulling et al.r Mutation Research 416 (1998) 115–124

The protein was suspended in assembly buffer Ž100 mM morpholinoethane sulfonic acid, 1 mM ethyl eneglycolbisŽb - aminoethylether. - N, N, N X , N X - tetraacetic acid, 0.5 mM MgCl 2 , pH 6.4. and stored in liquid nitrogen. MTP concentration was determined by the Bio-Rad protein assay based on the method of Bradford w23x. Male Chinese hamster V79 fibroblasts were grown in Dulbecco’s modified Eagle’s medium ŽDMEM. pH 7.5 containing 4500 mgrl glucose ŽLife Technologies, Cat. No. 41966. and supplemented with 100 IUrml penicillin, 100 mgrml streptomycin and 10% fetal calf serum ŽFCS, Life Technologies.. The cells were cultured at 378C in a 10% CO 2 atmosphere. Solutions of the test compounds in dimethylsulfoxide ŽDMSO, Sigma. were added to DMEM in amounts to yield a final DMSO concentration of 0.1% Žvrv.. Control experiments were carried out with equal DMSO concentrations but without test compounds. 2.2. Microtubule assembly assay The polymerization of microtubules ŽMT. was carried out as described by Pfeiffer and Metzler w24x. The test compounds dissolved in DMSO were added to assembly buffer pH 6.4 containing 10 mM freshly thawed MTP to yield concentrations ranging from 20–200 mM in a final volume of 0.5 ml containing 2% DMSO. After 20 min at 358C, MT assembly was started by adding 0.5 mM guanosine triphosphate ŽGTP. and the absorbance at 350 nm was measured for 30 min. Disassembly at 48C was followed by a second polymerization cycle at 358C. The control incubations contained all components plus 2% DMSO but no test compounds. 2.3. Growth inhibition and cytotoxicity Growth-inhibitory effects and cytotoxicity were tested by using the sulforhodamine B ŽSRB. assay according to Skehan et al. w25x and the Trypan blue assay. For the SRB assay, V79 cells were kept on 24-well culture dishes for 24 h, each well containing 6000 cells in 1 ml DMEM. Then the medium was replaced by DMEM containing the test compounds. After 48 h of exposure, 250 ml of cold 50% aqueous trichloroacetic acid were added to each well and the

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cells fixed for 1 h. After rinsing with water and air-drying, cell proteins were stained with a solution of 0.4% Žwrv. SRB in 1% aqueous acetic acid Ž250 ml per well.. Excess dye was removed after 30 min by rinsing with water. After air-drying, the proteinbound dye was extracted from each well with 1 ml cold 10 mM Tris buffer ŽpH 10.5. and the absorbance measured at 564 nm. Moreover, the morphology of the cells was examined after 48 h of exposure by phase contrast microscopy. For the trypan blue assay, V79 cells Ž10 6 cellsrflask. were incubated for 48 h in DMEM containing appropriate concentrations of the test compounds. The medium was then collected, the cells were washed with phosphate-buffered saline free of calcium and magnesium ŽPBS-CMF. and trypsinized with 5 ml PBS-CMF containing 0.25% trypsin and 0.02% EDTA. The cells were then collected from the combined supernatants in a small centrifuge at 1200 rpm for 5 min. The cell pellet was resuspended in 5 ml PBS of pH 7.5 at 378C and aliquots of 370 ml were added to 270 ml of a 0.4% solution of trypan blue in 0.9% saline. Following incubation at 378C for 3 min, the numbers of cells without the dye Žcolorless viable cells. and with the dye Žblue dead cells. were scored in a Neubauer chamber. 2.4. Mitotic arrest V79 cells were grown on microscope glass slides in a quadriperm vial Ž150,000 cells per slide. for 24 h before incubation with the test compounds for 6 h. Subsequently, the slides were fixed in methanolracetic acid 3:1 Žvrv. at y208C for 1 h. The air-dried slides were then stained with 30 ml each of antifade solution containing 1 mgrml 4X ,6X-diamidino-2-phenylindole dihydrochloride ŽDAPI, Sigma.. The number of cells arrested in metaphase was determined by fluorescence microscopy Žexcitation wavelength 365 nm.. 2.5. Tubulin staining As described in Section 2.4, V79 cells were grown on slides for 24 h and incubated with the test compounds for 6 h before fixation in methanol at y208C for at least 1 h. Slides were then kept in acetone at

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y208C for 10 min to increase the permeability of the cell membrane. After washing with PBS-CMF three times for 5 min each, the slides were incubated for 1 h at 378C with 100 ml goat serum ŽLife Technologies. in a water-saturated chamber, washed with PBS-CMF as before, incubated with 100 ml of a solution of mouse anti-a-tubulin antibody IgG1 ŽSigma; diluted 1:500 with PBS-CMF containing 1% BSA. as before, washed again and finally incubated as before with fluorescence-labelled Cy3e-goat anti-mouse antibody ŽJackson Immune Research; diluted 1:150 with PBS-CMF containing 1% BSA.. Slides were then stained with 30 ml antifade solution containing 2.5 mgrml DAPI and examined by fluorescence microscopy Žexcitation wavelengths 564 nm and 365 nm. for abnormalities in the morphology of cytoplasmic and spindle microtubules in interphase and metaphase cells, respectively. 2.6. Micronucleus assay and CREST staining V79 cells were spread on slides and exposed to test compounds for 6 h as described above. The slides were then briefly immersed in PBS-CMF and kept in fresh DMEM for various lengths of time Ž3, 6, 12 and 24 h. before the cells were washed with PBS-CMF and fixed in methanol at y208C for at least 1 h. Afterwards the CREST staining was carried out in analogy to the tubulin staining ŽSection 2.5. using goat serum, a solution of CREST antibody Žanti-centromere serum from Antibodies, diluted 1:10 with PBS-CMF containing 1% BSA. and fluorescein isothiocyanate ŽFITC.-conjugated goat anti-human IgG ŽSigma, diluted 1:200 with PBS-CMF containing 1% BSA.. Slides were then kept in Soerensen buffer pH 8.0 prior to staining with antifade solution containing 2.5 mgrml DAPI and 1 mgrml propidium iodide. Two thousand cells per slide were examined for micronuclei and CREST signals by fluorescence microscopy according to the criteria established by Countryman and Heddle w26x. 2.7. HPRT mutation assay From a batch of V79 cells tested for their spontaneous mutation frequency and the absence of mycoplasms, 1.5 = 10 6 cells in 10 ml DMEM fortified with 10% FCS were grown in a 250-ml cell culture

flask for each test compound and concentration. The medium was then removed and cells were briefly washed with PBS-CMF prior to incubation with the test compound in FCS-free DMEM for 3 h, followed by washing with PBS-CMF and trypsinization. Cytotoxicity was determined as the decrease in plating efficiency by plating 500 cells in 10-cm dishes. Seven days later, the colonies were fixed in ethanol, stained with 0.5% methylene blue and counted. Plating efficiency was calculated as the ratio of colonies over the number of seeded cells per dish. The remaining trypsinized cells were reseeded into 250-ml flasks at about 1 = 10 6 cells per flask and cultivated for another 6 days for phenotypic expression. Then exactly 10 6 cells each were plated on four 15-cm petri dishes in 30 ml DMEM containing 5% FCS and 7 mg 6-thioguanine Ž6-TG.rml for each substance concentration. Another 500 cells each were seeded into four 10-cm petri dishes containing 10 ml DMEM with 5% FCS but without 6-TG in order to determine cell viability at the time of mutant selection. The colonies were stained and counted after 7 days Žcell viability plates. or 10 days Ž6-TG plates.. Mutation frequency was calculated as the ratio of 6-TG-resistant mutant colonies over the number of seeded cells times cell viability. 3. Results 3.1. Inhibition of cell-free microtubule assembly Many aneuploidogenic agents interfere with the polymerization of microtubule proteins ŽMTP. to microtubules ŽMT., e.g., colchicine ŽCOL. or diethylstilbestrol ŽDES.. The inhibition of MT assembly can be detected under cell-free conditions w27x. In a typical assay, the MTP are incubated with the test compound for 20 min at 358C before the polymerization is started by the addition of GTP. The formation of MT causes light scattering and is measured by the increase in absorbance at 350 nm over 30 min. Subsequent cooling to 48C for 10 min causes depolymerization of MT. A second assembly of MT is then achieved by raising the temperature again to 358C. This depolymerizationrpolymerization cycle is important to distinguish MT formation from protein denaturation and aggregation. Control incubations are carried out in the absence of test compound.

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3.2. Disruption of microtubules in V79 cells In addition to the cell-free system, the effects of the lignans on MT were also studied in V79 cells. After treatment with the compounds for 6 h, the cells were fixed and stained with anti-a-tubulin antibodies. The cytoplasmic microtubule complex in interphase cells and the mitotic spindle in metaphase cells were not different from those of untreated cells even after exposure to 100 mM concentrations of the four lignans. This concentration represents the limit of solubility at the tolerable DMSO concentration of 0.1% as used in our cell experiments. In contrast to the lignans, 20 mM DES and 0.1 mM COL led to an almost complete loss of cytoplasmic microtubules and seriously damaged spindles Ždata not shown.. 3.3. Growth inhibition and cytotoxicity in V79 cells In order to select suitable concentrations for the genotoxicity assays in V79 cells, the cytotoxicity of the four lignans was determined in these cells by using the sulforhodamine B ŽSRB. test and the trypan blue assay. Fig. 2. Effect of the mammalian lignans ENL and END, the plant lignans MAT and SEC, and of the aneugens COL and DES on cell-free MT assembly. Concentrations were 200 mM for the lignans and 4 mM and 40 mM for COL and DES, respectively.

As depicted in Fig. 2, MT assembly was decreased to about 50% and 35% by 40 mM DES and 4 mM COL, respectively, whereas enterolactone ŽENL. and enterodiol ŽEND. did not inhibit MT polymerization even at a concentration of 200 mM, which is the limit of solubility under these conditions Ž2% DMSO, see Section 2.2.. Likewise, matairesinol ŽMAT. and secoisolariciresinol ŽSEC. were devoid of activity in the cell-free MT assembly assay ŽFig. 2.. With all compounds, MT assembly was completely reversible upon cooling to 08C, and a second assembly to the same extent as the first one was obtained by subsequent warming to 358C Ždata not shown..

Fig. 3. Effect of the four lignans and of DES on the growth of V79 cells as measured in the SRB assay. TrC represents the ratio of protein content in cells treated for 48 h over that in untreated controls. Data are the mean"S.D. of three independent experiments.

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After incubation with various concentrations of the compounds for 48 h, the total protein of the viable cells was measured in the SRB assay and compared with that of untreated cells. In addition, cell morphology was analyzed by phase contrast microscopy. The reference compound DES caused a pronounced reduction of cell growth at concentrations above 10 mM ŽFig. 3.. This growth inhibition was accompanied by marked alterations in the morphology of the cells which were no longer spindleshaped but round, indicating metaphase cells. The lignans ENL, END and SEC caused only a slight

inhibition of cell growth without any change in cell morphology even after treatment for 48 h with concentrations as high as 100 mM ŽFig. 3.. Interestingly, MAT inhibited cell proliferation to the same extent as DES ŽFig. 3.. However, in contrast to DES, MAT did not affect the shape of the cells at all. The result of the SRB test was confirmed by the trypan blue assay ŽTable 1.. With 20 mM DES, cell growth was completely inhibited and 20% of the cells counted after 48 h were dead. Likewise, the clastogenic agent 4-nitroquinoline-N-oxide ŽNQO. gave rise to a complete growth inhibition and to 36%

Table 1 Cytotoxicity of lignans in the trypan blue assay with V79 cells Compound Controla Controlb END ENL MAT SEC DES NQO

Concentration – – 100 m M 100 m M 100 m M 100 m M 20 m M 1 mM

Viable cells Count c

%

Dead cells Count c

%

Cell growth

41 " 6.0 600 " 28.3 609 " 37.4 471 " 25.2 155 " 7.2 399 " 25.5 10 " 2.1 10 " 7.3

98.8 99.1 99.6 98.7 99.9 99.4 80 64

0.5 " 0.58 6.0 " 1.4 2.3 " 0.5 6 " 2.4 1.5 " 1.3 2.3 " 1.5 2.6 " 0.5 5.5 " 3.7

1.2 0.9 0.4 1.3 0.1 0.6 20 36

100% 100.9% 77.0% 20.4% 63.8% none none

Data are mean " S.D. of three replicates. a Without test compound and solvent prior to incubation. b Incubated without test compound but with 0.1% DMSO. c Number of cells per 0.1 ml.

Fig. 4. Effect of the lignans and of COL on the number V79 cells arrested in metaphase after 6 h of treatment. Data are the mean of two independent experiments.

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dead cells. The reduction in cell number after 48 h treatment with END, ENL and SEC in relation to untreated cells ranged from 0 to 36%, and cell viability was not affected by these lignans. MAT caused an 80% inhibition of cell growth, but virtually no dead cells ŽTable 1.. Thus, the pronounced inhibitory effect on cell proliferation exerted by MAT is not due to cytotoxicity. 3.4. Metaphase arrest in V79 cells V79 cells were treated with various concentrations of the test compounds for 6 h and subsequently fixed and stained with DAPI to determine the number of metaphase cells per 1000 cells. COL and DES were taken as positive controls. The data are summarized in Fig. 4. Whereas COL and DES gave rise to a marked increase of metaphase cells, no significant effect was observed with ENL, END and SEC even at concentrations of 100 mM. MAT appeared to decrease the number of mitotic cells, which is consistent with its inhibitory effect on cell growth mentioned above. 3.5. Induction of micronuclei in V79 cells After incubation with the test compounds for 6 h, V79 cells were kept in fresh medium for various lengths of time ranging from 3 to 24 h. Subsequently cells were stained, using DAPI and propidiumiodide to detect micronuclei ŽMN. and CREST antikinetochore antibodies to distinguish between MN containing whole chromosomes ŽCREST-positive. and chromosomal fragments ŽCREST-negative.. The number of both types of MN was determined ŽFig. 5.. The clastogen NQO and the aneugen DES were used as positive controls. DES at 20 mM concentration caused a clear induction of CREST-positive MN with a maximum at 6 h ŽFig. 5.. No CREST-negative MN were induced by DES, but a marked increase in such MN was observed after treatment with 0.5 mM NQO; the highest yield was obtained at 24 h. When the lignans ENL, END, MAT and SEC were tested at 50 mM and 100 mM concentration, none of them induced MN of either type ŽFig. 5..

Fig. 5. Induction of micronuclei in V79 cells by the four lignans and by NQO and DES. Concentrations of compounds was 100 mM for the lignans, 0.5 mM for NQO and 20 mM for DES. Incubation period with the test compounds was 6 h followed by the postincubation time indicated. Data are the mean of two independent experiments.

3.6. Mutations at the HPRT locus The induction of forward mutations at the HPRT locus rendering male Chinese hamster V79 cells resistant against 6-thioguanine Ž6-TG. is a well validated assay for gene mutations w28x. The number of 6-TG-resistent cells Ž6-TG r mutants. was markedly increased over control values by 1 mM NQO ŽFig. 6., where the viability of cells was still high as

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Fig. 6. Mutation frequencies at the HPRT locus of V79 cells after treatment with various lignans and with NQO. Plating efficiencies were ) 80% for all compounds tested. Data are the mean " S.D. of three independent experiments.

indicated by a plating efficiency of ) 90%. When the four lignans were subject to this assay at concentrations of 50 and 100 mM, which did not affect the plating efficiency of the cells, none of them was able to induce a significant increase in 6-TG r mutants over controls ŽFig. 6.. For ENL, MAT and SEC, the number of 6-TG r mutants was not elevated at all at any of the test concentrations, whereas a marginal increase was observed for END at the higher concentration.

4. Discussion The mammalian lignans enterolactone ŽENL. and enterodiol ŽEND . and their plant precursors matairesinol ŽMAT. and secoisolariciresinol ŽSEC. reach high levels in humans after the ingestion of flaxseed, whole-grain products, vegetables and fruit. They are weak estrogens, and epidemiological and biochemical studies suggest that they may act as anticarcinogens. However, little attention has thus far been paid to their possible genotoxic effects, despite their structural similarity with the strong estrogen and established carcinogen diethylstilbestrol ŽDES.. DES has recently been shown to exhibit aneuploidogenic activity, which most likely contributes to its

carcinogenicity w29x. As a consequence of its aneuploidogenic potential, DES has been reported to inhibit cell-free MT assembly w30x, to disrupt the cytoplasmic microtubule complex and the mitotic spindle in intact cells w31x, and to induce CRESTpositive MN in various cell types in vitro w32x. These endpoints were chosen for the present study, and DES was used as a positive control together with the established aneugen COL. As recent studies from our laboratory with phytoestrogens of the coumestane and isoflavone type have revealed that coumestrol and genistein are strong clastogens leading to DNA strand breaks, CREST-negative MN and mutations at the HPRT locus in V79 cells w33x, the latter endpoints were also included and the clastogen NQO was employed as reference compound. Thus, our study was designed to clarify both the aneuploidogenic and the clastogenic potential of the major mammalian lignans ENL and END and their precursor plant lignans MAT and SEC. The results of our investigation, which are summarized in Table 2, suggest that all four lignans are devoid of aneuploidogenic, clastogenic and mutagenic potential at the endpoints and concentrations used in our study. The putative lack of aneuploidogenic potential is based on the observation that the lignans neither inhibit MT polymerization under

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Table 2 Comparison of the effects of the mammalian lignans ENL and END, the plant lignans MAT and SEC, and the reference compounds DES, COL and NQO at various endpoints for genotoxicity; data are mean" standard deviation of three replicates Compound

ENL

END

MAT

SEC

DES

COL

NQO

Inhibition of MT assembly Induction of mitotic arrest Tubulin staining in cells

y y y

y y y

y y y

y y y

q q q

q q q

n.t.a n.t. n.t.

Induction of micronuclei CREST-positive CREST-negative Induction of HPRT mutations

y y y

y y y

y y y

y y y

q y yb

q y n.t.

y q q

a

n.t., not tested. According to Barrett et al. w34x.

b

cell-free conditions nor affect cytoplasmic or spindle MT in V79 cells. Consistent with this behaviour is their failure to induce CREST-positive MN containing chromosomes with kinetochores in V79 cells. As expected, DES and COL are positive at all these endpoints. The lack of clastogenic potential for the lignans is demonstrated by their failure to induce CREST-negative MN containing chromosomal fragments, as does the established clastogen NQO. It is noteworthy that DES also shows no clastogenic effects. Furthermore, no mutagenicity was observed with the four lignans in the HPRT mutation assay in contrast to NQO.

5. Conclusion The data presented in this study provide consistent evidence that the lignans ENL, END, MAT and SEC are devoid of genotoxicity under the experimental conditions used in this study. This is important in view of the widespread exposure to these compounds. However, no information is yet available on the genotoxic potential of the oxidative metabolites of mammalian lignans. We have recently elucidated in our laboratory that ENL is biotransformed to twelve and END to six oxidative metabolites by rat hepatic microsomes, some of which are also formed with microsomes from human liver and excreted in the urine of humans after ingestion of flaxseed w35x. As V79 cells lack cytochrome P-450 dependent monooxygenase activity w36x, such oxidative products cannot be formed in this cell type. The structure

elucidation of the metabolites of mammalian and plant lignans and the assessment of their genotoxic potential is in progress in our laboratory. Acknowledgements We thank Sybille Mayer and Cornelia Hodapp for their help with parts of this work, and Dr. Kasper and Matthias Schottner for their generous gift of ¨ compounds. This study has been supported by the Deutsche Forschungsgemeinschaft ŽGrant Me 574r9-2.. References w1x K.D.R. Setchell, A.M. Lawson, F.L. Mitchell, H. Adlercreutz, D.N. Kirk, M. Axelson, Lignans in man and in animal species, Nature 287 Ž1980. 740–742. w2x N. Sathyamoorthy, T.T.Y. Wang, J.M. Phang, Stimulation of pS2 expression by diet-derived compounds, Cancer Res. 54 Ž1994. 957–961. w3x Y. Mousavi, H. Adlercreutz, Enterolactone and estradiol inhibit each other’s proliferative effect on MCF-7 breast cancer cells in culture, J. Steroid Biochem. Mol. Biol. 41 Ž1992. 615–619. w4x W.V. Welshons, C.S. Murphy, R. Koch, G. Calaf, V.C. Jordan, Stimulation of breast cancer cells in vitro by the environmental estrogen enterolactone and the phytoestrogen equol, Breast Cancer Res. Treat. 10 Ž1987. 169–175. w5x M. Axelson, K.D.R. Setchell, The excretion of lignans in rats-evidence for an intestinal bacterial source for this new group of compounds, FEBS Lett. 123 Ž1981. 337–343. w6x M. Axelson, J. Sjovall, B.E. Gustafsson, K.D.R. Setchell, ¨ Origin of lignans in mammals and identification of a precursor from plants, Nature 298 Ž1982. 659–660.

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