Introduction

Tamoxifen (Z)-1-{4-[2-(dimethylamino)ethoxy]phenyl}-1,2-diphenyl-1-butene (TAM), is a non-steroidal anti-estrogen used widely in the treatment of breast cancer. It is a first-generation selective estrogen receptor modulator and acts as an estrogen antagonist in mammary tissue but mimics the effects of estrogen in other tissues (Clemons et al. 2002; Shang and Brown 2002). The drug received FDA approval for adjuvant treatment in 1977 and for chemoprevention in healthy women “at high risk” in 1998. Using TAM therapy, the incidence of contralateral breast cancer was reduced by 47% and new breast cancers in women “at high risk” were reduced by 49% (Fisher et al. 1998; Poirier and Schild 2003). However, TAM treatment has the serious side effect of increasing the incidence of endometrial and, rarely, uterine cancer in breast cancer patients. For this reason, TAM has been classified as a group 1 carcinogen by IARC (1996).

Two potential mechanisms of endometrial/uterine tumour induction in women include an estrogenic pathway and a classical genotoxic pathway. As an estrogen agonist in the uterus, TAM is able to induce signal transduction resulting in promotion of cellular proliferation. As a genotoxin, TAM can be converted to reactive intermediates capable of binding to DNA; subsequent replication on a damaged DNA template may lead to mutagenesis in critical genes and a heritable loss of growth control (Poirier and Schild 2003). Recently, it was shown that a low level of TAM–DNA adducts in human uterus is unlikely to be involved with endometrial cancer development (Martin 2003). It is not yet clear whether the development of endometrial tumours in women treated with TAM is associated with a genotoxic or epigenetic mechanism(s).

TAM is well-recognized rat hepatocarcinogen (Greaves 1993). In rat liver TAM acts as a classical genotoxic chemical carcinogen by forming DNA adducts, which induce mutations in genes essential for growth control [8]. Rats exposed to TAM have parallel dose-related increases in hepatic TAM–DNA adducts and liver tumour incidence (Greaves 1993). In contrast, hepatic TAM–DNA adduct levels are low in TAM-exposed mice and mouse hepatocarcinogenicity does not occur (Phillips 2001).

We investigated the DNA-damaging potential of TAM in human peripheral blood lymphocytes and MCF-7 cells using the single-cell gel electrophoresis (comet assay). We employed the comet assay at three pHs: the alkaline (pH > 13) version revealing single and double strand breaks (SSBs and DSBs) as well as alkali labile sites (ALSs), the pH 12.1 version eliminating the expression of ALSs and the pH 9 version revealing only DSBs. In order to assess whether oxidative DNA damage may contribute to TAM-induced lesions, we employed two DNA repair enzymes: endonuclease III (Endo III) and formamidopyrimidine-DNA glycosylase (Fpg). Endo III converts oxidized pyrimidines into strand breaks, which can be detected by the comet assay (Collins et al. 1993). Fpg is a glycosylase initiating base excision repair in E. coli. It recognizes and removes 7,8-dihydro-8-oxoguanine (8-oxoguanine), the imidazole ring-opened purines 2,6-diamino-4-hydroxy-5-formamidopyrimidine (Fapy-Gua) and 4,6-diamino-5-formamido-pyrimidine (Fapy-Ade) as well as small amounts of 7,8-dihydro-8-oxoadenine (8-oxoadenine) (Krokan et al. 1997). The removing of specific modified bases from DNA by these enzymes leads to apurinic or apirymidinic sites, which are subsequently cleaved by their AP-lyase activity giving a gap in the DNA strand, which can be detected by the comet assay (Evans et al. 1995). The kinetics of repair of DNA damage induced by TAM was also measured. In order to evaluate the involvement of free radicals in the genotoxicity of TAM we pre-treated the cells with nitrone spin traps: DMPO and POBN. We also studied the genotoxic potential of the drug in the presence of vitamin C, amifostine and genistein.

Materials and methods

Chemicals

Tamoxifen citrate was a kind gift from HEXAL (Warsaw, Poland). Endo III and Fpg were kind gifts from Dr. Barbara Tudek of the Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland. TAM was taken from stock (0.1 M) ethanol solution. The final concentration of ethanol in samples was 0.01% for all drug concentrations and controls. Chemicals, culture media and related compounds, sodium ascorbate, amifostine [WR2721; 2-(3-aminopropyl)aminoethyl phosphorothioate], genistein (4′,5′, 7′-trihydroxyisoflavone), 5,5-dimethyl-1-pyrroline N-oxide (DMPO), α-(4-pyridyl 1-oxide)-N-tert-butylnitrone (POBN), low-melting-point (LMP) and normal-melting-point (NMP) agarose, phosphate-buffered saline (PBS), DAPI (4′,6-diamidino-2-phenylindole), dimethyl sulfoxide (DMSO) and hydrogen peroxide were purchased from Sigma-Aldrich Co. (St Louis, MO, USA). All other chemicals were of the highest commercial grade available.

Cells

Lymphocytes were isolated from peripheral blood obtained from young (23–25 years), non-smoking men by centrifugation in a density gradient of Gradisol L (15 min, 280 g, 4°C). The viability of the cells was measured after isolation by the trypan blue exclusion assay and was found to be about 99%. Each experiments on lymphocytes was performed on the cells obtained from blood of three different donors.

The breast cancer cell line was MCF-7 cells estrogen receptor positive (ER+). These cells were maintained in RPMI 1640 medium supplemented with 10% fetal calf serum, 100 U/ml penicillin and 100 μg/ml streptomycin. They were incubated at 37°C in a humidified incubator with 5% CO2 and 95% air. Cells were routinely passed by treatment with trypsin solution.

Cell viability

The viability of the cells was determined by trypan blue exclusion assay. The cells were incubated for 1 h at 37°C with TAM at 0.5, 1, 5 and 10 μM, washed and suspended in a RPMI 1640 medium. An equal volume of 0.4% trypan blue reagent was added to the cell suspension and the percentage of viable cells was evaluated under a field microscope. The viability of the cells was checked concurrently in all experiments at all tested concentrations of TAM.

DNA damage

TAM was added to the suspension of the cells to give final concentrations of 0.5, 1, 5 and 10 μM. Lymphocytes and MCF-7 cells were incubated with TAM for 1 h at 37°C. The experiment included a positive control, which was hydrogen peroxide at 20 μM applied for 10 min at 4°C. The cells, after treatment with TAM, were washed and resuspended in RPMI 1640 medium. A freshly prepared suspension of the cells in LMP agarose dissolved in PBS was spread onto microscope slides. The slides were processed as described below in “Comet assay”.

DNA repair

TAM was added to the suspension of the cells to give final concentrations of 0.5, 1, 5 and 10 μM. Lymphocytes and MCF-7 cells were incubated with TAM for 1 h at 37°C. Each experiment included a positive control, which was hydrogen peroxide at 20 μM applied for 10 min at 4°C. Lymphocytes and MCF-7 cells after the treatment with TAM were washed and resuspended in a fresh, RPMI 1640 medium preheated to 37°C. Aliquots of the cell suspension were taken immediately (time zero) and at 30, 60 and 120 min later. Placing the samples in an ice bath stopped DNA repair. The slides were processed as describe below in the “Comet assay”.

Treatment with DNA repair enzymes

Lymphocytes and MCF-7 cells were incubated with TAM for 1 h at 37°C at 1, 5 and 10 μM. Each experiment included a positive control, which was hydrogen peroxide at 20 μM applied for 10 min at 4°C. The slides after cell lysis were washed three times (5 min, 4°C) in an enzyme buffer containing 40 mM HEPES-KOH, 0.1 M KCl, 0.5 mM EDTA, 0.2 mg/ml bovine serum albumin, pH 8.0. The slides were then drained. Aliquots of 30 μl of Endo III or Fpg at 1 μg/ml in the buffer were applied to the agarose on slides, and incubation for 30 min at 37°C was performed (Collins et al. 1993; Evans et al. 1995). The control received only the buffer. The slides were processed as describe below in the “Comet assay”.

Spin trapping

In spin trapping experiments, the treatment with TAM was preceded by incubation with spin trap DMPO or POBN, at a final concentration of 100 μM, during a few seconds. Spin traps were derived from stock aqueous solutions at 1 mM. TAM was added to the suspension of the cells to give final concentrations of 0.5, 1, 5 and 10 μM without medium change. Lymphocytes and MCF-7 cells were incubated with TAM and spin traps for 1 h at 37°C. Each experiment included a positive control, which was hydrogen peroxide at 20 μM applied for 10 min at 4°C. The slides were processed as describe below in “Comet assay”.

Antioxidant treatments

Vitamin C was taken from stock solution (252 mM) in RPMI 1640 and added to the cell suspension to give a final concentration of 10 μM. Amifostine was taken from stock solution (233.43 mM) in 0.9% NaCl and added to the cell suspension to give a final concentration of 14 mM. Genistein was taken from stock (92.51 mM) solution in DMSO and added to the cell suspension to give a final concentration of 10 μM. The incubation with TAM was preceded by incubation with antioxidants during few seconds. TAM was added to the suspension of the cells to give final concentrations of 0.5, 1, 5 and 10 μM without medium change. Lymphocytes and MCF-7 cells were incubated with TAM and antioxidants for 1 h at 37°C. The slides were processed as describe below in “Comet assay”.

Comet assay

The comet assay was performed at pH > 13 essentially according to the procedure of Singh et al. (1988) with modifications (Collins et al. 1993; Evans et al. 1995; Klaude et al. 1996) as described previously (Blasiak and Kowalik 2000). A freshly prepared suspension of the cells (1–3 × 105 cells/ml) in 0.75% LMP agarose dissolved in PBS was spread onto microscope slides (Superior, Germany) precoated with 0.5% NMP agarose. The cells were then lysed for 1 h at 4°C in a buffer consisting of 2.5 M NaCl, 100 mM EDTA, 1% Triton X-100, 10 mM Tris, pH 10. After the lysis, the slides were placed in an electrophoresis unit, DNA was allowed to unwind for 20 min in the solution consisting of 300 mM NaOH and 1 mM EDTA, pH > 13. Electrophoresis was conducted in the solution consisting of 30 mM NaOH and 1 mM EDTA, pH > 13 at ambient temperature of 4°C (the temperature of the running buffer did not exceed 12°C) for 20 min at an electric field strength of 0.73 V/cm (28 mA). The slides were then washed in water, drained and stained with 2 μg/ml DAPI and covered with cover slips. To prevent additional DNA damage, all the steps described above were conducted under dimmed light or in the dark.

The assay at pH 12.1 was performed essentially according to the same procedure as the alkaline version except the pH value. In the version at pH 9, electrophoresis was run in a buffer consisting of 100 mM Tris and 300 mM sodium acetate at pH adjusted to 9.0 by glacial acetic acid (Singh and Stephens 1997). Electrophoresis was conducted for 60 min, after a 20-min equilibrium period, at 12 V (100 mA) at 4°C.

The comets were observed at 200× magnification in an Eclipse fluorescence microscope (Nikon, Tokyo, Japan) attached to COHU 4910 video camera (Cohu, San Diego, CA, USA) equipped with a UV-1 filter block (an excitation filter of 359 nm and a barrier filter of 461 nm) and connected to a personal computer-based image analysis system Lucia-Comet v. 4.51 (Laboratory Imaging, Praha, Czech Republic). Fifty images were randomly selected from each sample.

Three comet parameters were analysed: a measure of tail length (measured from the right edge of the comet head), percentage of DNA in the tail and tail moment (tail length × percentage of DNA in the tail). All these parameters are positively correlated with the level of DNA breakage and/or alkali labile sites and negatively correlated with the level of DNA cross-links (Ashby et al. 1995). Because our measurement system was not calibrated, tail length and tail moment were presented in arbitrary units.

Data analysis

The values of the comet assay in this study were expressed as mean ± SEM from three experiments, i.e., data from three experiments were pooled and the statistical parameters were calculated. The data were analysed using Statistica package (StatSoft, Tulsa, OK, USA). The values of the cell viability experiments were presented as mean ± SD from three experiments. If no significant differences between variations were found, as assessed by Snedecor-Fisher test, the differences between means were evaluated by applying the Student’s t test. Otherwise, the Cochran-Cox test was used.

Results and discussion

A number of studies have reported that TAM interacts with the estrogen receptor and this is generally considered to be the mechanism by which its pharmacological effects are mediated (Clemons et al. 2002; Shang and Brown 2002). Unfortunately, TAM has shown some serious side effects revealing its strong genotoxic activity (Greaves et al. 1993; IARC 1996; Phillips 2001; Martin et al. 2003; Poirier and Schild 2003). TAM appears to require metabolic activation to exert genotoxic action. It has been shown that TAM is converted to the genotoxic epoxide, as well as 4- and 2-hydroxy metabolites via enzymatic activation either by liver cytochrome P450 monooxidase or by different peroxidases or by hepatic α-oxidation of the TAM ethyl group (Poon et al. 1993; Lim et al. 1994; Phillips 2001). These metabolites could covalently bind to DNA, lipids and proteins inducing the irreversible damage to these biologically active molecules and membrane structures. TAM metabolites may also be oxidized by molecular oxygen giving rise to the ROS formation in the reduction/oxidation cycling process (Han and Liehr 1992; Pagano et al. 2001).

In this study, we found that TAM evoked a concentration-dependent decrease in the viability of human peripheral blood lymphocytes and MCF-7 cells (Fig. 1). At maximal applied concentration of the drug, 10 μM, the viability of lymphocytes was 70.23 ± 3.43% (P < 0.001) and MCF-7 cells was 75.0 ± 4.0% (P < 0.001).

Fig. 1
figure 1

Effect of TAM on viability of human lymphocytes (filled circle) and breast cancer MCF-7 cells (filled square) measured by trypan blue exclusion method. The figure shows mean results from three independent experiments. Error bars denote SD, ** P < 0.01, *** P < 0.001

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Table 1 presents numerical values for percentage of DNA in the tail, tail length and tail moment of comets of lymphocytes and MCF-7 cells. In both cell types, TAM induced a significant increase of these parameters at all concentrations except percentage of DNA in the tail and tail moment values for MCF-7 cells treated with TAM at 0.5 μM (P > 0.05). However, we observed a higher increase of DNA damage in lymphocytes (3.39 ± 0.3 versus 41.79 ± 1.81) than in MCF-7 cells (14.42 ± 2.35 versus 42.19 ± 4.77).

Table 1 Tail length, percentage of DNA in tail and tail moment of comets in pH > 13 version of comet assay on lymphocytes and MCF-7 cells incubated with TAM for 1 h at 37°C
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Figure 2 displays the tail DNA of lymphocytes (a) and MCF-7 cells (b) exposed to TAM and analysed by the comet assay in pH > 13, pH 12.1 and 9 versions. There were no differences in lymphocytes and MCF-7 cells between DNA damage in the pH > 13 and 12.1 versions. This result indicates that TAM damages DNA inducing mainly DNA strand breaks, but not ALSs. The drug, at all concentrations induced a significant (P < 0.001) increase in percentage of DNA in the tail in the pH 9 version of the comet assay in the case of lymphocytes but in MCF-7 cells this was a case only at 10 μM (P < 0.001). This indicates that TAM can induce DNA DSBs in human normal lymphocytes and breast cancer cells.

Fig. 2
figure 2

DNA damage, measured as the comet tail DNA of lymphocytes (a) and MCF-7 cells (b) incubated for 1 h at 37°C with TAM and analysed by the comet assay in the alkaline (empty bars), pH 12.1 (grey bars) or neutral (black bars) version. The number of cells scored in each treatment was 50. The figure shows mean results from three independent experiments. Error bars denote SEM; * P < 0.05, *** P < 0.001. The concentration axis is not linear and distances between points are chosen arbitrarily

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TAM may induce chromosomal breaks by two possible mechanisms: (a) directly by inducing DSBs by oxidative DNA damage or (b) defective translesion DNA synthesis (TLS) at sites of TAM-induced damage, which may result in an increase in the number of gaps, some of which may then be converted to DSBs (Miura et al. 2000; Pagano et al. 2001; Mizutani et al. 2004). It was observed that both TLS mutants and DSB repair mutants showed hypersensitivity to TAM (Mizutani et al. 2004).

Figure 3 shows DNA damage of lymphocytes (a) and MCF-7 cells (b) exposed to TAM and analysed by the comet assay in pH > 13 immediately after exposure and 30, 60 and 120 min thereafter. Lymphocytes were able to complete repair of TAM-induced DNA damage after exposure to concentration of 5 μM (Fig. 3a). At 10 μM TAM the repair was not completed until 120 min of repair incubation. MCF-7 cells were able to recover within 120 min after exposure at all TAM concentrations. The cells exposed to 20 μM H2O2 for 10 min at 4°C (positive control) were able to recover within the repair incubation time of 120 min (data not shown).

Fig. 3
figure 3

Time course of the repair of DNA damage, measured as the comet tail DNA in the comet alkaline version, in lymphocytes (a) and MCF-7 cells (b) treated for 1 h at 37°C with TAM at 0.5 μM (filled square), 1 μM (filled up triangle), 5 μM (filled down triangle) and 10 μM (filled diamond) compared with untreated control (filled circle). Error bars denote SEM; * P < 0.05, ** P < 0.01, *** P < 0.001. The number of cells scored and number of experiment repeats are the same as in Fig. 2

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Figure 4 shows DNA damage in lymphocytes (a) and MCF-7 cells (b) exposed to TAM and analysed by the comet assay in pH 9 immediately after exposure and 30, 60 and 120 min thereafter, showing the progress in DSBs repair. Lymphocytes were able to complete repair of TAM-induced DNA DSBs after exposure to concentration up to 5 μM (Fig. 4a). The repair of DSBs induced by TAM at 10 μM was incomplete on 120 min of repair incubation. However, MCF-7 cells were able to remove DNA DSBs within 120 min, even after exposure with TAM at 10 μM. The cells exposed to 20 μM H2O2 for 10 min at 4°C (positive control) were able to recover within the repair incubation time of 120 min (data not shown). Our data indicate that TAM-induced DSBs in normal cells were more persistent than those existing in cancer cells. Probably, it arises from DNA repair mechanisms, which are usually more effective in cancer cells than in normal cells (Belzile et al. 2006; Ding et al. 2006). It was shown that TAM damages DNA with slow kinetics in marked contrast to the rapid action of classical genotoxic agents such as UV, cross-linkers and alkylating agents (Mizutani et al. 2004).

Fig. 4
figure 4

Time course of the repair of DNA damage, measured as the comet tail DNA in the comet neutral version, in lymphocytes (a) and MCF-7 cells (b) treated for 1 h at 37°C with TAM at 0.5 μM (filled square), 1 μM (filled up triangle), 5 μM (filled down triangle) and 10 μM (filled diamond) compared with untreated control (filled circle). Error bars denote SEM; * P < 0.05, ** P < 0.01, *** P < 0.001. The number of cells scored and number of experiment repeats are the same as in Fig. 2

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Oxidative mechanism of DNA damage by TAM was confirmed in experiment with post-treatment with endonuclease III (Endo III) and formamidopyrimidine-DNA glycosylase (Fpg), repair enzymes recognizing oxidized DNA bases (Fig. 5). DNA originated from both types of the cells incubated with TAM and treated with Endo III and Fpg showed significant increase (P < 0.001) in DNA damage compared with the cells without treatment with these enzymes. 8-oxoguanine, which is recognized by Fpg enzyme, is one of the most frequent DNA base modifications observed with oxidative stress (Loft and Poulsen 1996). This lesion can be generated either by oxidation of the base in DNA, for example, by hydroxyl radicals or peroxynitrite, or by the incorporation of the oxidized nucleoside triphosphate into DNA during replication or repair synthesis. Generation of 8-oxoguanine in DNA is a mutagenic and potentially carcinogenic event since the oxidized base can alter hydrogen bonding or “coding specificity” preferentially forming base pairs with adenine rather than cytosine. That is why replication of the oxidatively modified DNA may result in transversion mutations (G to T) (Lindahl 1993; Loft and Poulsen 1996; McCall and Frei 1999).

Fig. 5
figure 5

DNA damage, measured as the comet tail DNA in the comet alkaline version, of human lymphocytes (a) and MCF-7 cells (b) incubated for 1 h at 37°C with TAM and treated with Endo III (grey bars) or Fpg (black bars) at 1 μg/ml as compared with the untreated cells (empty bars). Error bars denote SEM; *** p < 0.001. The concentration axis is not linear and distances between points are chosen arbitrarily. The number of cells scored and number of experiment repeats are the same as in Fig. 2

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DNA fragmentation, which we observed in the cells incubated with TAM, could be a result of apoptosis, which could underline a major mechanism of antitumor effect of TAM (Obrero et al. 2002; Salami and Karami-Tehrani 2003). Two distinct pathways of TAM-induced programmed cell death were described: (a) a caspase-dependent apoptotic cell death program inducible in the absence of ER at very high concentrations of TAM and (b) an ER-dependent and ligand-specific pathway induced at submicromolar concentrations of TAM bound to ER (Obrero et al. 2002).

In this study, we checked whether TAM was able to DNA damage directly in normal and breast cancer cells. To elucidate whether TAM induced free radicals we pre-incubated lymphocytes and MCF-7 cells with spin traps: DMPO and POBN, which can form adducts with free radicals and in this way reduce the extent of DNA damage (Kotamraju et al. 2000). We chose DMPO and POBN because they can form complex with a lot of reactive free radicals, including: hydroxyl radical, superoxide, radicals generated during lipid peroxidation, such as the carbon-centered pentadienyl radical (L•), the oxygen-derived peroxyl free radical (LOO•), the alkoxyl free radical (LO•) as well as thiyl radical.

To check the ability of spin traps to scavenge free radicals in our experimental conditions, incubations of lymphocytes and MCF-7 cells with H2O2 at concentration of 20 μM for 10°min at 4°C in the presence or in the absence of DMPO or POBN were performed. A significant decrease (P < 0.001) of DNA damage after incubation of lymphocytes with H2O2 and spin traps in comparison with cells not treated with the traps was observed. The decrease ranged from value 42.96 ± 3.69 to 17.80 ± 2.31 for DMPO and to 17.81 ± 2.43 for POBN. We also observed a significant decrease (P < 0.001) of DNA damage after incubation of MCF-7 cells with H2O2 and spin traps in comparison with cells not treated with the traps. This decrease ranged from 53.84 ± 4.16 to 38.26 ± 3.07 for DMPO and to 37.53 ± 2.97 for POBN.

We observed a decrease of TAM-induced DNA damage in human lymphocytes after incubation with the traps (Fig. 6a). In MCF-7 cells we also detected a significant decrease in the level of DNA damage in their presence (Fig. 6b). These results indicate that free radicals might account for DNA lesions evoked by TAM. However, observed decrease in the level of DNA damage was more pronounced in cancer cells. In these cells incubated with TAM at the maximal concentration 10 μM and pre-incubated with spin traps, the level of DNA damage decreased almost to the control level of DNA damage (P < 0.001) (Fig. 6b).

Fig. 6
figure 6

DNA damage, measured as the comet tail DNA in the comet alkaline version, of human lymphocytes (a) and MCF-7 cells (b) incubated for 1 h at 37°C with TAM in the presence of DMPO (filled square) or POBN (filled up triangle) at 100 μM as compared with cells incubated with tamoxifen only (filled circle). Error bars denote SEM; *** P < 0.001. The number of cells scored and number of experiment repeats are the same as in Fig. 2

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Figure 7 shows DNA damage of lymphocytes (a) and MCF-7 cells (b) exposed to TAM in the presence of vitamin C at 10 μM, amifostine at 14 mM and genistein at 10 μM. In both types of cells vitamin C and genistein significantly decreased of DNA damage induced by TAM. Amifostine also decreased such damage, but only in lymphocytes (Fig. 7a). In MCF-7 cells co-incubation with amifostine did not change TAM-induced DNA damage (Fig. 7b). Amifostine is a thiol-derivative used to protect normal cells in chemo- and radiotherapy of numerous cancer including solid tumours, leukaemias and lymphomas (Grdina et al. 2000). It undergoes activation by the interaction with alkaline phosphatase, which is scarce in neoplastic cells (Giatromanolaki et al. 2002). Therefore, different uptake of amifostine in normal and cancer cells may contribute to the diversity of the action of this compound. Recently, we have shown that amifostine decreased the DNA-damaging effect of idarubicin and amsacrine in normal human lymphocytes, but increased the effect in murine growth factor-dependent pro-B lymphoid cell line BaF3 expressing TEL/ABL or BCR/ABL, oncogenic fusion tyrosine kinases (Blasiak et al. 2002, 2003).

Fig. 7
figure 7

DNA damage, measured as the comet tail DNA in the comet alkaline version, of human lymphocytes (a) and MCF-7 cells (b) incubated for 1 h at 37°C with TAM in the presence of vitamin C at 10 μM (filled square), genistein at 10 μM (filled diamond) or amifostin at 14 mM (filled up triangle) as compared with cells incubated with tamoxifen only (filled circle). Error bars denote SEM; * P < 0.05, ** P < 0.01, *** P < 0.001. The number of cells scored and number of experiment repeats are the same as in Fig. 2

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Genistein is a phytoestrogen with in vitro anticancerogenic activity. It was shown to inhibit the growth of dysplastic and malignant epithelial breast cancer cells in vitro and the addition of TAM had a synergistic/additive inhibitory effect (Tanos et al. 2002). We did not observe any such effect in MCF-7 cells. From this point of view, genistein could not be recommended in the prevention of breast cancer. Probably, genistein acted as an antioxidant in our experimental conditions and protected normal and cancer cells against genotoxic potential of TAM.

Interestingly, vitamin C was reported to inhibit TAM–DNA adducts, formation in endometrial explant culture (Sharma et al. 2003). The level of α-hydroxytamoxifen, a metabolite of TAM, was also reduced in the presence of vitamin C. That report and our results with antioxidants and spin traps could support the role of oxidative biotransformation of TAM.

The results obtained suggest that TAM can induce DNA single- and double-strand breaks as well as oxidative modifications of purines and pyrimidines in lymphocytes and cancer breast cells. Free radicals may be involved in the formation of these DNA lesions. Therefore, TAM could be genotoxic for both types of cells and TAM-induced DNA damage is more persistent in lymphocytes than in cancer cells. It might suggest potential genotoxic side effect of the drug for lymphocytes, which can be diminished by cautious use of the combination of vitamin C, amifostine and genistein.