Lack of evidence for tamoxifen– and toremifene–DNA adducts in lymphocytes of treated patients

Helmut Bartsch3, David H. Phillips1, Jagadeesan Nair, Alan Hewer1, Gabriele Meyberg-Solomeyer2 and Eva-Maria Grischke2

Division of Toxicology and Cancer Risk Factors, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany,
1 Institute of Cancer Research, Haddow Laboratories, Sutton, Surrey, UK and
2 Frauenklinik, Ruprecht-Karls-Universität, Heidelberg, Germany


    Abstract
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 Abstract
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Tamoxifen (TAM) is used for the adjuvant treatment of women with breast cancer and has also been recommended as a chemopreventive agent. Among unwanted side effects, TAM was shown to increase endometrial cancer in treated women by mechanisms that are not yet clearly understood. We studied DNA adducts in lymphocytes of female breast cancer patients treated with TAM or toremifene (TOR), a TAM analogue and compared them with adducts formed by TAM in rat liver, where the drug induces tumours. DNA adducts were measured by TLC-32P-post-labelling assays. After TLC, all DNA samples including DNA from untreated healthy women showed a faint radioactive zone, where the positive control DNA adducts isolated from the liver of rats treated with TAM migrated. The relative adduct levels were calculated from the radioactivity present in this zone. Means ± SD of adduct levels per 108 nucleotides (associated with this area) were for untreated volunteers (control) 1.83 ± 1.41 (n = 13), for TAM treatment 2.17 ± 3.04 (n = 25) and for TOR treatment 1.18 ± 1.05 (n = 8). Most of the human samples were further analysed by HPLC after labelling with 32P in order to compare adducts in human DNA with those in liver DNA isolated from TAM-treated rats. None of the human samples showed any peaks at retention times where putative TAM–DNA adducts were eluted. In conclusion, lymphocyte DNA from female patients treated at therapeutic levels did not show evidence of the formation of TAM– or TOR–DNA adducts.

Abbreviations: TAM, tamoxifen; TOR, toremifene.


    Introduction
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 Abstract
 Introduction
 References
 
Tamoxifen (TAM) is used for the adjuvant treatment of breast cancer patients, increasing mean survival and reducing the incidence of contralateral breast cancer (1). It has also been recommended as a chemopreventive treatment for women with high risk of developing breast cancer (2,3). However, among unwanted side effects, TAM was shown to increase endometrial cancer in treated women by mechanisms that are not yet clear. Controversies exist on the occurrence of TAM-induced DNA adducts in human tissues that may be responsible for tumour initiation (reviewed in ref. 4). In this context, we studied DNA adduct patterns in lymphocytes of female patients treated with TAM or toremifene (TOR), a TAM analogue, and compared those with adducts formed by TAM in rat liver, where the drug induces tumours. After long-term treatment of rats with TAM, hepatocellular carcinoma developed, while animals receiving TOR at a similar or higher dose did not develop tumours (5). In this latter study, TAM did produce hepatic DNA adducts, while in another rat study TOR produced only insignificant adduct levels that differed by at least two orders of magnitude after short-term treatment (6). DNA adducts were detected by TLC- and HPLC-32P-post-labelling assays.

Blood samples (10 ml) were taken from post-menopausal female breast cancer patients who were receiving anti-oestrogen therapy as either TAM (20–30 mg per day) or TOR (60 mg per day) for at least 3 weeks, and from healthy female volunteers not receiving either treatment, hereafter referred to as controls. All samples were collected in the Women's Clinic at Heidelberg and transported to the German Cancer Research Center (DKFZ) within 2 h. All the patients had their breast cancer resected. Patients received TAM either as adjuvant therapy or for the treatment of metastasis. TOR-treated patients received the drug for treatment of metastasis. Lymphocytes were isolated using LymphoprepTM solution (Nycomed, Norway). The samples were stored at –80°C prior to analysis. The samples were coded, and the codes were broken only after receipt of all results. The three groups consisted of 13 controls aged 43–69 years (mean ± SD: 54.1 ± 8.6), 25 TAM-treated patients aged 47–71 years (55.6 ± 6.4) and eight TOR-treated patients aged 42–68 years (51.8 ± 9.1).

DNA was isolated by a phenol–chloroform method (7). Adequate care was taken to avoid excessive light exposure during isolation, and all buffers were kept to near neutral pH. Four micrograms of DNA were used for adduct analysis. DNA was hydrolysed to 2'-deoxynucleoside 3'-monophosphates by micrococcal endonuclease and spleen phosphodiesterase. The digests were enriched by nuclease P1 treatment and labelled with [{gamma}-32P]ATP by T4 polynucleotide kinase and resolved on polyethyleneimine-cellulose TLC plates, as described previously (8). In brief, multidirectional chromatography was carried out using the following solutions: D1, sodium phosphate (2.3 M, pH 5.8); D2, lithium formate (2.275 M):urea (5.25 M), pH 3.5; D3, lithium chloride (0.52 M):Tris–HCl (0.325 M):urea (5.525 M), pH 8.0. Most of the samples were also analysed by reverse-phase high-performance liquid chromatography (HPLC) (9) in order to verify the presence/absence of adducts. HPLC separation was achieved on a 5 µ C18 reverse-phase column eluted with 82% 2 M ammonium formate, pH 4 (solvent A) and 18% MeCN:MeOH (6:1) (solvent B) for 40 min followed by a linear gradient of 18–45% solvent B for 20 min. Flow rate was 1 ml/min. The detection limit of TLC-32P-post-labeling and HPLC methods was one to two adducts per 109 nucleotides. An adducted liver DNA sample, obtained by treating a rat with TAM (dose: 0.12 mmol/kg body wt, single dose by gavage; animal killed 24 h later) was used as a positive standard for analysis. The adduct level obtained, 1.1 adducts per 106 nucleotides, is typical of many experiments carried out in this laboratory.

The adduct levels were expressed as relative adduct labelling (RAL) per 108 unmodified nucleotides and calculated as described (8).

Figure 1Go shows the representative TLC chromatograms. Qualitatively, none of the human samples, except a single TAM-treated one (see area circled in Figure 1GGo) showed any distinct adduct spots in lymphocyte DNA from TAM- or TOR-treated patients. However, all the DNA samples including those from controls had a faint radioactive zone where DNA adducts in liver isolated from a rat treated with TAM migrated (Figure 1HGo). For comparison, the radioactivity in this zone was counted for each sample. The adduct levels associated with this zone for each study group are given in Figure 2Go. The adduct levels (RAL) associated with the radioactivity zone migrating at the area of putative TAM/TOR adducts in lymphocyte DNA of treated patients were not significantly different (analysed by the Mann–Whitney–Wilcoxon test) from those of untreated controls.



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Fig. 1. 32P-post-labelling analysis of lymphocyte DNA from breast cancer patients and controls. (A and B) Control subjects; (C and D) TOR-treated patients; (EG) TAM-treated patients; (H) positive control sample of liver DNA from a TAM-treated rat. DNA digests (4 µg) were labelled and subjected to thin-layer chromatography as described in the text. Radioactivity detection was carried out using an Instantimager detector (Packard Instruments, USA).

 


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Fig. 2. Box–Whisker plot of adduct levels associated with the zone on thin-layer chromatograms where TAM–DNA adducts in rat liver migrate (see Figure 1Go); boxes give 25th to 75th percentiles; in boxes solid lines represent median and dashed lines mean values, whiskers represent 10th and 90th percentiles, full circles are outliers. The probability (two-tailed) to exceed the means was P < 0.75 for control versus TAM, P < 0.35 for control versus TOR, and P < 0.61 for TAM versus TOR.

 
Most of the samples (33 out of 46) were further analysed by HPLC in order to compare the adducts in human DNA with DNA isolated from rats treated with TAM. Figure 3Go shows representative HPLC profiles of the samples analysed. None of the samples, including one which showed a distinct spot (Figure 1GGo), showed any peaks at the retention times where putative DNA adducts are eluted (Figure 3AGo). Thus, the distinct adduct spot detected in one TAM-treated patient, which amounted to an apparent adduct level of 15/108 nucleotides) was not verified further by HPLC to contain TAM-specific DNA adducts.



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Fig. 3. Radioactive HPLC profiles of 32P-post-labelled DNA samples, analysed as described in the text. (A) Aliquot of liver DNA (0.4 µg) from a TAM-treated rat; (B) lymphocyte DNA (4 µg) from a control woman; (C) lymphocyte DNA (4 µg) from a patient treated with TAM; (D) lymphocyte DNA (4 µg) from a patient treated with TOR.

 
The two major adducts formed in rat liver after TAM administration (Figure 3AGo) have been identified as deriving from the binding of the 2-amino group of guanine residues in DNA to the {alpha}-position of TAM (peak at 32.5 min) (10) while the earlier peak (at 30.1 min) derives from N-desmethyltamoxifen, presumably bonded to DNA in the same way (9,11,12). In both cases the TAM/N-desmethyltamoxifen moiety has trans configuration. Most of the DNA adducts with TAM are generated via {alpha}-hydroxylation followed by sulphate ester formation by sulphotransferases (10,1316). A recent in vitro study showed that rat, but not human, sulphotransferases can activate a TAM metabolite to produce DNA adducts and gene mutations in bacteria and mammalian cells in culture (17). Similarly, human recombinant hydroxysteroid sulphotransferase was able to catalyse the formation of TAM–DNA adducts in the presence of {alpha}-hydroxytamoxifen, but the activity of this enzyme was 3-fold lower than that of the equivalent rat enzyme (18).

In conclusion, analysis of lymphocytes from female breast cancer patients treated with the drugs at therapeutic levels did not demonstrate the formation of detectable TAM– or TOR–DNA adducts that are found in rat liver after TAM treatment, confirming our earlier negative results in white blood cells from TAM-treated breast cancer patients (19). However, positive results were reported in TAM-treated patients in another study (20). There is also controversy regarding the existence of damage to endometrial DNA in women after TAM administration (21). Carmichael et al. (2224) did not find evidence for drug-derived adducts in patients treated with either TAM or TOR, whereas Hemminki et al. (25) reported low levels of TAM adducts (<1 adduct per 108 nucleotides). Shibutani et al. (26) have recently reported the presence of varying relative amounts of cis- and trans-TAM–DNA adducts in the endometrium of some patients, a site where TAM is known to induce tumours in humans (3). Although the above and other reported data (discussed in ref. 4) suggest that the tumorigenicity of TAM may not be induced via direct DNA adduct formation, further analyses of endometrial epithelial tissue are clearly required to ascertain this mechanism.


    Acknowledgments
 
The authors wish to thank G.Bielefeldt for skilled secretarial help and I.Hofmann for technical assistance. This work was, in part, presented at the 10th Symposium of the Division of Experimental Cancer Research (AEK) of the German Cancer Society at Heidelberg, March 24–26, 1999 [Abstract #113: J. Cancer Res. Clin. Oncol. (1999) 125, S58]. We acknowledge the support of ASTA MEDICA AWD GmbH, Frankfurt, Germany.


    Notes
 
3 To whom correspondence should be addressed Email: h.bartsch{at}dkfz-heidelberg.de Back


    References
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 Introduction
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Received October 13, 1999; revised December 20, 1999; accepted December 20, 1999.