Idoxifene derivatives are less reactive to DNA than tamoxifen derivatives, both chemically and in human and rat liver cells
Martin R. Osborne3,
Alan Hewer,
Warren Davis,
Alastair J. Strain2,
Adrian Keogh2,
Ian R. Hardcastle1 and
David H. Phillips
Section of Molecular Carcinogenesis, Haddow Laboratories and
1 CRC Centre for Cancer Therapeutics, Institute of Cancer Research, 15 Cotswold Road, Sutton, Surrey SM2 5NG and
2 Liver Research Laboratories, Queen Elizabeth Hospital, Edgbaston, Birmingham B15 2TH, UK
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Abstract
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The drug tamoxifen shows evidence of genotoxicity, and induces liver tumours in rats. Covalent DNA adducts have been detected in the liver of rats treated with tamoxifen, and these arise through metabolism at the
-position to give an ester which reacts with DNA. (E)-1-(4-iodophenyl)-2-phenyl-1-[4-(2-pyrrolidinoethoxy)phenyl]-but-1-ene (idoxifene) is an analogue of tamoxifen in which formation of DNA adducts is greatly reduced; we could not detect any adducts in the DNA of cultured rat hepatocytes treated with 10 µM idoxifene, after analysis by the 32P-post-labelling method. The metabolite (Z)-4-(4-iodophenyl)-4-[4-(2-pyrrolidinoethoxy)phenyl]-3-phenyl-3-buten-2-ol (
-hydroxyidoxifene) gave adducts in rat hepatocytes, but far fewer than the corresponding tamoxifen metabolite. In human hepatocytes, neither idoxifene nor tamoxifen induced detectable levels of DNA adducts. We prepared the
-acetoxy ester of idoxifene as a model for the ultimate reactive metabolite formed in rat liver. It was less reactive than
-acetoxytamoxifen, as might be expected on mechanistic grounds. It reacted with DNA in the same way, to give adducts which were probably N2-alkyldeoxyguanosines, but to a lower extent. All these results indicate that idoxifene is much less genotoxic than tamoxifen, and should therefore be a safer drug.
Abbreviations:
-Hydroxyidoxifene, (Z)-4-(4-iodophenyl)-4-[4-(2-pyrrolidinoethoxy)phenyl]-3-phenyl-3-buten-2-ol; idoxifene, (E)-1-(4-iodophenyl)-2-phenyl-1-[4-(2-pyrrolidinoethoxy)phenyl]-but-1-ene.
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Introduction
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Tamoxifen (Figure 1A
) is an anti-oestrogen which is widely used in the treatment of breast cancer. It is also being tested as a tumour preventive agent in women who are at high risk of developing the disease (1). However, there are possible risks associated with its use. As well as anti-oestrogenic activity, it shows some oestrogenic effects and appears to be associated with an increased risk of carcinoma of the uterus (2,3). It has also been shown to form DNA adducts and induce liver cancer in rats (47). There is as yet no evidence for any such effect on liver in humans, and no tamoxifen adducts were detectable in human hepatocytes treated with tamoxifen (8). However, the possible genotoxic effects of tamoxifen remain a cause for concern and a number of analogues of tamoxifen have been prepared in an attempt to reduce the undesired side effects. One of these, (E)-1-(4-iodophenyl)-2-phenyl-1-[4-(2-pyrrolidinoethoxy)phenyl]-but-1-ene (idoxifene) (Figure 1C
) has undergone a phase I clinical trial (9) and there has been a preliminary report of its pharmacokinetics and metabolism as part of a phase II trial (10). It differs from tamoxifen in its terminal pyrrolidine substituent and, more significantly, in the iodine substituent at the p-position of one of the phenyl groups. It shows greater binding to the oestrogen receptor, but less oestrogenic activity in rat or mouse uterus than tamoxifen does (11). Both drugs give rise to DNA adducts in rat liver after subcutaneous injection, but idoxifene gives much fewer: mean level 0.06 per million DNA bases, compared with 7.1 for tamoxifen (12).
The purpose of the present work was to understand the reasons for the much reduced capacity of idoxifene to bond to rat liver DNA. The mechanism of bonding of tamoxifen to DNA is believed to be as follows (1317). It is oxidized by cytochrome P450 to
-hydroxytamoxifen, which is then conjugated by a sulfotransferase to form the sulfate ester. This ester is unstable under physiological conditions and loses the sulfate ion to yield a relatively stable carbocation, which alkylates DNA. We prepared
-acetoxytamoxifen (Figure 1B
) as a model compound for such a reactive ester metabolite, and showed that it reacts with DNA to give several tamoxifendeoxyguanosine and tamoxifen-deoxyadenosine adducts (18,19). Idoxifene forms fewer adducts than tamoxifen does, either because it is not metabolized by this pathway, or because the
-sulfate, once formed, reacts much less with DNA. Here we report an investigation of this latter possibility, by two methods. We prepared (Z)-4-(4-iodophenyl)-4-[4-(2-pyrrolidinoethoxy)phenyl]-3-phenyl-3-buten-2-ol (
-hydroxyidoxifene) by chemical synthesis and showed that it formed fewer adducts in rat liver cells than
-hydroxytamoxifen did. We then prepared the model ester
-acetoxyidoxifene (Figure 1D
) and found that it reacts with DNA to a considerably lower extent than the corresponding derivative of tamoxifen.
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Materials and methods
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Salmon testis DNA, tricaprylin, collagenase IV, proteinase K, RNase A, RNase T1 and water-saturated phenol were purchased from Sigma (Poole, UK).
-Acetoxytamoxifen (18) and
-hydroxyidoxifene (20) were prepared as described before. All operations with tamoxifen and idoxifene derivatives were carried out in darkness or subdued light where possible. Mass spectroscopy was carried out using a Finnigan TSQ 700 triple-quadrupole mass spectrometer fitted with an electrospray ion source as described before (19) and NMR spectroscopy with a Bruker AC250 spectrometer.
Reverse phase liquid chromatography was carried out on Waters (Watford, UK) apparatus, using a 250x4.6 mm column (Fisons, Loughborough, UK) packed with ODS (Nucleosil, 5 µm). The solvent (0.8 ml/min) was a wateracetonitrile mixture, containing 0.05 M ammonium formate throughout; either (A) 075% MeCN in 50 min or (B) 4575% MeCN in 20 min, then 75% MeCN. Tamoxifen and idoxifene derivatives were detected by their ultraviolet absorbance at 254 nm.
Preparation of
-acetoxyidoxifene
This was prepared by the same method as for
-acetoxytamoxifen (18). Six milligrams of
-hydroxyidoxifene was treated with 10 µl acetic anhydride in 100 µl pyridine for 18 h at 22°C; analysis by HPLC on an ODS column showed that acetylation was complete (system B;
-hydroxyidoxifene at 25 min,
-acetoxyidoxifene at 32 min). The product was purified by partition between ether (2 ml) and water (3x2 ml) and chromatography on a silica Sep-Pak, with hexaneethertriethylamine (11:8:1) as eluant. The mass spectrum showed a molecular ion at m/z = 582, as expected for MH+ = C30H33NO3I. Ms/ms gave daughter ions at m/z = 451 (MH+ less HOAc and pyrrolidine, 53%), 324 (?, 10%) and 98 (vinylpyrrolidine.H+, 100%). The proton NMR spectrum (in D6-acetone or D6-dimethylsulfoxide) showed peaks at
= 1.25 (3, d, J = 6.6, methyl), 1.7 (4, m, pyrrolidine), 1.9 (3, s, acetate), 2.42.5 (4, m, pyrrolidine), 2.7 (2, t, J = 6.0, CH2N), 3.95 (2, t, J = 6.0, CH2O), 5.7 (1, q, J = 6.6,
-H), 6.6 (2, d, J = 8.3) and 6.8 (2, d, J = 8.8, phenoxy-ring), 7.17.8 (9, m, phenyl and iodophenyl rings).
To determine the rate of hydrolysis, an HPLC-purified sample (0.2 mg) was incubated at 37°C in 15 ml water:acetonitrile (85:15, with 0.08 M sodium phosphate, pH 7). One millilitre samples were analysed by HPLC (system B) and the amount of remaining ester was estimated from the area of its peak on the ultraviolet absorption trace, compared with those of the hydrolysis products.
Reaction with DNA
The ester was dissolved in 5 ml ethanol and reacted with 20 mg salmon testis DNA in 10 ml water (unbuffered) for 40 h at 37°C. One millilitre of 2.5 M NaOAc was added, and the mixture extracted seven times with 15 ml ether to remove unreacted material. The DNA was precipitated with 2 vol ethanol, washed, dried and dissolved in 5 ml water. Degradation to nucleosides was with DNase 1 (0.5 mg; 20 h in 1 ml 10 mM Tris/10 mM MgCl2, pH 7), snake venom phosphodiesterase (Sigma type VIII-S, 0.05 U; 6 h in 0.1 M Tris, pH 9) and alkaline phosphatase (Sigma type III, 15 U, 17 h), all at 37°C. The hydrolysate was separated by reverse phase chromatography (system A) in 3 portions of 2 ml.
Preparation and treatment of hepatocytes
Rat hepatocytes were isolated from the livers of female Fischer F-344 rats, aged 810 weeks, according to standard procedures (21). They were established in primary culture as described before (13) and were allowed to attach to the flask for 3 h at 37°C in an atmosphere of 5% CO2 in the presence of 10% fetal calf serum. The medium was changed to exclude fetal calf serum and the agent added in dimethylsulfoxide solution, to a final concentration of 1 or 10 µM. Experiments were performed in duplicate flasks. After 18 h of incubation the cells were harvested and separated from the medium before DNA isolation.
Human hepatocytes were prepared from normal liver obtained from the paediatric transplant programme at Queen Elizabeth Hospital, Birmingham, UK, as described before (8). Four preparations were made from four donors: three females aged 5, 17 and 38 and one male aged 5. They were treated with the agent as for the rat hepatocytes.
DNA isolation and 32P-post-labelling
DNA was isolated and purified from hepatocytes by the phenolchloroform method (22). It was redissolved in NaCl (1.5 mM)/sodium citrate (0.15 mM) and stored at 20°C. In the nuclease P1 enrichment method of post-labelling analysis (23), aliquots (4 µg) were taken and evaporated to dryness using a Savant Speedvac SVC100 vacuum centrifuge. The DNA was digested overnight at 37°C with micrococcal nuclease (0.14 U) and spleen phosphodiesterase (1.2 µg, 1.2 µl) in sodium succinate (20 mM)/calcium chloride (10 mM) (pH 6.0, 0.8 µl) and water (2.8 µl). Samples were then further digested for 1 h at 37°C with nuclease P1 (0.15 U, 0.96 µl) in sodium acetate (62.5 mM, 2.4 µl) and ZnCl2 (0.3 mM, 1.44 µl). 32P-post-labelling of each sample was carried out by incubation at 37°C for 30 min with [
-32P]ATP (50 µCi, sp. act. ~4000 Ci/mmol; ICN Biomedicals, High Wycombe, UK), polynucleotide kinase (6 U, 0.6 µl) in bicine (14 mM, pH 9.0)/magnesium chloride (7 mM)/dithiothreitol (7 mM)/spermidine (0.7 mM) (1 µl).
Radiolabelled digests were applied to the origins of 10x10 cm polyethyleneiminecellulose TLC plates (Macherey-Nagel, Duren, Germany). Multidirectional chromatography was carried out using the following solutions: D1, sodium phosphate (2.3 M, pH 5.8) overnight onto a paper wick outside the tank; D2, lithium formate (2.275 M):urea (5.525 M), pH 3.5; D3, lithium chloride (0.52 M):TrisHCl (0.325 M):urea (5.525 M), pH 8.0. DNA adducts were detected as radioactive spots on the TLC plates, visualized and quantified using a Packard Instant Imager (Canberra Packard, Pangbourne, UK). The radioactivity of the spot, after background subtraction, was used to calculate the level of adducts; for this, the specific activity of the [
-32P]ATP was determined by labelling a known quantity of 3'-deoxyadenylic acid (23). Where no spot could be seen, the radioactivity was measured in an area of the plate where an adduct spot might be expected, in order to estimate a lower limit for the extent of DNA bonding. For idoxifene, this area was a rectangle just above the horizontal axis, corresponding to the spot of Figure 3B and C
; for tamoxifen, the area was further away from the origin, as determined by previous experiments (13).
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Results
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Reactivity of
-acetoxyidoxifene
The reactivity was estimated from its rate of hydrolysis in an aqueous medium (85:15 water:acetonitrile, pH 7). The disappearance of
-acetoxyidoxifene fitted a first-order curve, with half-life of 6 h. Under the same conditions the half-life of
-acetoxytamoxifen was 2.4 h, demonstrating its greater reactivity.
-Acetoxyidoxifene gave two products of hydrolysis, eluted in HPLC system B at 23 and 27 min (ratio 56:44). These were probably the trans- and cis-isomers of
-hydroxyidoxifene.
Reaction of
-acetoxyidoxifene with DNA
-Acetoxyidoxifene was dissolved in 5 ml ethanol and reacted with 20 mg DNA in 10 ml water. The DNA was isolated, then hydrolysed with enzymes to nucleosides. The mixture was analysed on an ODS column (system A); ultraviolet absorbing material was eluted as shown in Figure 2
. The pattern was similar to that obtained in a similar experiment with
-acetoxytamoxifen (19), except that the yield was lower (see below).

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Fig. 2. Elution of nucleosides from hydrolysates of DNA treated with -acetoxyidoxifene. The peaks at 1824 min corresponded to deoxycytidine, deoxyguanosine, thymidine and deoxyadenosine. The peaks labelled IG1 and IG2 corresponded to nucleosidedrug adducts. The material eluted before them, at 3540 min, probably consisted of undigested di- or oligonucleotide adducts.
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The principal nucleoside products were IG1, eluted at 43 min, and IG2, at 47 min. Both IG1 and IG2 had the ultraviolet spectrum expected for adducts of idoxifene with deoxyguanosine:
max 249 nm, shoulder at 276 nm. The mass spectrum of IG1 showed the following ions: m/z = 789 (MH+, 57%), 673 (MH+ deoxyribose, 44%), 522 (
,ß-dehydro-idoxifene.H+, 28%), 414 (MHK++, 32%), 406 (MHNa++, 47%) and 337 (673+H+, 100%). These are as expected for an idoxifene-deoxyguanosine adduct, C38H41N6O5I. There was insufficient adduct for further characterization. By analogy with tamoxifen (15,19) it is probable that IG1 has the
-position of idoxifene linked to the amino group of deoxyguanosine and IG2 is an isomer of IG1 in which the idoxifene has rotated from the trans to the cis configuration. The relative yields were ~60 IG1:40 IG2; the ratio with tamoxifen was ~74 TG1:26 TG2 (19).
Yield of adducts in reactions of acetates with DNA
The relative yields of products from the reactions of the acetates with DNA are shown in Table I
. From the ratios in column 3, it can be seen that
-acetoxytamoxifen gave 4.5 ± 0.5 times as many adducts as
-acetoxyidoxifene. The order of reactivity with DNA parallels the order of reactivity with water (above).
Adducts in rat hepatocytes
Rat hepatocytes in primary culture were treated with 10 µM idoxifene or
-hydroxyidoxifene and their DNA analysed for adducts by the 32P-post-labelling procedure. The adducts, as 32P-labelled nucleoside bisphosphates, were separated on thin-layer plates and typical results are shown in Figure 3
. Treatment with idoxifene produced no adduct spots (A); the radioactivity on the plate was no greater than that from untreated cells. DNA from cells treated with
-hydroxyidoxifene gave a major spot just above the horizontal D3-axis (B). For comparison, a sample of the
-acetoxyidoxifene-treated salmon DNA with 1800 adducts per million bases (above) was also analysed by the 32P-post-labelling procedure, giving rise to the pattern shown in Figure 3C
. The main spot was in the same position as that in Figure 3B
, consistent with the probability that the spot in each case corresponds to the adduct idoxifenedeoxyguanosine bisphosphate. It has not been possible to correlate it specifically with the trans- or cis-adduct (IG1 or IG2); it probably contains both.
The adduct levels are given in Table II
. This also includes results with tamoxifen and
-hydroxytamoxifen which were carried out in parallel as a positive control; these were similar to those obtained in an earlier experiment (13). It is evident that even the activated derivative
-hydroxyidoxifene forms <10% as many adducts as the corresponding tamoxifen derivative.
Adducts in human hepatocytes
Human hepatocytes were treated in the same way with idoxifene, and their DNA analysed for adducts. No spots which might correspond to idoxifenenucleoside bisphosphate adducts were seen (Figure 3D
). Nor were such DNA adduct spots obtained from human hepatocytes treated with tamoxifen, in accord with earlier results (8).
Although there was no visible difference between the plates from control and idoxifene-treated cells, we measured the radioactivity on each thin-layer plate in the region where the adduct IG1-bisphosphate should have been, in comparison with Figure 3B and C
, and calculated the number of adducts that this would correspond to. The results are given in Table II
, together with those for tamoxifen. They show clearly that neither drug gave a level of adducts that could be detected within the sensitivity of our method. It is possible that adducts may have been observed at higher drug concentrations, but substantially higher concentrations were impractical because of their toxicity. It is also possible that the hepatocytes had during their isolation lost their capablility of metabolizing drugs and forming DNA adducts, but this is unlikely, because similar preparations gave adducts after treatment with benzo[a]pyrene (unpublished data).
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Discussion
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Idoxifene is very similar to tamoxifen in its chemical structure and the points of difference are distant from the central butene moiety which is believed to be important in the mechanism of bonding to DNA. So it is remarkable that idoxifene forms <1% as many DNA adducts as tamoxifen in rat hepatocytes (in the present study), or in rat liver in vivo (12). We have shown here that the presumed intermediate metabolite
-hydroxyidoxifene forms only 10% as many adducts in rat hepatocytes as
-hydroxytamoxifen does. So it appears that in rat liver at least, this metabolite is probably produced to a much lower extent than with tamoxifen and, when formed, gives rise to much fewer adducts.
This second difference can be better understood by looking at
-acetoxyidoxifene, as a model for the ester involved in reaction with DNA. We have shown that it reacts with DNA as the tamoxifen analogue does and gives adducts which probably have structures similar to those of the tamoxifen adducts. However, it is only about a third as reactive as
-acetoxytamoxifen and gives only a fifth to a quarter as many adducts. This accords with the theoretical prediction (24) that the 4-iodo substituent in idoxifene should withdraw electrons from the aromatic groups and make the formation of an
-carbocation less favourable.
No DNA adducts were detected in human hepatocytes treated with tamoxifen, so that it was not surprising that no adducts were found in human hepatocytes treated with idoxifene either. Reaction with DNA in human cells is less probably because metabolism to the
-hydroxy-derivative is less; concentrations of
-hydroxytamoxifen in the culture medium of cells treated with tamoxifen were ~50x less in human than in rat liver cells (8).
The results described here, taken together with earlier experiments on DNA adducts in rat liver (12), indicate that idoxifene is unlikely to induce DNA adducts in humans to any significant extent. It should therefore be a safer alternative to tamoxifen as a tumour preventive agent.
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Acknowledgments
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We thank Dr Bernard Nutley for the mass spectra and Dr Derry Wilman for the NMR spectrum. The work was funded by the Cancer Research Campaign, UK, and in part by SmithKline Beecham plc.
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Notes
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3 To whom correspondence should be addressed Email: martino{at}icr.ac.uk 
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Received July 29, 1998;
revised October 5, 1998;
accepted October 5, 1998.