The mutational signature of
-hydroxytamoxifen at Hprt locus in Chinese hamster cells
Masoud Yadollahi-Farsani1,
Donald S. Davies and
Alan R. Boobis
Department of Health Toxicology Unit, Section on Clinical Pharmacology, Division of Medicine, Imperial College, London W12 0NN, UK
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Abstract
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The anti-oestrogen tamoxifen is very effective in the treatment and prevention of breast cancer. Tamoxifen is not a pure antagonist, but possesses weak oestrogenic activity that may contribute to a slightly increased risk of endometrial cancer. Whilst this can be incorporated into riskbenefit analysis for the use of the drug, residual concerns exist over the exact mechanism of formation of these tumours. Tamoxifen is a potent hepatocarcinogen in the rat, probably via a genotoxic mechanism. Whilst tamoxifen does not appear to cause liver tumours in humans, DNA adducts have been found in endometrial tissue of women receiving the drug. Hence, there is still a need to establish the mechanism of formation of these tumours. We have therefore determined the molecular nature of mutations induced in vitro by
-hydroxytamoxifen, the putative proximate genotoxic metabolite, in a mammalian cell line (V79-rHSTa) with stable expression of rat hydroxysteroid sulfotransferase a, which catalyses the further metabolism of
-hydroxytamoxifen to its ultimate genotoxic product. DNA sequence alterations were examined at the Hprt gene in 50 mutant clones. Simple base substitutions, mainly GC
TA transversions, predominated. However, single G:C base pair deletions and partial/complete exon skippings were also observed. All but one of the mutations involved guanine bases on the non-transcribed strand, probably indicating preferential repair of
-hydroxytamoxifen-induced guanine adducts from the transcribed strand. Nearest neighbour analysis of the mutations (on the non-transcribed strand) indicated that thymines (20/40) followed by guanines (13/40) were the most frequent 5' neighbours, with adenines or guanines the most frequent 3' neighbours. Many of the mutations occurred at TTGA/G sequences. Three mutational hot spots accounted for 11 GC
TA transversions and another site for two single G:C base pair deletions. A search for these characteristic mutations in tumour-related genes of treated rats and humans should help in understanding the mechanism(s) of tamoxifen-induced carcinogenicity.
Abbreviations: DMEM, Dulbeccos modified Eagle medium; Hprt, hypoxanthine-guanine phosphoribosyltransferase; rHSTa, hydroxysteroid sulfotransferase a.
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Introduction
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The non-steroidal anti-oestrogenic drug, tamoxifen has been used for the treatment of patients with advanced breast cancer for >20 years. It has also been used as adujuvant therapy for early stage breast cancer and its prophylactic use in healthy, high-risk women is increasing (1). The widespread therapeutic and newly emerging prophylactic use of tamoxifen has given rise to concerns about the side effects of the drug, particularly its carcinogenicity.
Tamoxifen is a potent hepatocarcinogen in rats (2) but not in humans. It gives rise to high levels of characteristic DNA adducts in rat liver (3,4) and hepatocytes (5) and induces cytogenetic damage in rat liver in vivo (6). Tamoxifen fails to form DNA adducts in human hepatocytes in vitro (7). This is because of major species differences in the enzymes involved in its activation and detoxification. Consistent with this, the balance of evidence suggests that tamoxifen is not hepatocarcinogenic in humans (8).
The formation of DNA adducts in rat liver is thought to proceed via the formation of
-hydroxytamoxifen (5,7,9) which undergoes phase II metabolism to a more reactive and DNA adduct-forming species by rat, but not human, sulfotransferase (10). These DNA adducts have been found to be pro-mutagenic in bacteria as well as in mammalian cells (10).
Although tamoxifen clearly forms DNA adducts, mutations and tumours in rat liver, the situation in other tissues is less clear. The drug increases the incidence of human endometrial cancer by a factor of 210, depending on the duration of treatment and the age of the patient, as well as other factors (1113). In addition, tamoxifen gives rise to an increased incidence of uterine adenocarcinoma when administered neonatally to rats or mice or transplacentally to mice (14,15). Squamous cell carcinoma of the endometrium has also been reported after a 6-month exposure of rats to tamoxifen (16,17). The mechanism of induction of uterine cancer by tamoxifen is not known. However, there are two possibilities: either tamoxifen acts as a tumour promoter because of its weak oestrogen agonist activity on the endometrium or it acts as a tumour initiator via a metabolite(s) that acts as a genotoxicant(s), damaging DNA (1).
In a study by Gamboa da Costa et al. (18), no increase in the frequency of mutations of the lacI transgene could be detected in the uterus of Big Blue rats treated with tamoxifen or
-hydroxytamoxifen (18). Also, the ability of tamoxifen to induce uterine adenocarcinoma in mice following neonatal treatment (14) but not following administration to adult animals (15) is suggestive of a mechanism involving hormonal perturbation of the developing organ (1) as does the time for tumour occurrence in women treated with the drug (19). However, there is residual concern because of some reports of tamoxifen-derived adducts in endometrial tissue of treated patients. TamoxifenDNA adducts have been detected in endometrium obtained from patients treated with the drug and are identified as the cis- and trans-forms of
-(N2-deoxyguanosinyl)tamoxifen (20,21). Although these results are disputed by others who have found no evidence of adduct formation in human endometrium (1), if present,
-(N2-deoxyguanosinyl)tamoxifen adducts have miscoding potential (22) and are subject to nucleotide excision repair (23).
From the foregoing, it is apparent that identification of a mutation fingerprint for tamoxifen could be of considerable value in the risk assessment of the compound. The aim of the present study was to determine the molecular nature of the mutations induced by
-hydroxytamoxifen at the hypoxanthine-guanine phosphoribosyltransferase (Hprt) gene in a Chinese hamster V79 cell line genetically constructed to express stably rat hydroxysteroid sulfotransferase a (rHSTa), an enzyme that can convert
-hydroxytamoxifen to the ultimate genotoxic species, with high efficiency (10).
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Materials and methods
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Chemicals
-Hydroxytamoxifen was generously provided by Prof. David Phillips, Institute of Cancer Research, Surrey. Dulbeccos modified Eagle medium (DMEM) and fetal bovine serum were from Gibco BRL (Paisley, UK) and Imperial (Andover, UK), respectively. AMV reverse transcriptase, ribonuclease inhibitor and oligo(dT)15 primer were from Promega (Southampton, UK). All other reagents, except where indicated, were from Sigma-Aldrich Co. (Poole, UK).
Cell lines
The recombinant Chinese hamster V79 cell lines, V79-rHSTa and V79hHST were generously provided by Professor H.Glatt (German Institute of Human Nutrition, Potsdom-Rehbrocke, Germany). These cell lines had been genetically constructed to express stably rat hydroxysteroid sulfotransferase a (V79-rHSTa) and the corresponding human sulfotransferase (V79hHST), respectively (24,25).
Culture conditions
The V79-rHSTa and V79hHST cell lines were cultured in DMEM supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 IU/ml penicillin and 100 µg/ml streptomycin and maintained at 37°C in a humidified atmosphere of 5% CO2/95% air.
-Hydroxytamoxifen treatment and mutant selection
Cells in exponential phase of growth (1.5x106) were plated on to 175 cm2 flasks in 30 ml of culture medium 18 h before treatment. The volume of medium was reduced to 20 ml, and
-hydroxytamoxifen (40 µM) dissolved in 40 µl of DMSO, or the solvent alone, was added. The choice of concentration of
-hydroxytamoxifen for use in these studies was based on published data using the same cell system (10). The exposure was terminated after 24 h by a change of medium. Two days later, the cells were detached by treatment with trypsin. Their number, expressed as a percentage of the corresponding value of the solvent-treated control cultures, was used as a measure of the cytotoxicity of the treatment. The cells were subcultured in normal medium for 3 days and then subcultured again using 6-thioguanine-supplemented medium for the selection of Hprt mutants (5x105/10 cm Petri dish; 12 dishes). The plating efficiency of cells at the time of selection was also determined in normal culture medium to correct the observed mutant frequencies. After 12 days, the colonies were counted and mutant frequencies were calculated. Ethyl methanesulphonate (50 mg/ml), a direct-acting mutagen, was used as a positive control compound. Viable colonies were randomly picked from the control and
-hydroxytamoxifen-treated cultures for sequence analysis. The Hprt mutant cells were grown in culture medium supplemented with 5 µg/ml 6-thioguanine for a further week and harvested for immediate sequence analysis.
Oligonucleotide primers
The sequence of oligonucleotide primers, used in PCR amplification and direct nucleotide sequencing of the Chinese hamster Hprt gene, were based on the cDNA sequence reported by Konecki et al. (26).
PCR
Forward: 895'-TAC CTC ACC GCT TTC TCG TG-3'70;
Reverse: 7025'-TGA ATG GGA CTC CTC GTG TT-3'683.
Sequencing
505'-GGC TTC CTC CTCACA CCG CT-5'29;
6965'-GGA CTC CTC GTG TTT GCA GA-5'677.
Reverse transcriptionPCR
Total cellular RNA was extracted from 2 to 5x106 cells. One microgram of this RNA was denatured at 70°C for 10 min and quick chilled on ice; reverse transcription was carried out in 20 ml of PCR buffer (10 mM TrisHCl, pH 9.0 at 25°C/50 mM KCl/1.5 mM MgCl2/10 ng BSA/ml) containing 0.5 µg of oligo(dT)15 primer, each dNTP at 1 mM, 20 U of ribonuclease inhibitor and 15 U of AMV reverse transcriptase. After 1 h of incubation at 50°C, the reaction was stopped by heating at 95°C for 5 min and was quick chilled. The first strand cDNA corresponding to the entire Hprt coding region was amplified by PCR by adjusting 10 µl of the reaction mixture to 80 µl with PCR buffer, 50 pmol each of forward and reverse primer and 2.5 U Taq DNA polymerase. Amplification was performed for 30 cycles with denaturation at 94°C for 1 min, annealing at 55°C for 1 min and polymerization at 72°C for 2 min. The initial denaturation step was extended by 4 min and the final polymerization step by 7 min. On completion of PCR, 10 µl of product were examined for successful amplification on 1.4% agarose gel stained with ethidium bromide in TBE [89 mM Trisborate, 2 mM EDTA (pH 8.0)] buffer.
Purification and direct sequencing of PCR products
PCR products were purified using the Wizard PCR Preps DNA Purification System (Promega) and used directly for nucleotide sequencing. Two sequencing primers, indicated above, were used to determine the nucleotide sequence of both strands of Hprt cDNA.
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Results
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Survival and mutant frequency
Exponentially growing populations of Chinese hamster V79 cells with stable expression of rat hydroxysteroid sulfotransferase a (V79rHSTa) or human hydroxysteroid sulfotransferase (V79hHST) were treated with 40 µM of
-hydroxytamoxifen for 24 h and assayed for survival and frequency of Hprt mutants as reported previously (10). In contrast to V79hHST cells, which showed no mutagenic or cytotoxic response to
-hydroxytamoxifen, V79rHSTa cells showed a reduction in survival (47% of control) and an increase in mutant frequency at the Hprt locus (Figure 1
). The frequency of Hprt mutants per million clonable V79rHSTa cells treated with 40 µM of
-hydroxytamoxifen was 109.3 ± 2.6, which was about 11 times higher than the background frequency of 9.4 ± 1. In contrast, the two cell lines showed similar survival (
93% of controls) and mutant frequency (Figure 1
) when treated for 24 h with 50 µg/ml of the direct-acting mutagen, EMS.
Determination of the molecular nature of hprt mutations
Individual Hprt mutant colonies were selected randomly from three independent experiments performed on different occasions using V79rHSTa cells treated with 40 µM of
-hydroxytamoxifen or with 0.2% DMSO as solvent control. The molecular nature of Hprt mutations in V79rHSTa cells was investigated using cDNA products, obtained by reverse transcriptionPCR, as template for nucleotide sequencing. Of the 50 mutants from
-hydroxytamoxifen-treated cells investigated, two failed to produce any Hprt cDNA PCR products. Of the remaining Hprt cDNAs, one contained a complex mutation comprising two closely spaced base substitutions and another eight contained partial or complete exon deletions (Table I
). The Hprt cDNA sequences from 33 mutants had single base alterations of missense type and four others of nonsense type (Table I
). The remaining two mutants had frameshifts caused by a single GC base pair deletion (Table I
).
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Table I. Types and positions of -hydroxytamoxifen-induced mutations in the Hprt coding region of V79-rHSTa cells and comparison with PhIP-induced mutations
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Table II
shows the types of spontaneous mutations found in the solvent-treated controls. Eight of nine spontaneous mutants that could be amplified by PCR of Hprt cDNA were subsequently sequenced. These comprised six simple base substitutions of missense type and two putative splice mutants with complete exon skip/exclusion.
Specificity of
-hydroxytamoxifen-induced mutations
Single base substitutions comprised the major class of mutation found at the Hprt locus of V79rHSTa cells treated with
-hydroxytamoxifen. Transversions predominated, with GC
TA accounting for the majority of base substitutions. All of the mutations identified to date at the Hprt locus of
-hydroxytamoxifen-treated cells involved GC base pairs, the guanine being on the non-transcribed strand (Table I
). This is in marked contrast to the spontaneous Hprt mutations in the solvent-treated control samples that involved A:T base pairs.
Site specificity of
-hydroxytamoxifen-induced mutations
Nearest neighbour analysis of
-hydroxytamoxifen-induced mutations at guanines on the non-transcribed strand indicated that thymines (20/40) followed by guanines (13/40) were the most frequent 5' neighbours. Adenines or guanines were the most frequent 3' neighbours. A large number of the mutations occurred at guanines in 5'-TTGA/G sequences (Table I
). Three mutational hotspots for GC
TA transversions and a site for two single G:C base pair deletions involved a guanine in a 5'-TTG sequence context (Table I
, Figure 2
).
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Discussion
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-Hydroxytamoxifen, a phase I metabolite of the anti-estrogenic drug tamoxifen, induces a significant increase in the frequency of Hprt mutations in genetically engineered V79rHSTa cells which stably express the rat hydroxysteroid sulfotransferase (rHSTa) gene.
-Hydroxytamoxifen is also cytotoxic to these cells. No cytotoxic or mutagenic response is, however, observed in V79hHST cells that express the human hydroxysteroid sulfotransferase (hHST) gene. In contrast, the two cell lines showed similar mutagenic and cytotoxic responses to a direct-acting alkylating agent, EMS indicating that the difference in the effects of
-hydroxytamoxifen in the two cell lines is due to metabolism by the rat sulfotransferase. This is consistent with a previous study indicating that in contrast to the rat hydroxysteroid sulfotransferase (rHSTa), the human enzyme shows very little ability to metabolize
-hydroxytamoxifen to a DNA adduct-forming, mutagenic species (10).
With the exception of a few partial/single exon skip mutations and two single G:C base pair deletions, the
-hydroxytamoxifen-induced mutations identified in this study involved simple base substitutions. Although GC
TA transversions were the most frequent, a significant number of GC
CG transversions and GC
AT transitions were also observed. However, similar to our previous study (27), which also involved a Chinese hamster V79 cell line, spontaneous Hprt mutations in the solvent-treated control samples were found to be predominantly transversions or transitions at A:T base pairs.
The spectrum of
-hydroxytamoxifen-induced mutations identified in the present study in the V79rHSTa cell line is broadly in line with those found at LacI and cII transgenes in the livers of transgenic rats administered tamoxifen (28,29). The major class of mutation induced by tamoxifen at the LacI and cII genes was GC
TA transversion, although a significant number of GC
AT transitions, GC
CG transversions and single or multiple guanine base deletions were also observed. GC
TA transversions are also the predominant mutations induced when
-(N2-deoxyguanosinyl)tamoxifen-adducted DNA is replicated in simian kidney (COS-7) cells (30).
Nearly all of the base substitutions in our study are at GC pairs suggesting that the pre-mutagenic adducts induced by
-hydroxytamoxifen involve guanine and/or cytosine. It has been shown that
-hydroxytamoxifen itself has only low reactivity with DNA in vitro (31) and the formation of tamoxifenDNA adducts is inhibited by sulfotransferase inhibitors (32). More recently, it has been shown that
-sulfate tamoxifens are highly reactive with DNA, forming four diastereoisomers of
-(N2-deoxyguanosinyl)tamoxifen (dG-N2-tamoxifen): the epimeric center of each trans- and cis-form of dG-N2-tamoxifen is at the
-carbon (33). The trans-forms of dG-N2-tamoxifen were found to be the major adducts in the liver of rats treated with tamoxifen (34). The miscoding specificities of each trans- and cis-form of dG-N2-tamoxifen adduct, during translational synthesis by mammalian DNA polymerases have been explored (33). The adducts allow high amounts of primer extension past the lesions and have strong miscoding potentials and predict G
T and G
C transversions and deletions in mammalian cells (33).
If the pre-mutagenic lesions associated with
-hydroxytamoxifen mutagenesis occur at guanine bases then nearly all mutations found in the present study must have arisen from the non-transcribed strand. This is in contrast to studies in lacI transgenic animals. The proportions of GC
TA transversions on the non-transcribed DNA strand for the LacI and cII genes were 45 and 53%, respectively, indicating that there was no strand bias for this mutation (28,29). These observations suggest that there is no strand preference for tamoxifen adduct formation and that there is no preferential repair of the tamoxifen adducts from one or the other of the strands of these genes. The difference between the present study and those using transgenic animals may arise from the fact that, in contrast to artificially incorporated transgenes, Hprt is a transcriptionally active housekeeping gene that is subject to preferential repair of the transcribed strand by nucleotide excision repair (3537). dG-N2-tamoxifen adducts have been shown to be removed by the mammalian nucleotide excision repair system, although with only moderate to poor efficiency (23). Whilst species differences in overall repair capacities could also explain the observed difference, strand bias has been observed in several species and for several transcribed genes (3541).
The majority of GC
TA transversions in the LacI gene were found at CpG sites. However, in our study, similar to that of the cII gene, none of the GC
TA transversions induced by
-hydroxytamoxifen are at CpG sites. Furthermore, in the study of tamoxifen-induced mutations in the LacI gene, for 75% of the GC
TA transversions, a G:C base pair was found at the 3' position relative to the mutated site (29). No such strong near neighbour 3' G:C effect was observed in the present study. However, a disproportionately large number of mutated guanines on the non-transcribed strand had thymines followed by guanines on the 5' side and a large number of base substitutions and the guanine base deletions involved a guanine in 5'-TTG sequences (Table I
, Figure 2
).
Comparison of the spectrum of
-hydroxytamoxifen-induced mutations found in the present study with that we identified previously for the dietary carcinogen PhIP (27) reveals similarities, but some major differences in site specificity (Table I
and Figure 2
). The targeting of mutations at guanine residues on the non-transcribed strand of Hprt by the two agents is to be expected, as both produce bulky adducts with guanines that are preferentially repaired from the transcribed strand of this functionally active housekeeping gene by the nucleotide excision repair pathway. The similarity of the types of mutations induced by these two agents can be explained by the similarity in their 3D structure [and both have oestrogenic activity (42)], the nature of the genotoxic electrophiles and that, according to current understanding, such bulky guanine adducts represent powerful promutagenic lesions, which promote nucleotide miss-insertion, preferably of adenines.
There are significant differences in the sequence context of mutations induced by
-hydroxytamoxifen compared with those induced by PhIP. Nearest neighbour analysis of mutations induced by
-hydroxytamoxifen reveals that thymines rather than guanines are the most frequent 5' neighbours and that most mutations occur in 5'-TTGA/G sequences rather than in 5'-GGA ones. Furthermore, in contrast to the PhIP signature G:C base pair deletion mutation that occurs at 5'-GGGA sites in endogenous marker genes (27), transgenes (43,44) and tumour-related genes of the rodents (45), the observed G:C base pair deletion with tamoxifen involves a 5'-TTG sequence. This indicates that the two agents exhibit different sequence preferences for adduct formation, even for adjacent guanine residues. Even closely located guanine bases have different electrostatic potentials that largely determine their reactivity. Another important factor in reactivity is steric accessibility. Neighbouring bases affect accessibility by influencing local DNA helix geometry. Modelling of the interaction with guanine residues in different sequence contexts may be of value. Despite the differences found in the context of guanine residues mutated by
-hydroxytamoxifen and PhIP, there are some striking similarities in the regions of the Hprt gene targeted by the two compounds (Figure 2
). Presumably, the local structure/activity of the DNA at these sites is such that ready access of bulky aromatic electrophiles is possible, whereas the reactivity and steric accessibility of the specific guanine residues dictate the actual site of adduction.
Vancutsem et al. (46) have reported a high frequency of p53 tumour suppresser gene mutations in tamoxifen-induced hepatocellular carcinomas of female SD rats. Of the 24 hepatocellular carcinomas examined nine contained an A
G transition in the second base of codon 231, resulting in a miscoding mutation. A further four contained a non-miscoding C
T transition in the third base of codon 294 and one tumour contained both mutations. No similar findings have been reported since the original publication. The mutations reported are quite different from those found in all other studies of tamoxifen-induced mutations. There is no obvious explanation for this. It is perhaps worth noting that, based on the published methodology, the possibility that the two apparent mutational hotspots were the result of erroneous amplification of p53 pseudogene sequences cannot be entirely ruled out (47). There are several other possible explanations to account for the observed differences between the tamoxifen-induced mutational spectrum at transgenes in the liver of treated rats and that observed in the eventual liver tumours (29).
A thorough search for tamoxifen signature mutations in genes known to be involved in the development and progression of relevant tumours in rats and humans could provide valuable information regarding its mechanism(s) of carcinogenicity and would contribute substantially to the risk-benefit analysis of the prophylactic use of tamoxifen and related compounds.
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Notes
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1 To whom correspondence should be addressed Email: y.farsani{at}ic.ac.uk 
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Acknowledgments
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We thank Prof. David Phillips, Institute of Cancer Research, Surrey, for the gift of
-hydroxytamoxifen. We are also very grateful to Prof. H.Glatt, German Institute of Human Nutrition, Potsdam-Rehbrocke, for the gift of V79-rHSTa and V79hHST cells. This study was executed with the financial support of The Department of Health (UK) and The UK Food Standard Agency.
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Received June 26, 2002;
revised August 29, 2002;
accepted August 30, 2002.