Tamoxifen induces G:C->T:A mutations in the cII gene in the liver of lambda/lacI transgenic rats but not at 5'-CpG-3' dinucleotide sequences as found in the lacI transgene

Reginald Davies, Timothy W. Gant, Lewis L. Smith and Jerry A. Styles1

MRC Toxicology Unit, Hodgkin Building, University of Leicester, PO Box 138, Lancaster Road, Leicester LE1 9HN, UK


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Tamoxifen, a rat liver carcinogen, can induce mutations in the lacI gene in the livers of lambda/lacI transgenic rats. However, the presence of persistent tamoxifen adducts on the liver DNA raises the possibility that some contribution to the mutagenesis from ex vivo mutations during the in vitro lacI assay cannot be ruled out. To address this issue, mutagenesis at the cII gene of the transgenic shuttle vector was determined using a selection based assay which is unaffected by the presence of tamoxifen–DNA adducts. Female lambda/lacI transgenic rats were dosed orally with tamoxifen (20 mg/kg body wt) daily for 6 weeks, causing a 3.2-fold increase in the mutant frequency (MF) in the cII gene compared with that obtained with solvent treated animals. This was similar to the MF found previously at the lacI gene and confirms that tamoxifen is mutagenic in vivo. The major class of mutation induced by tamoxifen in the cII gene was G:C->T:A transversions as was found previously in the lacI gene. However, in the one unreplicated study of mutations in the p53 gene of liver tumours induced by tamoxifen, no G:C->T:A transversions were found; possible differences between mutagenesis in normal and tumour tissues are explored. The major proportion of the G:C->T:A transversions occurred at 5'-CpG-3' dinucleotide (CpG) sites in the lacI gene, but not at such sites in the cII gene. The methylation of CpG sites greatly enhances the targeting of deoxyguanosine by carcinogens, thus this finding might be explained by differences in the methylation patterns at their respective CpG sites; however, nothing is known about the methylation status of either the lacI nor the cII gene in this transgenic rat. This study raises the important issue of which target genes (mammalian or transgenic) should be used as endpoints in mammalian mutagenesis assays.

Abbreviations: CpG, 5'-CpG-3' dinucleotide; MF, mutant frequency.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Tamoxifen is a non-steroidal antiestrogen that is widely used as adjuvant therapy for breast cancer (1), and has recently been shown to reduce the incidence of breast cancer in healthy women who have a higher than normal risk of getting the disease (2). Although tamoxifen is linked to increased rates of endometrial cancer in women (3), there is no current evidence to suggest that tamoxifen causes liver cancer in humans. However, the compound causes liver cancer in rats (4), and has been shown to induce mutations at the lacI gene in the livers of lambda/lacI transgenic rats (5). Transgenic rodents carrying chromosomally integrated bacteriophage lambda shuttle vectors offer a powerful tool for studying mutagenesis in vivo (6,7). The relevance of lambda/lacI transgenic rodents in toxicological studies depends on the relationship between the mutagenicity found for a particular compound and its subsequent carcinogenesis or pathogenesis in the species. Of some interest then is the relationship between the mutational spectrum caused by a compound in these short term mutagenicity studies and the mutational spectrum found in the tumours. The major class of mutation induced by tamoxifen at the lacI gene was G:C->T:A transversions (5), however, no G:C->T:A transversions were found in the p53 gene in rat liver tumours induced by tamoxifen (11). We thought that it was important to examine mutations in a separate gene in the shuttle vector to see if the mutational spectrum was comparable with that found at the lacI gene.

Tamoxifen treatment of rats gives rise to persistent adducts on liver DNA (8), and, from theoretical considerations, adducts might give rise to ex vivo mutations as a result of mutational mechanisms operating during replication of the lambda phage in the bacterial assay for mutations in the lacI gene. Such ex vivo mutations can be recognised by a characteristic `sectored' plaque morphology, and whilst such plaques were a negligible proportion of the tamoxifen-induced mutant plaques found previously (5), it was considered essential to establish that mutations arising as a result of treatment with tamoxifen originated in vivo rather than ex vivo.

Recently a novel selection-based assay for mutants in the cII gene of the lambda phage shuttle vector was described which circumvents the problem of persistent DNA adducts (9). The cII protein is a positive regulator of gene transcription that plays a pivotal role controlling the lytic versus lysogenic development pathways of the phage in infected cells (10). Using hfl negative Escherichia coli host cells, the cII protein is stabilised and, under different growth conditions, phages carrying respectively mutant and non-mutant cII genes give rise to plaques, and hence the MF at the cII gene may be determined. The lytic or lysogenic decision takes place before any DNA replication, therefore pre-existing adducts on the recovered lambda phage DNA do not become fixed unless there is already a mutation in the cII gene that allows lytic growth. Thus, ex vivo mutations due to the presence of tamoxifen adducts are unlikely. Therefore, a study of mutagenesis in the cII gene of the shuttle vector could confirm our previous findings at the lacI gene, and give new information on the mutational spectrum induced by tamoxifen in vivo.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chemicals
Tamoxifen citrate (Z-2-[4-(1,2-diphenyl-1-butenyl)phenoxyl]-N,N-dimethyl ethanamine) was a generous gift from Zeneca Pharmaceuticals, Alderley Park, Macclesfield, Cheshire, UK.

Animal studies
In study 1, 6–8-week-old female lambda/lacI transgenic rats, (Big BlueTM, Stratagene, La Jolla, CA) rats (four or five per group) were treated with tamoxifen (dissolved in tricaprylin) at a dose of 20 mg/kg body wt/day p.o. for 6 weeks, and a control group received tricaprylin (1 ml/kg/day p.o.) for 6 weeks. The animals were killed 2 weeks after the last dose and livers stored at –80°C until DNA isolation. An additional group of rats received a single dose of aflatoxin B1 (0.5 mg/kg p.o.) 2 weeks before being killed. In study 2 the tamoxifen dose was lowered to 10 mg/kg/day p.o. and the study carried out as study 1. (Full details can be found in ref. 5.)

Mutagenesis assay at the cII gene.
High molecular weight DNA was prepared from liver using a RecoverEaseTM DNA isolation kit (Stratagene). The bacteriophage {lambda} transgene was recovered from the DNA by incubating with in vitro {lambda} packaging extract (Transpack, Stratagene). The cII selection assay was a modification of that described by Jacubczak et al. (9). The cII protein regulates the transcription of genes necessary for lysogenic growth (10); in the absence of the cII protein the cell undergoes lytic growth. The intracellular concentration of cII protein is controlled not only at the level of transcription, but at the level of protein stability. The E.coli host strain (G1225) used for the selection assay carries mutant hflA and hflB protease genes that facilitate the lysogenic response by increasing the stability of the cII protein (12). On infection of the hfl host strain and incubation at 24°C, lambda phages bearing non-mutant cII genes will undergo lysogenic growth, but phages with mutant cII genes will undergo lytic growth and give rise to plaques. Because the lambda LIZ shuttle vector contains a temperature sensitive mutation (c1857) in the cI gene, the cI protein (which is essential for lysogeny) is not functional at 37°C, so all phages give rise to plaques under the non-selecting conditions at 37°C. To determine the total titer of packaged phage, 50 µl of a 200-fold dilution of the packaged phages was preadsorbed on to 200 µl G1225 hfl E.coli cells (Epicentre Technologies, Madison, WI) (OD600 = 0.5), mixed with top agar and poured on to 90 mm TB1 (tryptone–vitamin B1) plates and incubated at 37°C for 24 h. Phages containing mutations in cII were identified by preadsorbing 100 µl of packaged phage particles on to G1225 cells (as above) and incubated at 24°C for 48 h. At least 630 000 plaques were screened per rat for each group. Mutant plaques were scored and stored in 0.5 ml SM buffer and confirmed by replating under the selecting conditions. The ratio of mutant cII genes to non-mutant cII genes determines the mutant frequency (MF) at the cII gene.

DNA sequence analysis
cII mutant plaques
. A proportion of mutant cII plaques were sequenced from each animal. {lambda} cII mutant plaques were picked at random and purified on G1225 cells by single plaque isolation. To prepare phage template DNA some of the plaque was removed and boiled in 40 µl H2O. The cII gene was PCR-amplified with the ExpandTM High Fidelity PCR system (Roche Diagnostics, Lewes, UK) using 10 µl template. The primers used were 5'-CTTGTCTGCGACAGATTCCT-3' and 5'-CCTCTGCCGAAGTTGAGTAT-3'. A touchdown PCR procedure was used with the annealing temperature decreasing from 62 to 53°C over 10 cycles, followed by a further 25 cycles using 52°C as annealing temperature. The PCR products were purified on Centricon 100 columns (Millipore). Sequencing reactions were performed with the Applied Biosystems prism dye-terminator cycle sequencing kit (Perkin Elmer) and analysed on an Applied Biosystems model 373 automated sequencer. The primers used were 5'-CCGCTCTTACACATTCCAGC-3' and 5'-CCTCTGCCGAAGTTGAGTAT-3'. To correct for an overestimate of mutagenesis due to clonal expansion of cells bearing mutant cII genes, duplicated mutations arising in a single animal but not observed in other animals were subtracted from the total. The mutation frequency is the MF minus these `jackpot' mutations. Thus, for control cII mutant plaques, 59 out of 60 mutants were included in the analysis; and for the tamoxifen-induced cII mutant plaques, 61 out of 62 mutants were included in the analysis.

lacI mutant plaques
. A proportion of mutant lacI plaques were sequenced from each animal. Template DNA was prepared as above and the lacI gene was PCR-amplified as above using 5'-GTACCCGACACCATCGAATG-3' and 5'-GAGTCACGACGTTGTA-3' as primers. A touchdown PCR procedure was used with the annealing temperature decreasing from 59 to 50°C over 10 cycles, followed by a further 26 cycles using 49°C as annealing temperature. The PCR products were purified and sequencing was carried out as above using appropriate primers until a mutation was detected on the lacI gene. The mutation frequency was determined as above. For control lacI mutant plaques, 53 out of 58 mutants were included in the analysis; and for the tamoxifen-induced lacI mutant plaques, 52 out of 54 mutants were included in the analysis.

Statistical analyses of MF were carried out using a one-way ANOVA using Minitab version 10 (Minitab, State College, PA), and data are presented as means ± SD. Data on the spectrum of mutations were analysed using a Monte Carlo estimate of Fisher's Exact Test.


    Results
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 Materials and methods
 Results
 Discussion
 References
 
Tamoxifen treatment of rats at 20 mg/kg body wt orally, daily, for 6 weeks caused a 3.2-fold increase in MF at the cII gene from liver compared with solvent treated controls (Figure 1Go) (P < 0.0005). This compares with a 2.9-fold increase at the lacI gene in the same experiment (5). Aflatoxin B1 treatment caused a significant increase in MF at the cII gene compared with that obtained from solvent treated controls as it did at the lacI gene (5). In a second study, tamoxifen treatment of rats at the lower dose of 10 mg/kg body wt, daily, for 6 weeks caused a 1.9-fold increase in MF at the cII gene compared with solvent treated controls, however this was not statistically significant (P = 0.053). This is a smaller fold increase than that found at the lacI gene (5) where tamoxifen caused a statistically significant 3.4-fold increase in MF at the lacI gene in the same study.



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Fig. 1. MF at the cII gene in the livers of lambda/lacI transgenic rats treated with various compounds. The animals were treated for 6 weeks at the doses indicated and killed 2 weeks after the last dose. The data are shown as means. AB1, aflatoxin B1; bars, SD; *P < 0.0005.

 
The type and location of mutations at the lacI gene have been published elsewhere (5) and additional new data are shown in Table IGo. To compare mutagenesis at both the lacI and cII genes the contribution of the various types of mutations to the overall mutation frequency were determined. The lacI data from Davies et al. (5) and Table IGo was combined and reanalysed and Figure 2Go shows the contribution of the various mutations to the overall mutation frequency at the lacI gene. Tamoxifen caused a significant 7.9-fold increase in G:C->T:A transversions compared with that found in control mutant plaques (P = 0.0037).


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Table I. The type and location of mutations in the lacI gene from control-derived mutants
 


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Fig. 2. The contribution of various mutational classes in the lacI gene to the mutation frequency of control and tamoxifen-treated rats. DNA sequence analysis of 53 control (white bars) and 52 tamoxifen (black bars) derived lacI mutants were included in the analysis. f+1, Frameshift as a result of the addition of a single base pair; f–1, frameshift as a result of the deletion of a single base pair; *P < 0.0037.

 
Table IIGo shows the type and location of mutations on the cII gene. Figure 3Go shows the contribution of the various types of mutations in the cII gene to the overall mutation frequency. Tamoxifen caused a significant 7.6-fold increase in G:C->T:A transversions compared with that found in control mutant plaques (P = 0.033). A common mutational event found in both control and tamoxifen-derived mutant plaques was insertion or deletion of a single G:C base pair at a run of six consecutive G:C base pairs which start at nucleotide number 179 in the cII gene. 10.2% of control and 16.4% of tamoxifen-derived mutant cII plaques did not have mutations in the cII gene. Tamoxifen did not cause a significant increase in insertions of single base pairs or deletions of G:C base pairs at the cII gene as was found in the lacI gene (5).


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Table II. The type and location of mutations in the cII gene
 


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Fig. 3. The contribution of various mutational classes in the cII gene to the mutation frequency of control and tamoxifen treated rats. DNA sequence analysis of 59 control (white bars) and 61 tamoxifen (black bars) derived cII mutants were included in the analysis. (G:C)6, frameshift mutations between positions 179 and 185 of the gene; *P < 0.033; other details as for Figure 2Go.

 
Table IIIGo shows the sequence context of the G:C->T:A transversions induced in control and tamoxifen-derived plaques at both the lacI and cII genes. For control and tamoxifen treatments 86% and 65%, respectively, of the G:C->T:A transversions in the lacI gene were found at CpG sites. However, none of the G:C->T:A transversions induced by tamoxifen at the cII gene were at CpG sites. Thus, tamoxifen caused significantly more G:C->T:A transversions at CpG sites in the lacI gene than in the cII gene (P < 0.00005). The major difference between the control and tamoxifen-induced G:C->T:A transversions at the lacI gene was that 75% of the tamoxifen-induced transversions had a G:C base pair at the 3' position relative to the transversion. No such G:C base pair (at the 3' position relative to the transversion) was found for the G:C->T:A transversions induced by tamoxifen at the cII gene. The proportions of G:C->T:A 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; this suggests that there was no strand preference for tamoxifen adduct formation.


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Table III. Sequence context of G:C->T:A transversion mutations
 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Tamoxifen induces mutations in the lacI gene recovered from the livers of lambda/lacI transgenic rats (5), and we now show that the MF induced by tamoxifen at the cII gene in the lambda shuttle vector was similar to that induced at the lacI gene. As DNA synthesis takes place subsequent to the transcription of the cII gene it seems unlikely that the mutations detected at this gene were due to the bacterial fixation of tamoxifen adducts known to be present on rat liver DNA. Thus, it seems certain that the mutations induced by tamoxifen occurred in the liver and were not ex vivo artefacts.

The cII selection assay is a promising alternative to the lacI mutagenesis assay as it can be performed more rapidly, and is relatively inexpensive. However, a significant contributor to mutagenesis at the cII gene was frameshift mutations at a run of six G:C base pairs between nucleotide numbers 179 and 185 of the gene. This may be due to slippage of DNA polymerase at nucleotide repeats during DNA synthesis (13). This frameshift mutation was the largest single mutational group amongst the mutants derived from control animals, and may, by increasing the apparent spontaneous mutation frequency, have the effect of reducing the sensitivity of the cII assay, compared with the lacI assay, for mutagenicity studies. This may explain the low increase in MF induced by treating rats with 10 mg tamoxifen/kg body wt compared with the control values. Indeed, recently it was shown that dimethylnitrosamine treatment of mice gave a 6.3-fold increase in MF at the lacI gene but only a 2.7-fold increase at the cII gene, the spontaneous MF at the cII gene being 5.6-fold higher than the corresponding MF at the lacI gene (14).

The major difference between the control and tamoxifen-induced G:C->T:A mutations in the lacI gene was the finding that, for 75% of the tamoxifen-induced mutations, a G:C base pair was found at the 3' position relative to the transversion. A possible explanation for this observation is that adjacent G:C base pairs may influence the position of tamoxifen adducts on the rat liver DNA. Alternatively, the location of tamoxifen adducts at the 3' position relative to the transversion may direct the mutagenesis. In this context it has been shown that a significant fraction of aflatoxin B1-induced G:C->T:A mutations occurred at the base 5' to the adducted guanine (15).

The major mutational event induced by tamoxifen in both the lacI and the cII genes was G:C->T:A transversions which could have arisen by the fixation of tamoxifen adducts which can be formed on deoxyguanosine residues (16). An important issue raised in transgenic mutagenesis studies is the relationship between the mutational spectrum induced at the transgene in the target tissue and the mutational spectrum seen in the eventual tumours. Indeed, do compounds induce a mutational `fingerprint' that could be used to determine which agent may have caused a human tumour of unknown aetiology? It has been shown that 50% of rat hepatocellular carcinomas induced by tamoxifen had mutations in the p53 gene (11). Seventy-five percent of these mutations were A:T->G:C transitions in the second base of codon 231 and the remainder were non-miscoding G:C->A:T transitions in codon 294. These data have not been replicated elsewhere and some questions have been raised concerning the two apparent mutational hot spots found in this study in so far as erroneous amplification of pseudogene sequence could not be rigorously excluded based on the published methodology (17). Nevertheless, there are several possible explanations for the difference between the mutational spectra found in the tumour tissue and that which we observed in liver tissue in transgenic animals:

The DNA sequence context of the G:C->T:A transversions induced by tamoxifen showed that, whereas 65% of the transversions in the lacI gene were found at CpG sites, the G:C->T:A transversions induced at the cII gene were not at such sites. The lacI gene has, proportionally, a greater number of CpG sites (88 per 1000 bp) compared with the cII gene (51 per 1000 bp). In the cII gene 53% of the CpG sites are found in the last third part of the gene, and there is some evidence that mutations in the C-terminal region of the cII protein do not affect protein function severely (10). Thus, it appears that the lacI gene is more susceptible than the cII gene to mutagenesis at CpG sites. Mutagenesis is likely to be directed by the position of the tamoxifen–DNA adducts on the respective genes, for which there is no current information. Deoxycytosine methylation was found to greatly enhance guanine alkylation at all CpG dinucleotide sites in the human p53 gene by a variety of carcinogens such as benzo(a)pyrene diol epoxide, aflatoxin B1 8,9-epoxide and N-acetoxy-2-acetylaminofluorene (2225). Most CpG dinucleotides in the DNA binding domain of the lacI gene are methylated to a high extent in all of the lambda/lacI transgenic mouse tissues examined (26), however nothing is known about the methylation of CpG sites at the lacI or cII genes in the lambda/lacI transgenic rat. Thus, the apparent differences in mutation distribution at the CpG sites in the two genes could be explained if the lacI gene was methylated and the cII gene was not methylated in the rat liver.

CpG sites are important regulatory sequences for several tumour suppressor genes (27) and the major mutational hot spots at the p53 gene in human cancers occur at CpG sequences. A relative excess of spontaneous G:C->A:T mutations at the hprt locus occur at CpG sequences in human T-lymphocytes in vivo (28), and the predominant class of spontaneous mutations in the lacI transgene in a variety of tissues in lambda/lacI mice was G:C->A:T transitions most of which occurred at CpG sequences (29). These mutations are thought to arise from deamination of 5-methylcytosine residues located at CpG sites (30). Thus, methylated CpG sites are both an endogenous promutagenic factor and a preferential target for attack by exogenous chemical carcinogens (24). As the lacI gene contains proportionally more CpG sites than mammalian genes, the lacI gene may be an inappropriate mutational target since mutations at this gene could be an overestimate of both spontaneous (31) and chemical mutagenesis in the rat genome. However, recently it was shown that a reduction in the CpG content of the lacI gene did not reduce the rate of spontaneous mutation or the contribution of CpG related events (32). Conversely, if compounds did preferentially bind to CpG sites and mutations at these sites were causal events in tumourigenesis, then the use of a `sensitive' gene such as lacI in in vivo mutagenesis studies could be a very appropriate target for detecting potential carcinogens.

A recent report relevant to this study was the finding that the hydroxysteroid sulfotransferase a from rat liver metabolises {alpha}-hydroxytamoxifen (a major phase I metabolite of tamoxifen) to a form which makes DNA adducts and mutations; similar studies with all six known human xenobiotic-metabolising sulphotransferases failed to show similar mutagenic transformation (33). The authors conclude that the risk of DNA adduct formation, and cancer, in the human liver is low and explains why tamoxifen is a powerful carcinogen in the rat liver. With regard to the human endometrium, cytochrome P450 mRNAs transcripts which are potentially capable of metabolising tamoxifen–DNA adducts have been detected (34) and tamoxifen-induced DNA adducts have been found in human endometrium (35). In our hands tamoxifen failed to induce mutations at the lacI gene or adducts in rat uterus DNA derived from the animals used in this study (J.A.Styles, R.Davies, S.Fenwick, J.Walker, E.A.Martin, I.N.H.White and L.L.Smith, in preparation).


    Acknowledgments
 
We acknowledge Philip Carthew and Michael Festing for stimulating discussions, and Stuart Baylis, Joseph Walker and Heather Piercy for excellent technical assistance.


    Notes
 
1 To whom correspondence should be addressed Email: jas12{at}le.ac.uk Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received January 26, 1999; revised March 31, 1999; accepted April 1, 1999.