Mutations induced by
-hydroxytamoxifen in the lacI and cII genes of Big Blue transgenic rats
Tao Chen1,4,
Goncialo Gamboa da Costa2,
M.Matilde Marques2,
Sharon D. Shelton1,
Frederick A. Beland3 and
Mugimane G. Manjanatha1
1 Division of Genetic and Reproductive Toxicology, National Center for Toxicological Research, Jefferson, AR 72079, USA,
2 Centro de Química Estrutural, Complexo I, Instituto Superior Técnico, Av. Rovisco Pais, 1049-001 Lisboa, Portugal,
3 Division of Biochemical Toxicology, National Center for Toxicological Research, Jefferson, AR 72079, USA
 |
Abstract
|
---|
The antiestrogen tamoxifen is widely used for the treatment of breast cancer and more recently for the prevention of breast cancer. A concern over the use of tamoxifen as a chemopreventive agent is its carcinogenicity in rat liver, through a genotoxic mechanism involving
-hydroxylation, esterification, and DNA adduct formation, primarily by reaction with dG. In a recent study [Gamboa da Costa et al., Cancer Lett., 176, 3745 (2002)], we demonstrated a significant increase in the mutant frequency in the lacI gene of Big Blue rats treated with tamoxifen, and a further increase in rats administered
-hydroxytamoxifen. In the present study, we have assessed mutation induction by tamoxifen and
-hydroxytamoxifen in the liver cII gene of Big Blue rats and have characterized the types of mutations induced by
-hydroxytamoxifen in the liver lacI and cII genes. The mutant frequencies in the liver cII gene were 80 ± 13 x 106 in the control, 112 ± 13 x 106 in the tamoxifen-treated group (P < 0.01 vs. control), and 942 ± 114 x 106 in the
-hydroxytamoxifen-treated animals (P < 0.001 vs. control; P < 0.001 vs. tamoxifen). Molecular analysis of the mutants indicated that the
-hydroxytamoxifen-induced mutational spectrum differed significantly from the control spectrum, but was very similar to the spectrum induced by tamoxifen for both the lacI and cII genes [Davies et al., Environ. Mol. Mutagen., 28, 430433 (1996); Davies et al., Carcinogenesis, 20, 13511356 (1999)]. G:C
T:A transversion was the major type of mutation induced by
-hydroxytamoxifen and tamoxifen, while G:C
A:T transition was the main type of mutation in the control. These results support the hypothesis that
-hydroxytamoxifen is a major proximate tamoxifen metabolite causing the initiation of tumors in the liver of rats treated with tamoxifen.
 |
Introduction
|
---|
Tamoxifen has been widely used for adjuvant therapy in the treatment of patients with breast cancer for nearly 30 years, and many millions of women have been treated with this drug successfully. More recently the drug has been shown to reduce the incidence of breast cancer in healthy women who are at high risk of developing breast cancer (1,2). A concern over the use of tamoxifen as a chemopreventive agent is the induction of endometrial cancer in women, and liver and endometrial tumors in rats (37). The induction of liver tumors by tamoxifen in rats has been associated with a genotoxic mechanism involving DNA adduct formation and mutation induction (811). The mechanism by which endometrial tumors are induced by tamoxifen in women is not clear. Our recent data suggest that induction of endometrial tumors in rats is not due to the genotoxicity of tamoxifen (11,12); however, a recent study with monkeys demonstrated that non-human primates metabolize tamoxifen into genotoxic intermediates and that tamoxifenDNA adducts are detectable in various tissues including the uterus (13,14).
The hepatic metabolism of tamoxifen gives rise to a variety of metabolites. Although many putative reactive intermediates of tamoxifen have been proposed (reviewed in ref. 15), it is now generally believed that a substantial portion of the DNA adducts arise from a minor metabolite,
-hydroxytamoxifen, after conversion to a reactive sulfate ester by sulfotransferases (1619). The major DNA adduct resulting from this pathway is (E)-
-(deoxyguanosin-N2-yl)-tamoxifen, which is accompanied by minor amounts of the Z diastereomer and deoxyadenosine adducts (8,20).
Tamoxifen is a gene mutagen in rat liver, although it is not mutagenic in several regulatory short-term tests. Tamoxifen gave negative responses in the Salmonella typhimurium reversion assay and in the in vitro human lymphocyte chromosome aberration assay. It was only weakly positive in a modified in vivo/in vitro unscheduled DNA synthesis assay in rat hepatocytes (2123). In contrast to these short-term tests, tamoxifen increased mutant frequencies
3-fold over the background and induced mainly G:C
T:A transversion mutations in the liver lacI and cII genes of Big Blue rats (9,24,25). Short-term dosing of
-hydroxytamoxifen in a previous experiment, however, resulted in only a 1.8-fold increase in mutant frequency compared with the controls, with no significant increase in G:C
T:A transversion in the liver lacI gene of Big Blue rats (10).
In a recent study (11), we used a subchronic dosing regimen for treating Big Blue rats with tamoxifen or
-hydroxytamoxifen. Intraperitoneal administration of 21 daily doses of 54 µmol
-hydroxytamoxifen/kg body weight increased the lacI mutant frequency in the liver to a value 77-fold higher than background and 24-fold greater than that observed with tamoxifen. In the present study, we examined the mutation induction by tamoxifen and
-hydroxytamoxifen in the liver cII gene, and characterized the types of mutations induced by
-hydroxytamoxifen in the lacI and cII genes of rat livers.
 |
Materials and methods
|
---|
Treatment of animals and lacI mutation assay
Tamoxifen was purchased from Sigma (St Louis, MO).
-Hydroxytamoxifen was prepared by the method of Foster et al. (26). Details of the treatment of the rats and the lacI mutation assay are given elsewhere (11). Briefly, 8-week-old female Big Blue rats were treated intraperitoneally with 21 daily doses of 54 µmol tamoxifen/kg body wt, 54 µmol
-hydroxytamoxifen/kg body wt, or the solvent (trioctanoin). Six rats from each treatment group were killed one month after the last treatment. The livers were isolated, frozen, and stored at 80°C. DNA extracted from the liver samples was packaged into
vectors, and the infectious phages with vectors were plated to assay lacI mutant plaques as described previously (27,28).
cII Mutation assay
High-molecular-weight genomic DNA was extracted from the rat livers using the RecoverEase DNA Isolation Kit (Stratagene, La Jolla, CA) and stored at 4°C until DNA packaging was performed. The packaging of the
phage, plating the packaged DNA samples, and determination of mutant frequency were carried out following the manual for
select-cII mutation detection system for Big Blue rodents (Stratagene). The
shuttle vector containing the cII target gene was rescued from total genomic DNA with
phage packaging extract (Transpack; Stratagene). The plating was performed with the Escherichia coli host strain G1250. To determine the total titer of packaged phages, G1250 bacteria were mixed with 1:100 dilutions of phage, plated on TB1 plates, and incubated overnight at 37°C (nonselective conditions). For mutant selection, the packaged phages were mixed with G1250, plated on TB1 plates and incubated at 24°C for 42 h (conditions for
cII- selection). After incubation at 24°C,
phages with wild-type cII genes undergo lysogenization and become part of the developing bacterial lawn, whereas phages with mutated cII genes undergo lytic growth and give rise to plaques. When incubated at 37°C,
phages with wild-type cII genes also undergo a lytic cycle, resulting in plaque formation. The cII mutant frequency was the ratio of the number of mutant plaques (as determined at 24°C) to the total number of plaques screened (as determined at 37°C).
Sequence analysis of lacI and cII mutants
The template DNA preparation from lacI mutants was performed as previously described (27,29). Briefly, in vivo excision of LIZ was carried out by mixing a verified mutant phage stock with XL1-Blue cells and ExAssist helper phages. The pLIZ phagemids recovered from in vivo excision were selected by plating out with SOLR cells on LB plates containing ampicillin. A single ampicillin-resistant SOLR E.coli colony was selected and grown overnight in 3 ml of medium. The phagemid DNA containing the entire lacI gene was extracted using an ABI PRISM Miniprep Kit.
The cII target DNA for sequencing was amplified by PCR with two primers: 5'-AAAAAGGGCATCAAATTAACC-3' and 5'-CCGTTGAGTATTTTTGCTG-3'. The cII mutant plaques were selected at random from different animals and replated at low density to verify the mutant phenotype. Single well-isolated plaques were selected and transferred to a microcentrifuge tube containing 25 µl of autoclaved distilled water. The tube was placed in boiling water for 5 min and centrifuged at 12 000 g for 3 min. For PCR amplification, 10 µl of the supernatant was added to 40 µl of a PCR mastermix such that the final amounts of the reagents were 1x Taq polymerase reaction buffer, 10 pmol of each primer, 12.5 nmol of each dNTP, and 2.5 U of Taq polymerase. The PCR reaction was performed with the following cycling parameters: a 3 min denaturation at 95°C, followed by 30 cycles of 30 s at 95°C, 1 min at 60°C, and 1 min at 72°C, with a final extension of 10 min at 72°C. The PCR products were purified using PCR purification kits (Qiagen, Chatsworth, CA).
The lacI and cII mutant DNA was sequenced with an ABI Prism Big Dye Terminator Cycle Sequencing Kit and a 377 DNA Sequencer (Applied Biosystems, Foster City, CA). The primers for sequencing lacI mutations have been described (27,29); the primers for cII mutation sequencing were the same as those used for the PCR. The sequence data were analyzed with the help of Sequence Navigator software (Applied Biosystems).
Statistical analyses
One-way ANOVA followed by StudentNewmanKeuls test was used to evaluate the differences in mutant frequencies among groups. Since the variance increased with the magnitude of the mutant frequency, the data were log-transformed before conducting the analysis. Mutational spectra were compared using the computer program written by Cariello et al. (30) for the Monte Carlo analysis developed by Adams and Skopek (31).
 |
Results
|
---|
cII Mutant frequency
Six female Big Blue rats per group were treated for 21 days by i.p. injection with 54 µmol tamoxifen, 54 µmol
-hydroxytamoxifen, or the solvent only. One month after the last treatment, the cII mutant frequency was assessed in the liver (Table I
). Compared with rats administered the solvent alone (80 ± 13 x 106), there was a significant increase in the cII mutant frequency in the livers of Big Blue rats treated with tamoxifen (112 ± 13 x 106; P < 0.01) or
-hydroxytamoxifen (942 ± 114 x 106; P < 0.001). In addition, the response observed with
-hydroxytamoxifen was significantly greater than that induced by tamoxifen (P < 0.001).
Mutations in the lacI gene induced by
-hydroxytamoxifen
The liver lacI mutant frequency from
-hydroxytamoxifen-treated rats was 770 ± 270 x 106, which is 77-fold greater than the control frequency (Figure 1
) (11).
-Hydroxytamoxifen-induced lacI mutations were evaluated by DNA sequence analysis of 54 mutants isolated from the six treated rats (Table II
and summarized in Table IV
). Mutations that were found more than once in the mutants isolated from a single animal were assumed to be siblings and were considered to represent a single independent mutation. Accordingly, a total of 52 independent mutations were identified. Of the 52 mutations, 90% (47) were single base pair substitutions and 10% (5) were frameshifts. Among the single base pair substitutions, 98% (46/47) involved G:C base pairs and 2% (1/47) occurred at A:T base pairs. The most commonly occurring base pair substitution was G:C
T:A transversion (61%, 32/52), followed by G:C
A:T transition (17%, 9/52).
View this table:
[in this window]
[in a new window]
|
Table IV. Comparison of cII and lacI independent mutations in livers of Big Blue rats treated with solvent, tamoxifen or -hydroxytamoxifen.
|
|
Mutations in the cII gene induced by
-hydroxytamoxifen
A total of 94 cII mutants from six
-hydroxytamoxifen-treated rats were successfully sequenced and 71 of them were found to be independent (Table III
and summarized in Table IV
). Among the independent mutations, 76% (54/71) were base pair substitutions, 18% (13/71) were frameshift mutations and 6% (4/71) were complex mutations (i.e. double base substitutions). Among the base substitutions, 98% (53/54) were mutated at G:C base pairs while only 2% (1/54) occurred at A:T base pairs. The predominant base substitution was G:C
T:A transversion (52%, 37/71), followed by G:C
A:T transition (14%, 10/71).
Comparison of mutational spectra
Table IV
lists the independent cII and lacI mutations isolated from livers of Big Blue rats treated with vehicle, tamoxifen and
-hydroxytamoxifen. G:C
T:A transversion was the major type of mutation induced by
-hydroxytamoxifen and tamoxifen, while G:C
A:T transition was the main type of mutation in the control. There was no significant difference between the mutational spectra induced by
-hydroxytamoxifen in the liver lacI and cII genes (P = 0.3), while the overall patterns of mutations in the control and
-hydroxytamoxifen-treated rats differed significantly (P < 0.0001 for both the lacI and cII genes). Although the cII mutational spectrum found for
-hydroxytamoxifen-treated rats was not significantly different from that of tamoxifen-treated rats (P = 0.1), a significant difference was found between the spectra of lacI mutation in the livers of tamoxifen-treated and
-hydroxytamoxifen-treated rats (P < 0.0001). This was mainly due to the higher induction of G:C
T:A transversion in the
-hydroxytamoxifen-treated rats (61 vs. 43%).
 |
Discussion
|
---|
-Hydroxytamoxifen is a much stronger mutagen than tamoxifen in rat liver
The mutant frequencies in the lacI and cII genes were 10 ± 10 x 106 and 80 ± 13 x 106 in the control, 32 ± 18 x 106 and 112 ± 13 x 106 in the tamoxifen-treated rats and 770 ± 270 x 106 and 942 ± 114 x 106 in the
-hydroxytamoxifen-treated animals (Figure 1
). The net increases of mutant frequency induced by tamoxifen over the control were 22 x 106 in the lacI gene and 32 x 106 in the cII gene, while the net increases by
-hydroxytamoxifen were 760 x 106 in the lacI gene and 862 x 106 in the cII gene. Therefore, the net induction of mutant frequencies by
-hydroxytamoxifen was 35-fold greater than tamoxifen in the lacI gene and 27-fold greater in the cII gene. It has been suggested that a dose of 103 µmol/kg tamoxifen would be metabolized to the equivalent of 1 µmol/kg
-hydroxytamoxifen (10) or
1% of the administered dose. Given the low rate of conversion of tamoxifen to
-hydroxytamoxifen, it is not unexpected that administration of tamoxifen and
-hydroxytamoxifen at equimolar doses will result in far more mutation induction by
-hydroxytamoxifen.
In a previous study, oral treatment with 10 daily doses of 103 mmol/kg
-hydroxytamoxifen resulted in
1.8-fold increase in mutation frequency but no significant increase in G:C
T:A transversions (10). The lack of efficient induction of mutations by oral administration of
-hydroxytamoxifen may be due to inactivation of the chemical at the acidic pH of the stomach. For example, in earlier studies we found that with
-hydroxytamoxifen the binding to hepatic DNA was four-fold higher when administered by i.p. injection compared with gavage (12). Since the tamoxifen and
-hydroxytamoxifen gave similar DNA adduct profiles when given by either route (12), we elected to use i.p. dosing to maximize the chance for mutant induction. Our results indicate that
-hydroxytamoxifen induces mutations much more efficiently through i.p. injection administration than oral treatment.
G:C
T:A transversion induced by
-hydroxytamoxifen reflects the mutational specificity of tamoxifenDNA adducts
The results from this study indicate that the mutation profiles in the lacI and cII genes of liver from
-hydroxytamoxifen-treated rats were significantly different from the corresponding profiles in control rats (Table IV
). Compared with the controls, the mutations detected in
-hydroxytamoxifen-treated rats had lower percentages of G:C
A:T transitions (17% in the lacI and 14% in the cII for
-hydroxytamoxifen-treated rats vs. 41% in the lacI and 42% in the cII for the control rats) and higher percentages of G:C
T:A transversions (61% in the lacI and 52% in the cII for
-hydroxytamoxifen-treated rats vs. 18% in the lacI and 15% in the cII for the control rats).
The types of mutations in the liver cII gene from
-hydroxytamoxifen-treated rats were very similar to those from tamoxifen-treated animals. With the lacI gene there was a significant difference between the tamoxifen- and
-hydroxytamoxifen-induced spectra; nonetheless, with both compounds, the cII and lacI spectra shared common features: most base substitutions occurred at G:C base pairs, with G:C
T:A being the major type of mutation.
A mechanism by which tamoxifen induces G:C
T:A transversions in rat liver has been suggested. Tamoxifen is known to be metabolized to
-hydroxytamoxifen by cytochrome P450 3A, and then converted into
-sulfoxytamoxifen by sulfotransferases.
-Sulfoxytamoxifen can bind to DNA to form the major DNA adduct, (E)-
-(deoxyguanosin-N2-yl)tamoxifen (1619). This is a bulky DNA adduct and preferentially gives rise to G:C
T:A transversion mutation like many other carcinogens, such as N-hydroxy-2-acetylaminofluorene and benzo[a]pyrene, which also form bulky DNA adducts (33,35). Our finding that the types of mutations induced by
-hydroxytamoxifen are similar to those induced by tamoxifen further supports the hypothesis that
-hydroxytamoxifen is a major proximate tamoxifen metabolite causing mutations in rat liver.
The cII gene is an acceptable alternative to the lacI gene of Big Blue rats in the determination of mutations
The cII assay is performed using a positive selection of mutants. Compared with the lacI assay, it is faster and less expensive to perform. In addition, the cII gene is 294 base pairs in length (vs. 1080 base pairs for lacI), offering potentially easier analysis of mutations (36). A primary purpose for using the cII mutation assay and subsequent analysis of the cII mutations was to confirm the results on mutant frequencies and types of mutations induced by tamoxifen and
-hydroxytamoxifen in the lacI gene. An additional reason was to compare the characteristics of the two systems by measuring mutant frequencies and mutational spectra in the two genes from the same DNA samples.
Our results indicate that the overall results provide some interesting comparisons and contrasts between the two mutational systems. The net increase in mutation induction by tamoxifen and
-hydroxytamoxifen was similar in the two systems (22 x 106 in the lacI and 32 x 106 in the cII for tamoxifen; 760 x 106 in the lacI and 862 x 106 in the cII for
-hydroxytamoxifen). In addition, the mutational spectrum in the liver cII gene from
-hydroxytamoxifen-treated rats was not significantly different from that in the liver lacI gene (P = 0.3). Our results confirm the finding of Harbach et al. (34) that the smaller size of the cII coding region is a promising alternative to the larger target of the lacI gene. Although
-hydroxytamoxifen induced a higher mutant frequency in the cII gene, there was a higher spontaneous background mutation frequency in the liver cII gene (80 x 106) than that in the liver lacI gene (10 x 106). The differences in the background mutant frequencies resulted in a much greater fold- increase in mutant frequency by
-hydroxytamoxifen in the lacI gene as compared to the cII gene (77-fold vs. 12-fold). The packaging efficiency was higher using the strain G1250 in the cII assay than the strain SCS-8 in the lacI assay. The plaque-forming units (PFU) per packaging reaction in the cII assay were
180 000, whereas PFU per packaging reaction in the lacI assay were
100 000. This increase in PFU per packaging reaction allowed more plaques to be screened in the cII assay compared with the lacI assay, with a concomitant decrease in the standard deviation. Consequently, although the increase in the cII mutant frequency induced by tamoxifen was only 1.4-fold, it was statistically significant (P < 0.01).
 |
Conclusion
|
---|
-Hydroxytamoxifen induced a much greater mutant frequency than tamoxifen in the liver lacI and cII genes of Big Blue rats. The spectra of mutations induced by
-hydroxytamoxifen in the lacI and cII genes were similar to those induced by tamoxifen but distinct from the control spectra. The main type of mutations induced by
-hydroxytamoxifen was consistent with the major tamoxifen-induced DNA adduct. These results support the hypothesis that
-hydroxytamoxifen is a major proximate tamoxifen metabolite causing induction of mutations and the initiation of tumors in the liver of rats treated with tamoxifen.
 |
Notes
|
---|
4 To whom correspondence should be addressed at HFT 130, NCTR, 3900 NCTR Road, Jefferson, AR 72079, USA Email: tchen{at}nctr.fda.gov 
 |
References
|
---|
- Early Breast Cancer Trialists Collaborative Group (1998) Tamoxifen for early breast cancer: an overview of the randomised trials. Lancet, 351, 14511467.[ISI][Medline]
- Fisher,B., Costantino,J.P., Wickerham,D.L., et al. (1998) Tamoxifen for prevention of breast cancer: report of the National Surgical Adjuvant Breast and Bowel Project P-1 Study. J. Natl Cancer Inst., 90, 13711388.[Abstract/Free Full Text]
- Stearns,V. and Gelmann,E.P. (1998) Does tamoxifen cause cancer in humans? J. Clin. Oncol., 16, 779792.[Abstract]
- Hard,G.C., Williams,G.M. and Iatropoulos,M.J. (1993) Tamoxifen and liver cancer. Lancet, 342, 444445.[ISI][Medline]
- Williams,G.M., Iatropoulos,M.J., Djordjevic,M.V. and Kaltenberg,O.P. (1993) The triphenylethylene drug tamoxifen is a strong liver carcinogen in the rat. Carcinogenesis, 14, 315317.[Abstract]
- Mäntylä,E., Nieminen,L. and Karlsson,S. (1995) Endometrial cancer induction by tamoxifen in the rat. Eur. J. Cancer, 31A (Suppl. 5), S14.
- Mäntylä,E.T.E., Karlsson,S.H. and Nieminen,L.S. (1996) Induction of endometrial cancer by tamoxifen in the rat. In: Li,J.J., Li,S.A., Gustafsson,J.Å., Nandi S. and Sekely,L.I. (eds.) Hormonal Carcinogenesis. II. Proceedings of the Second International Symposium, Springer, New York, pp. 442445.
- Osborne,M.R., Hewer,A., Hardcastle,I.R., Carmichael,P.L. and Phillips,D.H. (1996) Identification of the major tamoxifen-deoxyguanosine adduct formed in the liver DNA of rats treated with tamoxifen. Cancer Res., 56, 6671.[Abstract]
- Davies,R., Oreffo,V.I.C., Martin,E.A., Festing,M.F.W., White,I.N.H., Smith,L.L. and Styles,J.A. (1997) Tamoxifen causes gene mutations in the livers of lambda/lacI transgenic rats. Cancer Res., 57, 12881293.[Abstract]
- White,I.N.H., Carthew,P., Davies,R., Styles,J., Brown,K., Brown,J.E., Smith,L.L. and Martin,E.A. (2001) Short-term dosing of
-hydroxytamoxifen results in DNA damage but does not lead to liver tumours in female Wistar/Han rats. Carcinogenesis, 22, 553557.[Abstract/Free Full Text]
- Gamboa da Costa,G., Manjanatha,M.G., Marques,M.M. and Beland,F.A. (2002) Induction of lacI mutations in Big Blue rats treated with tamoxifen and
-hydroxytamoxifen. Cancer Lett., 176, 3745.[ISI][Medline]
- Gamboa da Costa,G., McDaniel-Hamilton,L.P., Heflich,R.H., Marques,M.M. and Beland,F.A. (2001) DNA adduct formation and mutant induction in Sprague-Dawley rats treated with tamoxifen and its derivatives. Carcinogenesis, 22, 13071315.[Abstract/Free Full Text]
- Schild,L.J., Divi,R.L. and Poirier,M.C. (2002) Formation of tamoxifen-DNA adducts in organs of monkeys dosed orally with tamoxifen for 30 days. Environ. Mol. Mutagen., 39 (Suppl. 33), 56.
- Beland,F.A., Churchwell,M.I., Doerge,D.R., Malejka-Giganti,D., Zhang,X., Divi,R.L., Poirier,M.C., Gamboa da Costa,G. and Marques,M.M. (2002) High performance liquid chromatography electrospray ionization mass spectrometry analysis of tamoxifen DNA adducts in rats, monkeys and humans. Proc. Am. Assoc. Cancer Res., 43, 343.
- Phillips,D.H. (2001) Understanding the genotoxicity of tamoxifen? Carcinogenesis, 22, 839849.[Abstract/Free Full Text]
- Davis,W., Venitt,S. and Phillips,D.H. (1998) The metabolic activation of tamoxifen and
-hydroxytamoxifen to DNA-binding species in rat hepatocytes proceeds via sulphation. Carcinogenesis, 19, 861866.[Abstract]
- Shibutani,S., Dasaradhi,L., Terashima,I., Banoglu,E. and Duffel,M.W. (1998)
-Hydroxytamoxifen is a substrate of hydroxysteroid (alcohol) sulfotransferase, resulting in tamoxifen DNA adducts. Cancer Res., 58, 647653.[Abstract]
- Glatt,H., Bartsch,I., Christoph,S., Coughtrie,M.W.H., Falany,C.N., Hagen,M., Landsiedel,R., Pabel,U., Phillips,D.H., Seidel,A. and Yamazoe,Y. (1998) Sulfotransferase-mediated activation of mutagens studied using heterologous expression systems. Chem.-Biol. Interact., 109, 195219.[ISI][Medline]
- White,I.N.H. (1999) The tamoxifen dilemma. Carcinogenesis, 20, 11531160.[Abstract/Free Full Text]
- Osborne,M.R., Hardcastle,I.R. and Phillips,D.H. (1997) Minor products of reaction of DNA with
-acetoxytamoxifen. Carcinogenesis, 18, 539543.[Abstract]
- Tucker,M..J., Adam,H.K. and Patterson,J.S. (1984) Tamoxifen. In: Laurence,D.R., McLean,A.E.M. and Weatherall,M. (eds.) Safety Testing of New Drugs. Academic Press, New York, pp. 125161.
- White,I.N.H., de Matteis,F., Davies,A., Smith,L.L., Crofton-Sleigh,C., Venitt,S., Hewer,A. and Phillips,D.H. (1992) Genotoxic potential of tamoxifen and analogues in female Fischer F344/n rats, DBA/2 and C57BL/6 mice and in human MCL-5 cells. Carcinogenesis, 13, 21972203.[Abstract]
- Styles,J.A., Davies,A., Lim,C.K., de Matteis,F., Stanley,L.A., White,I.N.H., Yuan,Z.-X. and Smith,L.L. (1994) Genotoxicity of tamoxifen, tamoxifen epoxide and toremifene in human lymphoblastoid cells containing human cytochrome P450s. Carcinogenesis, 15, 59.[Abstract]
- Davies,R., Oreffo,V.I.C., Bayliss,S., Dinh,P.-A., Lilley,K.S., White,I.N.H., Smith,L.L. and Styles,J.A. (1996) Mutational spectra of tamoxifen-induced mutations in the livers of lacI transgenic rats. Environ. Mol. Mutagen., 28, 430433.[ISI][Medline]
- Davies,R., Gant,T.W., Smith,L.L. and Styles,J.A. (1999) 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. Carcinogenesis, 20, 13511356.[Abstract/Free Full Text]
- Foster,A.B., Jarman,M., Leung,O.-T., McCague,R., Leclercq,G. and Devleeschouwer,N. (1985) Hydroxy derivatives of tamoxifen. J. Med. Chem., 28, 14911497.[ISI][Medline]
- Chen,T., Aidoo,A., Manjanatha,M.G., Mittelstaedt,R.A., Shelton,S.D., Lyn-Cook,L.E., Casciano,D.A. and Heflich,R.H. (1998) Comparison of mutant frequencies and types of mutations induced by thiotepa in the endogenous Hprt gene and transgenic lacI gene of Big Blue® rats. Mutat. Res., 403, 199214.[ISI][Medline]
- Manjanatha,M.G., Shelton,S.D., Culp,S.J., Blankenship,L.R. and Casciano,D.A. (2000) DNA adduct formation and molecular analysis of in vivo lacI mutations in the mammary tissue of Big Blue® rats treated with 7,12-dimethylbenz[a]anthracene. Carcinogenesis, 21, 265273.[Abstract/Free Full Text]
- Manjanatha,M.G., Chen,J.B., Shaddock,J.G., Jr., Harris,A.J., Shelton,S.D. and Casciano,D.A. (1996) Molecular analysis of lacI mutations in Rat2TM cells exposed to 7,12-dimethylbenz[a]anthracene: evidence for DNA sequence and DNA strand biases for mutation. Mutat. Res., 372, 5364.[ISI][Medline]
- Cariello,N.F. (1994) Software for the analysis of mutations at the human hprt gene. Mutat. Res., 312, 173185.[ISI][Medline]
- Adams,W.T. and Skopek,T.R. (1987) Statistical test for the comparison of samples from mutational spectra. J. Mol. Biol., 194, 391396.[ISI][Medline]
- Farabaugh,P.J., Schmeissner,U., Hofer,M. and Miller,J.H. (1978) Genetic studies of the lac repressor. VII. On the molecular nature of spontaneous hotspots in the lacI gene of Escherichia coli. J. Mol. Biol., 126, 847863.[ISI][Medline]
- Chen,T., Mittelstaedt,R.A., Shelton,S.D., Dass,S.B., Manjanatha,M.G., Casciano,D.A. and Heflich,R.H. (2001) Gene- and tissue-specificity of mutation in Big Blue® rats treated with the hepatocarcinogen N-hydroxy-2-acetylaminofluorene. Environ. Mol. Mutagen., 37, 203214.[ISI][Medline]
- Harbach,P.R., Zimmer,D.M., Filipunas,A.L., Mattes,W.B. and Aaron,C.S. (1999) Spontaneous mutation spectrum at the lambda cII locus in liver, lung, and spleen tissue of Big Blue® transgenic mice. Environ. Mol. Mutagen., 33, 132143.[ISI][Medline]
- Skopek,T.R., Kort,K.L., Marino,D.R., Mittal,L.V., Umbenhauer,D.R., Laws,G.M. and Adams,S.P. (1996) Mutagenic response of the endogenous hprt gene and lacI transgene in benzo[a]pyrene-treated Big BlueTM B6C3F1 mice. Environ. Mol. Mutagen., 28, 376384.[ISI][Medline]
- Jakubczak,J.L., Merlino,G., French,J.E., Muller,W.J., Paul,B., Adhya,S. and Garges,S. (1996) Analysis of genetic instability during mammary tumor progression using a novel selection-based assay for in vivo mutations in a bacteriophage
transgene target. Proc. Natl Acad. Sci. USA, 93, 90739078.[Abstract/Free Full Text]
Received May 2, 2002;
revised June 27, 2002;
accepted July 8, 2002.