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The present study compares the formation of DNA adducts, determined by 32P-postlabelling, in the livers of rats given tamoxifen and the N-demethylated metabolites N-desmethyltamoxifen and N,N-didesmethyltamoxifen. Results show that after 4 days treatment (0.11 mmol/kg i.p.), similar levels of DNA damage were seen
after treatment with either tamoxif...
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Context 1
... anti-oestrogen tamoxifen {trans-(Z)-1-[4-[2-(dimethylam- ino)ethoxy]phenyl]-1,2-diphenyl-1-butene} is currently under- going evaluation as a chemopreventive agent in women at increased risk of developing breast cancer (1). Recent results in healthy, high risk women from the NSABP P1 study demonstrated that this drug resulted in a 49% reduction in breast cancer incidence (2). However, tamoxifen treatment is associated with an increased incidence of endometrial cancer (3,4) and long-term administration to rats results in a dose- dependent increase in hepatic tumours (5,6). Tamoxifen requires metabolic activation to electrophilic species before it can react with DNA, forming adducts detectable using the 32 P- postlabelling assay (7,8). The major phase I metabolites of tamoxifen in both rat and human liver are N-desmethyltamox- ifen and 4-hydroxytamoxifen (Figure 1; 9,10). However, one of the main DNA adducts detected in the livers of tamoxifen- dosed rats is thought to arise as a consequence of α-hydroxylation, followed by hydroxysteroid sulphotransfer- ase-mediated sulphate conjugation (11)(12)(13). This adduct, which co-elutes with the major product from the in vitro reaction between α-sulphate tamoxifen or α-acetoxytamoxifen and DNA, contains tamoxifen in the trans form covalently linked via the α carbon to the exocyclic amino group of deoxyguano- sine (dG-N 2 -tam) (14,15). This same adduct has recently been identified in DNA extracted from the livers of tamoxifen-treated rats (16). A second major product was N-desmethyltamoxifen linked covalently to the amino group of deoxyguanosine in a similar manner (16). Previously we have shown that 4- hydroxytamoxifen also forms other minor adducts in rat liver ...
Citations
... The tamoxifen (Cat-T708, Lot#057k2378, Sigma, USA) was administered to pregnant female rats during the preimplantation period. Preparation of the tamoxifen with the vehicle (tricaprylic acid) and the dose administered to the pregnant females was done following standard method [21] . A group of pregnant females (TMX group, n=5) was administered tamoxifen for 3 d beginning from day 1 of pregnancy. ...
... Threefold fewer Tam-DNA adducts were formed by the human sulfotransferase than by the rat sulfotransferase under the same conditions (Shibutani et al., 1998), which provides evidence of species-differences in this activation step. Bioactivation may also occur with N-desmethylTam, N,N-didesmethylTam and Tam-N-oxide via a-hydroxylation and/or sulfation (Brown et al., 1999;Phillips et al., 1999b). Another bioactivation mechanism is via metabolism of 4-OHTam to a highly electrophilic quinone methide by a P450-catalyzed, direct 2-electron oxidation . ...
... Possible explanations for the differences include use of different rat strains, the higher dose used in the latter study, and better resolution of the HPLC method. The predominant adducts in rat liver, formed via the a-hydroxylation/sulfation pathway, have been identified as a-(deoxyguanosine-N2-yl)-tamoxifen (dG-N 2 -Tam) and a-(deoxyguanosine-N2-yl)-N-desmethyltamoxifen (dG-N 2 -desMeTam), and a minor adduct as a-(deoxyguanosine-N2yl)-N,N-didesmethyltamoxifen (dG-N 2 -didesMeTam) (Brown et al., 1999;Marques & Beland, 1997;Osborne et al., 1996;Phillips et al., 1999b;Rajaniemi et al., 1999) (Figure 2). Guanine adducts represent the major products formed in vivo, with a minor adenine adduct, (a-deoxyadenosine-N 6 -yl)-tamoxifen, identified only in vitro (Osborne et al., 1997). ...
... Where detected, they were seen only at extremely low levels in leucocytes, colon, and uterus. Han & Liehr 1992;Phillips et al., 1996Phillips et al., , 1999bBrown et al., 1999Brown et al., , 2007Osborne et al., 1996;Rajaniemi et al., 1999;Marques & Beland 1997;Hashiba et al., 2006;Williams et al., 1997;White et al., 1992White et al., , 1997Carthew et al., 2000;Hemminki et al., 1996Hemminki et al., , 1997Bartsch et al., 2000;Martin et al., 1995Martin et al., , 1998Martin et al., , 2003Carmichael et al., 1999;Beland et al., 2004;Shibutani et al., 1999Shibutani et al., , 2000a Dose-response for adduct levels In rat liver, Tam DNA adducts accumulate in a dose and time dependent manner. In mice, Tam DNA adducts are formed at much lower levels than in similarly treated rats and decrease with time (with continued dosing) to non-detectable levels by 2 years. ...
Abstract The framework analysis previously presented for using DNA adduct information in the risk assessment of chemical carcinogens was applied in a series of case studies which place the adduct information into context with the key events in carcinogenesis to determine whether they could be used to support a mutagenic mode of action (MOA) for the examined chemicals. Three data-rich chemicals, aflatoxin B1 (AFB1), tamoxifen (Tam) and vinyl chloride (VCl) were selected for this exercise. These chemicals were selected because they are known human carcinogens and have different characteristics: AFB1 forms a unique adduct and human exposure is through contaminated foods; Tam is a pharmaceutical given to women so that the dose and duration of exposure are known, forms unique adducts in rodents, and has both estrogenic and genotoxic properties; and VCl, to which there is industrial exposure, forms a number of adducts that are identical to endogenous adducts found in unexposed people. All three chemicals produce liver tumors in rats. AFB1 and VCl also produce liver tumors in humans, but Tam induces human uterine tumors, only. To support a mutagenic MOA, the chemical-induced adducts must be characterized, shown to be pro-mutagenic, be present in the tumor target tissue, and produce mutations of the class found in the tumor. The adducts formed by AFB1 and VCl support a mutagenic MOA for their carcinogenicity. However, the data available for Tam shows a mutagenic MOA for liver tumors in rats, but its carcinogenicity in humans is most likely via a different MOA.
... The tamoxifen (Cat-T708, Lot#057k2378, Sigma, USA) was administered to pregnant female rats during the preimplantation period. Preparation of the tamoxifen with the vehicle (tricaprylic acid) and the dose administered to the pregnant females was done following standard method [21] . A group of pregnant females (TMX group, n=5) was administered tamoxifen for 3 d beginning from day 1 of pregnancy. ...
Objective To study the immunohistochemical localization of insulin-like growth factor-
I (IGF-I) in rats’ uteri to determine if expression of the growth factor is ovarian steroid
dependent.
Method The study was carried out in presence and absence of ovary in situ of non
pregnant females and during early gestation (day 1 to day 5.5). Cyclic females were
tested to observe the effect of native steroids on IGF-I expression. Adult females were
ovariectomized (OVX) and injected (s.c) with estradiol-17β in a dose of 0.1 μg/ml per
day for three consecutive days at interval of 24 h prior to the collection of uterine horns.
During the early pregnancy, studies were carried out on day 3.5 and day 5.5 of
geatation respectively to determine the steroids’ effects during pre- and post-implantation
period. Tamoxifen was administered (s.c) in a dose of 250 μg/ml per day from day 1 to
day 3 of gestation while, the prostaglandin F2α (PGF2α) was administered (s.c) from
day 3 of gestation onward for three consecutive days at interval of 24 h in a dose of
150 μg/ml per day. Expression of IGF-I was immunohistochemically localised using
IGF-I antibody in paraffin embedded sections.
Results IGF-I was expressed in rat uterus during estrus phase as well as during pre
and post-implantation period. The ovariectomized females’ uteri lost the expression of
IGF-I. Exogenous administration of tamoxifen and PGF2α reduce the expression of the
growth factor.
Conclusion Expression of IGF-I in rat uterus during cyclic stage and early gestation
depends upon the availability of circulating estrogen and progesterone. Uterine
... The tamoxifen (Cat-T708, Lot#057k2378, Sigma, USA) was administered to pregnant female rats during the preimplantation period. Preparation of the tamoxifen with the vehicle (tricaprylic acid) and the dose administered to the pregnant females was done following standard method [21] . A group of pregnant females (TMX group, n=5) was administered tamoxifen for 3 d beginning from day 1 of pregnancy. ...
Objective
To study the immunohistochemical localization of insulin-like growth factor-I (IGF-I) in rats' uteri to determine if expression of the growth factor is ovarian steroid dependent.
Method
The study was carried out in presence and absence of ovary in situ of non pregnant females and during early gestation (day 1 to day 5.5). Cyclic females were tested to observe the effect of native steroids on IGF-I expression. Adult females were ovariectomized (OVX) and injected (s.c) with estradiol-17β in a dose of 0.1 μg/ml per day for three consecutive days at interval of 24 h prior to the collection of uterine horns. During the early pregnancy, studies were carried out on day 3.5 and day 5.5 of geatation respectively to determine the steroids' effects during pre- and post-implantation period. Tamoxifen was administered (s.c) in a dose of 250 μg/ml per day from day 1 to day 3 of gestation while, the prostaglandin F2α (PGF2α) was administered (s.c) from day 3 of gestation onward for three consecutive days at interval of 24 h in a dose of 150 μg/ml per day. Expression of IGF-I was immunohistochemically localised using IGF-I antibody in paraffin embedded sections.
Results
IGF-I was expressed in rat uterus during estrus phase as well as during pre and post-implantation period. The ovariectomized females' uteri lost the expression of IGF-I. Exogenous administration of tamoxifen and PGF2α reduce the expression of the growth factor.
Conclusion
Expression of IGF-I in rat uterus during cyclic stage and early gestation depends upon the availability of circulating estrogen and progesterone. Uterine expression of IGF-I can be modulated by manipulating circulating ovarian steroid either during cyclic stage or during gestation.
... Other phase I metabolites of tamoxifen such as N-desmethyl and N,N-didesmethyltamoxifen are also substrates for a-hydroxylation and sulphation; consequently, the most abundant adduct formed in rat liver with long-term dosing corresponds to an N-demethylated version of dG-N 2 -tamoxifen, whilst dG-N 2 -N,N-didesmethytamoxifen constitutes a minor lesion in rat liver (93)(94)(95). The trans forms of these adducts predominate, with the cis isomers contributing only a small fraction; after 8 weeks of continuous dietary intervention with 20 mg/kg tamoxifen, trans dG-N 2 -tamoxifen and dG-N 2 -N-desmethyltamoxifen accounted for 49 and 44%, respectively, of the total adducts formed in rat liver whilst the cis forms each constituted 3-4% (96). ...
The anti-oestrogen tamoxifen, which is widely used in the treatment of breast cancer and is also approved for the prevention of this disease, causes an increased incidence of endometrial cancer in women. The ability of tamoxifen to induce endometrial tumours and the underlying carcinogenic mechanisms have been a subject of intense interest over the last approximately 20 years. They are central to the assessment of risks versus benefits for the drug, especially in a chemopreventive context. This review outlines the clinical justification for using tamoxifen as a chemopreventive agent and describes the genotoxic mechanisms considered responsible for tamoxifen-induced tumours in rat liver and how these might relate to women. In rat hepatic tissue, tamoxifen is metabolically activated via alpha-hydroxylation and sulphate conjugation to give a reactive species that binds to DNA predominantly at the N(2)-position of guanine, producing pro-mutagenic lesions. Whether tamoxifen-DNA adducts contribute similarly to the development of cancers in women depends on whether they can be formed in human tissues and the type of specific molecular and cellular responses they induce, if present. This review discusses the current data relating to these issues and highlights areas where further research is needed.
... Treatment of plasmid with α-acetoxytamoxifen resulted in the formation of one major peak (Figure 1B) consistent with the dG-N2-tam adduct detected in hepatic DNA from a tamoxifen-treated rat (Figure 1D). An additional minor peak is also observed eluting just prior to the major adduct, which has previously been suggested to be a diastereoisomer of trans-dG-N2-tamoxifen (29). Following incubation of 4-OHtamQM with plasmid DNA, two main 32P-post-labelled adduct peaks are detected, presumably cis- and trans-isomers of dG-N2-4-OHtam (Figure 1C) (17). ...
... To quantify the number of tamoxifen-DNA adducts formed on the treated plasmids, aliquots of DNA (10 μg) were analysed using our established 32P-post-labelling assay, with HPLC separation of 32P-post-labelled nucleotides and online radiochemical detection (18). A positive control sample, liver DNA from a tamoxifen treated rat, was analysed in parallel to enable adduct identification (18,29). ...
Tamoxifen elevates the risk of endometrial tumours in women and α-(N2-deoxyguanosinyl)-tamoxifen adducts are reportedly present in endometrial tissue of patients undergoing therapy. Given the
widespread use of tamoxifen there is considerable interest in elucidating the mechanisms underlying treatment-associated cancer.
Using a combined experimental and multivariate statistical approach we have examined the mutagenicity and potential consequences
of adduct formation by reactive intermediates in target uterine cells. pSP189 plasmid containing the supF gene was incubated with α-acetoxytamoxifen or 4-hydroxytamoxifen quinone methide (4-OHtamQM) to generate dG-N2-tamoxifen and dG-N2-4-hydroxytamoxifen, respectively. Plasmids were replicated in Ishikawa cells then screened in Escherichia coli. Treatment with both α-acetoxytamoxifen and 4-OHtamQM caused a dose-related increase in adduct levels, resulting in a damage-dependent
increase in mutation frequency for α-acetoxytamoxifen; 4-OHtamQM had no apparent effect. Only α-acetoxytamoxifen generated
statistically different supF mutation spectra relative to the spontaneous pattern, with most mutations being GC→TA transversions. Application of the LwPy53 algorithm to the α-acetoxytamoxifen spectrum predicted strong GC→TA hotspots at codons 244 and 273. These signature alterations do not correlate with current reports of the mutations observed
in endometrial carcinomas from treated women, suggesting that dG-N2-tam adduct formation in the p53 gene is not a prerequisite for endometrial cancer initiation in women.
... 15,16), which are proven mutagenic lesions in mammalian cells (17,18). Analogous reactions may also occur with other phase I metabolites of tamoxifen (19,20). ...
... [1-Phenyl-U-14 C]tamoxifen (2.03 GBq/mmol) of >98% radiochemical purity was from Cambridge Research Biochemicals. N-Desmethyltamoxifen and a-hydroxytamoxifen were synthesized according to published methods (19,28). Gelatin capsules containing 1.85 MBq of [ 14 C]-tamoxifen and 20 mg unlabeled drug were prepared in the Radiopharmacy Department at Leicester Royal Infirmary. ...
Tamoxifen is widely prescribed for the treatment of breast cancer and is also licensed in the United States for the prevention of this disease. However, tamoxifen therapy is associated with an increased occurrence of endometrial cancer in women, and there is also evidence that it may elevate the risk of colorectal cancer. The underlying mechanisms responsible for tamoxifen-induced carcinogenesis in women have not yet been elucidated, but much interest has focused on the role of DNA adduct formation. We investigated the propensity of tamoxifen to bind irreversibly to colorectal DNA when given to 10 women as a single [(14)C]-labeled therapeutic (20 mg) dose, approximately 18 h before undergoing colon resections. Using the sensitive technique of accelerator mass spectrometry, coupled with high-performance liquid chromatography separation of enzymatically digested DNA, a peak corresponding to authentic dG-N(2)-tamoxifen adduct was detected in samples from three patients, at levels ranging from 1 to 7 adducts/10(9) nucleotides. No [(14)C]-radiolabel associated with tamoxifen or its major metabolites was detected. The presence of detectable CYP3A4 protein in all colon samples suggests that this tissue has the potential to activate tamoxifen to alpha-hydroxytamoxifen, in addition to that occurring in the systemic circulation, and direct interaction of this metabolite with DNA could account for the binding observed. Although the level of tamoxifen-induced damage displayed a degree of interindividual variability, when present, it was approximately 10 to 100 times higher than that reported for other suspect human colon carcinogens such as 2-amino-1-methyl-6-phenyimidazo[4,5-b]pyridine. These findings provide a mechanistic basis through which tamoxifen could increase the incidence of colon cancers in women.
Tamoxifen‐DNA adducts detected in the liver of mice Among such anti-cancer drugs, the treatment period of treated with tamoxifen have not yet been identified. In the tamoxifen is exceptionally long (3–5 years). Therefore, careful present study a new type of tamoxifen‐DNA adduct, four evaluation of safety for chronic use of tamoxifen is required. stereoisomers of α-(N 2 -deoxyguanosinyl)tamoxifen N-oxide Tamoxifen is a potent carcinogen targeting the liver in rats 3-monophosphate (dG3P-N 2 -TAM N-oxide) were prepared (9,10). Carcinogenic actions of tamoxifen toward rat liver as standard DNA adducts by reacting 2-deoxyguanosine are not related to its estrogen antagonist activity but occur 3-monophosphate with trans-α-acetoxytamoxifen N-oxide through DNA adduct formation (11). When liver DNA from in addition to four stereoisomers of α-(N 2 -deoxyguano- tamoxifen-treated mice and rats were analyzed by 32 P-postsinyl)tamoxifen 3-monophosphate (dG3P-N 2 -TAM) that labeling HPLC analysis, many tamoxifen–DNA adduct peaks was reported previously. Liquid chromatography-electro- were observed (12). Osborne et al. (13) indicated that trans-αspray ionization‐mass spectrometry of the reaction (N 2 -deoxyguanosinyl)tamoxifen (trans-dG-N 2 -TAM) is a major products gave the most abundant ion at m/z 731 ([M ‐ H] ‐ ), tamoxifen–DNA adduct in rat liver. Using the in vitro reaction which corresponded to dG3P-N 2 -TAM N-oxide. The modi- of DNA with tamoxifen α-sulfate or α-acetoxytamoxifen, cisfied products digested by alkaline phosphatase corre- dG-N 2 -TAM was also identified as a minor adduct by mass sponded to the isomers of dG-N 2 -TAM N-oxide whose spectrometry and nuclear magnetic resonance (14,15). Howstructures were identified previously by mass spectrometry ever, no information was available on whether such minor and nuclear magnetic resonance. Using these standard adducts are formed in tissues of tamoxifen-treated animals. In markers, we analyzed the hepatic DNA adducts of female theory, reaction of α-acetoxytamoxifen or tamoxifen α-sulfate DBA/2 mice treated with tamoxifen at a dosage of 120 mg/ with DNA or 2-deoxyguanosine (dG) yielded four stereokg/day for 7 days by 32 P-post-labeling coupled with an isomers of dG-N 2 -TAM (two epimers at α-position of the trans HPLC/radioactive detector. Mixtures of eight isomers of form and two epimers of the cis form) (14). Therefore, to dG3P-N 2 -TAM and dG3P-N 2 -TAM N-oxide were separated identify the epimers of dG-N 2 -TAM adducts in tamoxifeninto six peaks, since each of the cis epimers were not treated animals, authentic isomers and high-resolution HPLC separated under the present HPLC conditions. Nine conditions are required.
