Formation of tamoxifen-DNA adducts in human endometrial explants exposed to alpha-hydroxytamoxifen

An increased risk of developing endometrial cancer has been observed in women receiving tamoxifen (TAM) endocrine therapy and chemoprevention. The genotoxic damage induced by TAM metabolites may be involved in the development of endometrial cancer. To investigate the capability of endometrial tissues to form TAM-DNA adducts, primary cultured human endometrial explants were exposed to alpha-hydroxytamoxifen (alpha-OHTAM) and used for quantitative analysis of TAM-DNA adducts, using (32)P-postlabeling/HPLC analysis. A trans isoform of alpha-(N(2)-deoxyguanosinyl)tamoxifen (dG-N(2)-TAM) was detected as the major adduct in eight of nine endometrial explants exposed to 100 microM alpha-OHTAM at levels of 7.7 +/- 5.3 (mean +/- SD) adducts/10(7) nucleotides. Approximately 25- and 37-fold lower amounts of the cis form of dG-N(2)-TAM and another trans isoform were also detected. The dG-N(2)-TAM adduct (3.3 adducts/10(7) nucleotides) was detected in one of three endometrial explants exposed to 25 microM alpha-OHTAM. No TAM-DNA adducts were detected in any unexposed tissues. These results indicate that TAM-DNA adducts are capable of forming through O-sulfonation and/or O-acetylation of alpha-OHTAM in the endometrium. The endometrial explant culture can be used as a model system to explore the genotoxic mechanism of antiestrogens for humans.     

Tamoxifen-DNA adducts detected in the endometrium of women treated with tamoxifen.

Women treated for breast cancer with tamoxifen are at increased risk of developing endometrial cancer. This carcinogenic effect has been attributed to estrogenic stimulation and/or to a genotoxic effect of this drug. To examine genotoxicity, we developed a (32)P-postlabeling TLCL/HPLC procedure for quantitative analysis of tamoxifen-DNA adducts in endometrial tissue. This assay is several orders of magnitude more sensitive than those previously used for this purpose; with it, we can detect five tamoxifen-DNA adducts in 10(11) bases. Endometrial tissue was obtained from women undergoing tamoxifen therapy and from untreated control subjects. DNA adducts, identified as trans and cis epimers of alpha-(N(2)-deoxyguanosinyl)tamoxifen, were detected in six of thirteen patients in the tamoxifen-treated group. Levels of trans and cis adducts ranged from 0.5 to 8.3 and from 0.4 to 4.8 adducts/10(8) nucleotides, respectively. Tamoxifen-DNA adducts were not detected in endometrial tissue obtained from the control subjects. We conclude from this study that one or more tamoxifen metabolites react with endometrial DNA to form covalent adducts, establishing the potential genotoxicity of this drug for women and suggesting the use of TAM-DNA adducts as biomarkers for investigations of tamoxifen-induced endometrial cancer.     

Identification and quantification of tamoxifen-DNA adducts in the liver of rats and mice

A new HPLC gradient system was developed for (32)P-postlabeling analysis to identify and quantify hepatic tamoxifen-DNA adducts of rats and mice treated with tamoxifen. Four stereoisomers of alpha-(N(2)-deoxyguanosinyl)tamoxifen (dG(3')(P)-N(2)-TAM), alpha-(N(2)-deoxyguanosinyl)-N-desmethyltamoxifen (dG(3')(P)-N(2)-N-desmethyl-TAM), and alpha-(N(2)-deoxyguanosinyl)tamoxifen N-oxide (dG(3')(P)-N(2)-TAM N-oxide) were prepared by reacting either alpha-acetoxytamoxifen, alpha-acetoxy-N-desmethyltamoxifen or alpha-acetoxytamoxifen N-oxide with 2'-deoxyguanosine 3'-monophosphate, and used as standard markers for (32)P-postlabeling/HPLC analysis. Our HPLC gradient system can separate the above 12 nucleotide isomers as nine peaks; six peaks representing two each trans epimers (fr-1 and fr-2) of dG(3')(P)-N(2)-TAM, dG(3')(P)-N(2)-N-desmethyl-TAM and dG(3')(P)-N(2)-TAM N-oxide, and three peaks representing a mixture of two cis epimers (fr-3 and fr-4) of nucleotides. Tamoxifen was given to female F344 rats and DBA/2 mice by gavage at doses of 45 mg/kg/day and 120 mg/kg/day, respectively, for 7 days. Totally 15 and 17 tamoxifen-DNA adducts were detected in rats and mice, respectively; among them 13 adducts were observed in both rats and mice. trans-dG-N(2)-TAM (fr-2) and trans-dG(3')(P)-N(2)-N-desmethyl-TAM (fr-2) were two major adducts in both animals. Except for these two adducts, trans-dG-N(2)-TAM N-oxide (fr-2) was the third abundant adduct that accounted for 6.4% of the total adducts in mice, while this accounted for only 0.3% in rats. A trans-isomer (fr-1) and cis-isomers (fr-3 and -4) of dG(3')(P)-N(2)-TAM, dG(3')(P)-N(2)-N-desmethyl-TAM and dG(3')(P)-N(2)-TAM N-oxide were also detected as minor adducts in both animals except for cis-form of dG-N(2)-TAM N-oxide in rats. Although the administered dose for rats was 2.7-fold less than that for mice, the total adduct level of rats (216 adducts/10(8) nucleotides) were 3.8-fold higher than mice (56.2 adducts/10(8) nucleotides). Thus, these three types of tamoxifen adducts accounted for 95.0 and 92.5% of the total DNA adducts of the rats and mice, respectively. The formation of tamoxifen adducts primarily resulted from alpha-hydroxylation of tamoxifen.     

Preparation of oligodeoxynucleotides containing a diastereoisomer of alpha-(N(2)-2'-deoxyguanosinyl)tamoxifen by phosphoramidite chemical synthesis

Women treated with an antiestrogen tamoxifen (TAM) for endocrine therapy or prevention of breast cancer show an increased risk of developing endometrial cancer. TAM-DNA adducts have been detected in the liver of rodents treated with TAM and in the endometrium of women taking TAM. The major TAM adducts have been identified as diastereoisomers of trans- and cis-forms of alpha-(N(2)-deoxyguanosinyl)tamoxifen (dG-N(2)-TAM) and alpha-(N(2)-deoxyguanosinyl)-N-desmethyltamoxifen. In the study presented here, we prepared oligodeoxynucleotides containing a diastereoisomer of dG-N(2)-TAM by phosphoramidite chemical synthesis. Initially, the trans- and cis-forms of alpha-aminotamoxifen (alpha-NH(2)-TAM) were synthesized from alpha-hydroxytamoxifen using the Mitsunobu reaction followed by hydrolysis. Thereafter by coupling the trans- and cis-form of alpha-NH(2)-TAM with the DMT-derivative of 2-fluoro-(O(6)-2-(trimethylsilyl)ethyl)-2'-deoxyinosine, the trans- and cis-forms of DMT-dG-N(2)-TAM, respectively, were prepared in high yield and used in the preparation of the phosphoramidite precursors. Large quantities of oligodeoxynucleotides containing a trans- or a cis-form of dG-N(2)-TAM were prepared efficiently by automated DNA synthesizer. The incorporation of dG-N(2)-TAM adduct into the oligodeoxynucleotides was confirmed using (32)P-postlabeling/polyacrylamide gel electrophoresis analysis. These site-specifically modified oligodeoxynucleotides will be used for exploring biological properties and three-dimensional structure of TAM-DNA adducts.     

Tamoxifen-DNA adducts formed by alpha-acetoxytamoxifen N-oxide

DNA adduct formation is assumed to be a major carcinogenic event, leading to the development of endometrial cancer in breast cancer patients taking tamoxifen and healthy women enrolled in a tamoxifen chemopreventive trial. To determine whether DNA adducts were formed by tamoxifen, trans- and cis-alpha-acetoxytamoxifen N-oxides were synthesized as model-activated forms via major tamoxifen metabolites, tamoxifen N-oxide and alpha-hydroxytamoxifen N-oxide. When alpha-acetoxytamoxifen N-oxide was reacted with human DNA, at least three DNA adducts were detected by (32)P-postlabeling coupled with HPLC. The total amount of DNA adducts formed by trans-alpha-hydroxytamoxifen N-oxide was 1.5-fold higher than that formed by the cis form. Both trans- and cis-alpha-acetoxytamoxifen N-oxide reacted with 2'-deoxyguanosine, resulting in the formation of three adducts (fr-1, fr-2-1, and fr-2-2). These products were studied using mass spectroscopy and proton magnetic resonance spectroscopy. fr-1 was identified as a mixture of the epimers of trans-alpha-(N(2)-deoxyguanosinyl)tamoxifen N-oxide. fr-2-1 and fr-2-2 were determined to be epimers of cis-alpha-(N(2)-deoxyguanosinyl)tamoxifen N-oxide.     

Determination of tamoxifen--DNA adducts in leukocytes from breast cancer patients treated with tamoxifen

Tamoxifen (TAM), a widely used antiestrogen for breast cancer therapy and chemoprevention, increases the incidence of endometrial cancer in women. The formation of DNA adducts induced by tamoxifen may initiate endometrial cancer. To evaluate the genotoxic risk of TAM, the formation of DNA adducts in leukocytes was examined. Blood samples were collected from 47 breast cancer patients (61.7 +/- 12.5 years) taking TAM (20 mg/day; average duration until sampling, approximately 37 months) and 20 untreated patients (58.2 +/- 12.3 years), and their leukocyte DNA was analyzed by 32P-postlabeling/HPLC analysis. This assay resolves synthetic standards, trans- and cis-diastereoisomers of alpha-(N2-deoxyguanosinyl)tamoxifen 3'-monophosphate (dG3'P-N2-TAM), alpha-(N2-deoxyguanosinyl)-N-desmethyltamoxifen 3'-monophosphate (dG3'P-N2-N-dMeTAM), and alpha-(N2-deoxyguanosinyl)tamoxifen N-oxide 3'-monophosphate', and is capable of determining TAM adducts quantitatively. The detection limit of this assay is 0.6 adducts/10(9) nucleotides. trans-dG3'P-N2-TAM (fr-2; one of the two trans-isomers) was detected in six of 47 breast cancer patients treated with TAM. Among them, trans-dG(3'P-N2-N-dMeTAM (fr-2) was also detected in two patients. The total amounts of TAM-DNA adducts in the positive patients were 2.6 +/- 3.0 adducts/10(9) nucleotides. No adducts were detected in the controls. The presence of TAM-DNA adducts in the leukocyte DNA samples was confirmed using several 32P-postlabeling/HPLC systems.     

Synthesis of oligodeoxynucleotides containing a single 6alpha- or 6beta-diastereoisomer of N2-(estradiol-6-yl)-2'-deoxyguanosine

DNA damage induced by estrogens is associated with developing breast, ovary, and endometrial cancers. The quinone of 2-hydroxyestrogen (2-OHE), a major estrogen metabolite, produces 2-OHE-derived dG and dA adducts in DNA. N(2)-[Estradiol-6(alpha or beta)-yl]-2'-deoxyguanosine [dG-N(2)-6(alpha or beta)-E(2)] lacking a 2-OH moiety may also be formed through sulfonation of 6-hydroxyestrogen. To explore the biological properties of such estrogen-DNA adducts, oligodeoxynucleotides modified by estrogen-derived DNA adduct were prepared by chemical synthesis. Initially, 6alpha- and 6beta-aminoestradiol 17-acetate (6alpha- and 6beta-NH(2)-E(2) 17Ac) were prepared by reductive amination of 6-oxo-estradiol 3,17-diacetate. The DMT-phosphoramidite derivative of N(2)-(3,17-diacetoxyestradiol-6alpha-yl)-2'-deoxyguanosine and its 6beta-isomer were prepared by coupling 5'-O-(4,4'-dimethoxytrityl)-2-fluoro-O(6)-[2-(4-nitrophenyl)ethyl]-2'-deoxyinosine separately with 6alpha- and 6beta-forms of NH(2)-E(2) 17Ac, respectively, followed by selective acetylation of the steroidal 3-hydroxyl group. The desired oligodeoxynucleotide containing a single dG-N(2)-6alpha-E(2) or dG-N(2)-6beta-E(2) was prepared efficiently by an automated DNA synthesizer. Synthesis of these site-specifically modified oligodeoxynucleotides will benefit further research into the biological properties and three-dimensional structure of 6alpha- and 6beta-diastereoisomers of estrogen-DNA adducts.     

Characterization of DNA adducts formed in vitro by reaction of N-hydroxy-2-amino-3-methylimidazo[4,5-f]quinoline and N-hydroxy-2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline at the C-8 and N2 atoms of guanine.

The covalent binding of the carcinogenic N-hydroxy metabolites of 2-amino-3-methylimidazo-[4,5-f]quinoline (IQ) and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) to deoxynucleosides and DNA was investigated in vitro. Two major adducts were formed by the reaction of the N-acetoxy derivatives of IQ and MeIQx with deoxyguanosine (dG); however, no adducts were formed with deoxycytidine, deoxyadenosine, or thymidine. From proton NMR and mass spectroscopic characterization the adducts were identified as 5-(deoxyguanosin-N2-yl)-2-amino-3-methylimidazo[4,5-f]quinoline (dG-N2-IQ),N-(deoxyguanosin-8-yl)-2-amino-3-methylimidazo-[4,5-f]q uinoline (dG-C8-IQ), 5-(deoxyguanosin-N2-yl)-2-amino-3,8-dimethylimidazo[4,5-f]qu inoxaline (dG-N2-MeIQx), and N-(deoxyguanosin-8-yl)-2-amino-3,8-dimethylimidazo[4,5-f]qui noxaline (dG-C8-MeIQx). The level of dG-C8 adducts was approximately 8-10 times greater than the amount of dG-N2 adducts formed from the reaction of dG with the N-acetoxy derivatives of IQ and MeIQx. The C-8-substituted dG adduct was also the major adduct formed from reactions of DNA with N-acetoxy-IQ and N-acetoxy-MeIQx. Approximately 60-80% of the bound carcinogens were recovered from DNA as dG-C8 adducts upon enzymatic digestion. The dG-N2 adducts also were detected and accounted for approximately 4% of the bound IQ and 10% of the bound MeIQx. These results suggest that the relative contributions of the nitrenium and carbenium ion resonance forms as well as DNA macromolecular structure are major determinants for DNA adduct substitution sites. Investigations on adduct conformation of 1H NMR spectroscopy revealed that the anti form is preferred for the dG-N2 adducts of IQ and MeIQx, while the syn form is preferred for the dG-C8 adducts. The possible role of these adducts in the initiation of carcinogenesis is discussed.     

Genotoxic mechanism of tamoxifen in developing endometrial cancer

Increased risk of developing endometrial cancers has been observed in women treated with tamoxifen (TAM), a widely used drug for breast cancer therapy and chemoprevention. The carcinogenic effect may be due to genotoxic DNA damage induced by TAM. In fact, TAM-DNA adducts were detected in the endometrium of women treated with this drug. TAM is alpha-hydroxylated by cytochrome P450 3A4 followed by O-sulfonation by hydroxysteroid sulfotransferase, and reacts with guanine residues in DNA, resulting in the formation of alpha-(N2-deoxyguanosinyl)tamoxifen adducts. During this metabolic process, short-lived carbocations are produced at the ethyl moiety of TAM as reactive intermediates. TAM-DNA adducts promote primarily G -->T transversions in mammalian cells. The same mutations have been frequently detected at codon 12 of the K-ras gene in the endometrial tissue of women treated with this drug. TAM-DNA adducts, if not readily repaired, may act as initiators, leading to development of endometrial cancers. The reactivity of TAM metabolites with DNA is inhibited in toremifene, where the hydrogen atom has been replaced by a chlorine atom at the ethyl moiety. Therefore, toremifene may be a safer alternative to TAM. This article describes an overview of the mechanism of TAM-DNA adduct formation, mutagenic events of this adduct, and detection of TAM-DNA adducts in the endometrium of women treated with TAM.     

Characterization of the major DNA adduct formed by alpha-hydroxy-N-desmethyltamoxifen in vitro and in vivo

Tamoxifen is hepatocarcinogenic in rats and has been associated with an increased risk of endometrial cancer in women. Recent reports suggest that it may be genotoxic in humans. N-Desmethyltamoxifen is a major tamoxifen metabolite that has been proposed to be responsible for one of the major adducts detected in liver DNA of rats treated with tamoxifen. The metabolic activation of N-desmethyltamoxifen to DNA binding products may involve oxidation to alpha-hydroxy-N-desmethyltamoxifen followed by esterification. In the study presented here, we report the synthesis of alpha-hydroxy-N-desmethyltamoxifen and the characterization of the major adduct obtained from alpha-sulfoxy-N-desmethyltamoxifen in vitro as (E)-alpha-(deoxyguanosin-N(2)-yl)-N-desmethyltamoxifen. In addition, we use (32)P-postlabeling in combination with HPLC to compare the adducts formed in the livers of female Sprague-Dawley rats treated by gavage with tamoxifen or equimolar doses of alpha-hydroxy-N-desmethyltamoxifen. We conclude that one of the major adducts formed in vivo and previously suggested to derive from N-desmethyltamoxifen is chromatographically identical to alpha-(deoxyguanosin-N(2)-yl)-N-desmethyltamoxifen.     

Benzo[a]pyrene diol epoxide forms covalent adducts with deoxycytidylic acid by alkylation at both exocyclic amino N(4) and ring imino N-3 positions

The carcinogen 7r,8t-dihydroxy-9t,10t-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE) alkylates DNA at dGuo, dAdo, and dCyd. dCyd adducts, formed in small amounts, elute near the more abundant dGuo adducts. We isolated the dCyd adducts formed with dCMP. Each BPDE enantiomer forms three major adducts with dCMP, two cis and one trans. The trans adduct and one of the cis adducts form by alkylation at exocyclic N(4), while the second cis adduct is a dUrd adduct formed by alkylation at ring N-3 followed by deamination. Epoxide ring-opening geometries were assigned on the basis of halide and temperature effects on adduct yield, the sign of the major CD band, and benzo ring proton NMR coupling constants. One of each set of cis adducts is fluorescent (FL), and the other is nonfluorescent (NF). The trans and FL cis adducts have fluorescence quantum yields 40-50% of that of the BPDE hydrolysis product. The long wavelength UV maxima of the FL and NF cis adducts are red-shifted 1 and 3 nm relative to the trans adduct. (1)H NMR deuterium exchange experiments indicate that in the trans and FL cis adducts N(4)-H is coupled to C10-H. Adduct formation experiments with methyl-protected Cyd derivatives show that NF cis adducts result from alkylation at N-3. MS results, pK(a) measurements, and dUrd alkylation experiments indicate that the N-3 dCyd adducts spontaneously deaminate to dUrd adducts. NMR coupling constants show that in the NF cis adduct the C7 and C8 substituents are quasi equatorial and the C9 substituent is quasi axial, unlike in other cis BPDE adducts. (1)H NOESY spectra of the (-)-BPDE NF cis adduct reveal that it exists in two conformers. Molecular modeling shows that the conformers result from two low-energy conformations of very similar energies with the pyrimidine in opposite orientations, separated by significant barriers to rotation of the uracil moiety.     

Analysis and quantification of DNA adducts of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline in liver of rats by liquid chromatography/electrospray tandem mass spectrometry

Liquid chromatography with electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) was used to measure DNA adducts of the carcinogen 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) with a microbore C-18 reversed-phase column. Quantification of the isomeric adducts N-(deoxyguanosin-8-yl)-2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (dG-C8-MeIQx) and 5-(deoxyguanosin-N(2)-yl)-2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (dG-N(2)-MeIQx) was achieved using synthetic, isotopically labeled internal standards. The reaction of the N-acetoxy ester of 2-(hydroxyamino)-3,8-dimethylimidazo[4,5-f]quinoxaline (HONH-MeIQx) with calf thymus DNA (ct DNA) resulted in formation of these adducts in a ratio of 5:1 (dG-C8-MeIQx:dG-N(2)-MeIQx). The detection limit by LC/ESI-MS/MS in the selected reaction monitoring (SRM) mode ([MH(+) --> MH - 116](+)) (loss of deoxyribose) approached 500 fg (1 fmol) of adduct standard, and 1 adduct per 10(8) DNA bases using 100 microg of DNA following solid-phase extraction. The SRM analysis of rat liver DNA 24 h after an oral dose of MeIQx (10 and 0.5 mg/kg) revealed the presence of isomeric dG-MeIQx adducts at levels of 3.07 +/- 0.84 and 0.45 +/- 0.27 adducts per 10(7) bases, respectively. LC/ESI-MS/MS product ion spectra were acquired on both adducts from the elevated dose of MeIQx for unambiguous adduct identification. The contribution of dG-N(2)-MeIQx to the total adducts in vivo was significantly more important than that observed in vitro. dG-C8-MeIQx was the principal adduct formed at the 10 mg/kg dose, (dG-C8-MeIQx:dG-N(2)-MeIQx (3:2)); however, dG-N(2)-MeIQx was the major lesion detected at the 0.5 mg/kg dose (dG-C8-MeIQx:dG-N(2)-MeIQx 1:10). The striking differences between the relative amounts of dG-C8-MeIQx and dG-N(2)-MeIQx formed in vivo as a function of dose suggest that reactive esters of HONH-MeIQx other than N-acetoxy-MeIQx may be formed in vivo and react preferentially with the N(2) atom of guanine, or that dG-C8-MeIQx is removed at a significantly more rapid rate than dG-N(2)-MeIQx. The dG-N(2)-MeIQx adduct, previously thought to be a minor adduct, is likely to be an important contributor to the genotoxic damage of MeIQx.     

Identification and characterization of deoxyguanosine-crotonaldehyde adducts. Formation of 7,8 cyclic adducts and 1,N2,7,8 bis-cyclic adducts.

Crotonaldehyde, a chemically reactive alpha,beta-unsaturated carbonyl compound, is an important industrial chemical and a ubiquitous environmental pollutant. It has been shown to be carcinogenic and mutagenic. We have studied the reaction of crotonaldehyde with nucleosides and 5'-mononucleotides and found three different types of adducts with deoxyguanosine and 2'-deoxyguanosine 5'-monophosphate. No adducts could be isolated either with nucleosides other than deoxyguanosine or with nucleotides other than 2'-deoxyguanosine 5'-monophosphate. With crotonaldehyde, deoxyguanosine produced 1,N2 and 7,8 adducts as well as 1,N2/7,8 bis-adducts. The 1,N2 adducts were mixtures of diastereomers: one pair in which the substituents in the newly formed ring were trans [adduct Ia (6S,8S) and (6R,8R)], about 94%, and another pair Ib in which they were cis. In the case of the 7,8-adducts IIa,b, the ribose was cleaved and a mixture of isomers in which the substituents were cis-IIa and trans-IIb (2:1) in the newly formed tetrahydropyrrole ring was observed. A 3:2 cis-IIIa and trans-IIIb mixture of 1,N2,7,8 bis-adducts was found with the isomerism in the newly formed tetrahydropyrrole ring in analogy to the 7,8 adducts IIa,b. The corresponding bis-adduct with the cis form in the newly formed tetrahydropyrimidine ring was not observed.     

Inefficient repair of tamoxifen-DNA adducts in rats and mice.

A long-term treatment with tamoxifen (TAM) to women increases the risk of developing endometrial cancer. The cancer may result from genotoxic damage induced by this drug. In fact, TAM-DNA adducts were detected in the liver of rats treated with TAM and initiated to develop hepatocellular carcinomas. To explore the distribution and repair rate of TAM-DNA adducts, the level of TAM-DNA adducts in all tissues of rats and mice was monitored for 28 days and 7 days, respectively, after the termination of TAM treatment, using 32P-postlabeling/polyacrylamide gel electrophoresis and 32P-postlabeling/HPLC analyses. TAM-DNA adducts were formed specifically in the liver of rodents. In rats, the level of hepatic TAM-DNA adducts was decreased only to 43% in 28 days, indicating that the half-life of adducts was approximately 25 days. Among trans [fraction (fr)-1 and fr-2]- and cis (fr-3 and fr-4)-isoforms of TAM-DNA adducts, a trans-form (fr-1) was removed much more slowly than other adducts, indicating that the repair rate of TAM-DNA adducts varied depending on the structure of isoforms. The repair rate of TAM-DNA adducts was also compared between nucleotide excision repair-deficient (Xpc knockout) and wild mice. Although the level of hepatic TAM-DNA adducts observed with Xpc knockout mice was slightly higher than that of the wild type, the removal of TAM-DNA adducts in both mice was only 20% in 7 days. Thus, TAM-DNA adducts are not efficiently repaired from the targeted tissue, leading to the development of cancer.     

Synthesis and reactivity of a potential carcinogenic metabolite of tamoxifen: 3,4-dihydroxytamoxifen-o-quinone

Although tamoxifen is approved for the treatment of hormone-dependent breast cancer as well as for the prevention of breast cancer in high-risk women, several studies in animal models have shown that tamoxifen is heptocarcinogenic, and in humans, tamoxifen has been associated with an increased risk of endometrial cancer. One potential mechanism of tamoxifen carcinogenesis could involve metabolism of tamoxifen to 3,4-dihydroxytamoxifen followed by oxidation to a highly reactive o-quinone which has the potential to alkylate and/or oxidize cellular macromolecules in vivo. In the study presented here, we synthesized the 3,4-dihydroxytamoxifen, prepared its o-quinone chemically and enzymatically, and studied the reactivity of the o-quinone with GSH and deoxynucleosides. The E (trans) and Z (cis) isomers of 3,4-dihydroxytamoxifen were synthesized using a concise synthetic pathway (four steps). This approach is based on the McMurry reaction between the key 4-(2-chloroethoxy)-3,4-methylenedioxybenzophenone and propiophenone, followed by selective removal of the methylenedioxy ring of (E, Z)-1-[4-[2-(N,N-dimethylamino)ethoxy]phenyl]-1-(3, 4-methylenedioxyphenyl)-2-phenyl-1-butene with BCl(3). Oxidation of 3,4-dihydroxytamoxifen by activated silver oxide or tyrosinase gave 3,4-dihydroxytamoxifen-o-quinone as a mixture of E and Z isomers. The resulting o-quinone has a half-life of approximately 80 min under physiological conditions. Reaction of the o-quinone with GSH gave two di-GSH conjugates and three mono GSH conjugates. Incubation of 3,4-dihydroxytamoxifen with GSH in the presence of microsomal P450 gave the same GSH conjugates which were also detected in incubations with human breast cancer cells (MCF-7). Reaction of 3, 4-dihydroxytamoxifen-o-quinone with deoxynucleosides gave only thymidine and deoxyguanosine adducts; neither deoxyadenosine nor deoxycytosine adducts were detected. Preliminary studies conducted with human breast cancer cell lines showed that 3, 4-dihydroxytamoxifen exhibited cytotoxic potency similar to that of 4-hydroxytamoxifen and tamoxifen in an estrogen receptor negative (ER(-)) cell line (MDA-MB-231); however, in the ER(+) cell line (MCF-7), the catechol metabolite was about half as toxic as the other two compounds. Finally, in the presence of microsomes and GSH, 4-hydroxytamoxifen gave predominantly quinone methide GSH conjugates as reported in the previous paper in this issue [Fan, P. W., et al. (2000) Chem. Res. Toxicol. 13, XX-XX]. However, in the presence of tyrosinase and GSH, 4-hydroxytamoxifen was primarily converted to o-quinone GSH conjugates. These results suggest that the catechol metabolite of tamoxifen has the potential to cause cytotoxicity in vivo through formation of 3,4-dihydroxytamoxifen-o-quinone.     

Halogenated analogues of tamoxifen: synthesis, receptor assay, and inhibition of MCF7 cells.

This study was conducted to develop a ligand for imaging estrogen-receptor-positive breast tumors by positron emission tomography or single photon emission computed tomography. We synthesized fluoro and iodo analogues of tamoxifen, and these halogenated analogues produced greater affinity for binding to the receptor than tamoxifen. Values of the inhibition affinity constants were as follows: tamoxifen, 15,000 nM; fluoromethyl-N,N-diethyltamoxifen, 2500 nM for the cis isomer and 500 nM for the trans isomer; and iodomethyl-N,N-diethyltamoxifen, 1500 nM for the cis isomer and 1000 nM for the trans isomer. In studies of human MCF7 breast tumor cell growth, concentrations that inhibited tumor growth in 50% of the cases were as follows: tamoxifen, 11 microM; fluoromethyl-N,N-diethyltamoxifen, 4.5 and 11.8 microM for the cis and trans isomers, respectively; and iodomethyl-N,N-diethyltamoxifen, 2.4 and 6.3 microM for the cis and trans isomers, respectively. These studies suggest that both fluoro and iodo analogues of tamoxifen may be useful diagnostic compounds for predicting the response of estrogen-receptor-positive breast tumors to tamoxifen analogues used in chemotherapy.     

Incorporation of N2-deoxyguanosine metabolic adducts of 2-aminonaphthalene and 2-aminofluorene into oligomeric DNA

In previous work we described an efficient procedure for the synthesis of the respective N2 and N6 adducts of 2'-deoxyguanosine (dG) and 2'-deoxyadenosine (dA) derived from a series of aminoaryl compounds. We now outline methods for the site-specific introduction into oligomeric DNA of the adducts dG-N2-AN (6), dG-N2-AAN (7), dG-N2-AF (8), and dG-N2-AAF (9) derived from 2-aminonaphthalene (2-AN) or 2-aminofluorene (2-AF). For the 2-AN adduct 7, containing an acetylamino group, the 5'-O-4,4'-dimethoxytrityl- (DMT-) 3'-O-phosphoramidite (14) required for automated DNA synthesis was synthesized in high yield via the sequence 10-->11-->14. On the other hand, introduction of the desacetyl adduct 6 into oligomeric DNA was accomplished via the N-trifluoroacetyl-DMT-phosphoramidite derivative 18. This involved a similar sequence (10-->15-->18) except that the order of the reactions was changed to avoid a decomposition that occurred when the silyl-protected amino derivative 11 was treated with trifluoroacetic anhydride. In the 2-AF series the 5'-O-DMT-3'-O-phosphoramidites 27a and 27b, related to 8 and 9, were prepared by similar methods. Again, however, the order of the reactions was changed to avoid the extreme insolubility associated with the N2-[3-(2-acetylaminofluoren-3-yl)]dG (dG-N2-AAF, 9) adduct that we had noted previously. The incorporation into oligomeric DNA of the acetylamino compounds 7 and 9 proceeded smoothly and in high yield (95-100%). By contrast, the trifluoroacetyl analogues led in both the naphthyl and fluorenyl series to a mixture of oligomers containing the desired free amino adduct (6 or 8) accompanied by the N-acetyl adduct (7 or 9, respectively, after the deprotection step), indicating secondary acetylation by the capping agent acetic anhydride.     

Supercoiled DNA promotes formation of intercalated cis-N2-deoxyguanine adducts and base-stacked trans-N2-deoxyguanine adducts by (+)-7R,8S-dihydrodiol-9S,10R-epoxy-7,8,9,10-tetra- hydrobenzo[a]pyrene

The highly reactive and mutagenic benzo[a]pyrene metabolite, (+)-7R,8S-dihydroxy-9S,10R-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE), forms predominantly N2-deoxyguanine DNA adducts in two stereoisomeric configurations (cis and trans). In previous in vitro assays using oligonucleotide substrates site specifically modified with cis- and trans-BPDE adducts, the nucleotide excision repair (NER) systems of eukaryotes and prokaryotes incise cis-BPDE adducts more efficiently than trans-BPDE adducts [Hess, et al. (1997) Mol. Cell Biol 17, 7069; Zou, et al. (2001) Biochemistry 40, 2923). We investigated the influence of DNA secondary structure on stereospecificity of BPDE adduct formation, and incision of BPDE adducts by the prokaryotic UvrABC NER endonuclease was examined. BPDE adducts formed at low density on supercoiled plasmids were incised 6-7-fold better by the thermoresistant Bacillus caldotenaxUvrABC than were BPDE adducts formed on linear DNA. Linearizing supercoiled plasmid DNAs after BPDE adduct formation did not diminish incision efficiency. These results suggested that configuration and/or conformation of adducts formed on linear and supercoiled DNAs differed. This hypothesis was confirmed by low temperature fluorescence spectroscopy of adducted supercoiled and linear DNAs. Spectroscopic results indicated that intercalated cis-BPDE adducts as well as base-stacked trans-BPDE adducts formed more abundantly in supercoiled DNA than in linear DNA. A higher cis to trans adduct ratio in supercoiled DNA was confirmed by high resolution [32P]postlabeling analyses. These results demonstrate that DNA secondary structure influences both configuration and conformation of BPDE adducts formed at low density (approximately 1 adduct/kbp) and suggests that the ratio of cis- to trans-BPDE adducts and amount of base-stacked trans adducts formed under physiological exposure conditions may be higher than inferred from high dose experiments.     

DNA adduct formation in the livers of female Sprague-Dawley rats treated with toremifene or alpha-hydroxytoremifene

Toremifene, an analogue of tamoxifen in which the ethyl side chain has been replaced with a 2-chloroethyl substituent, is used as a chemotherapeutic agent in postmenopausal women with advanced breast cancer. Toremifene is metabolized in a manner similar to that of tamoxifen, with alpha-hydroxytoremifene being a predominant metabolite in incubations in vitro. DNA adducts have been detected previously in liver DNA upon the administration of toremifene to rats; however, the identity of these adducts is unknown. In the present study, we have characterized the DNA adducts produced by alpha-hydroxytoremifene and have compared the extent of hepatic DNA adduct formation in rats administered toremifene, alpha-hydroxytoremifene, or tamoxifen. alpha-Hydroxytoremifene was synthesized, further activated by sulfation, and then reacted with salmon testis DNA. After enzymatic hydrolysis to deoxynucleosides, HPLC analysis indicated the formation of two major DNA adducts, which were characterized as (E)- and (Z)-alpha-(deoxyguanosin-N2-yl)toremifene on the basis of 1H NMR and mass spectral analyses. To assess the formation of toremifene DNA adducts in vivo, female Sprague-Dawley rats were treated intraperitoneally with toremifene, alpha-hydroxytoremifene, or tamoxifen. 32P-Postlabeling analyses of hepatic DNA from the tamoxifen-treated rats indicated three DNA adducts at a total level of 2,200 +/- 270 adducts/108 nucleotides. DNA adducts were not detected (<5 adducts/108 nucleotides) in the livers of rats treated with toremifene. Two DNA adducts, of which the major one coeluted with the 3',5'-bis-phosphate of (E)-alpha-(deoxyguanosin-N2-yl)toremifene, were present at a level of 57 +/- 12 adducts/108 nucleotides in hepatic DNA from rats administered alpha-hydroxytoremifene. The low level of hepatic DNA adduct formation observed with both toremifene and alpha-hydroxytoremifene, as compared to that with tamoxifen, may be due to the limited esterification of alpha-hydroxytoremifene and/or the poor reactivity of alpha-sulfoxytoremifene.