Anti-breast cancer potential of SS5020, a novel benzopyran antiestrogen.

Treatment with tamoxifen (TAM) increases the risk of developing endometrial cancer in women. The carcinogenic effect is thought to involve initiation and/or promotion resulting from DNA damage induced by TAM as well as its estrogenic action. To minimize this serious side-effect while increasing the anti-breast cancer potential, a new benzopyran antiestrogen, 2E-3-{4-[(7-hydroxy-2-oxo-3-phenyl-2H-chromen-4-yl)-methyl]-phenyl}-acrylic acid (SS5020), was synthesized. Unlike TAM, SS5020 exhibits no genotoxic activity to damage DNA. Furthermore, SS5020 does not present significant uterotrophic potential in rats; in contrast, the structurally related compounds, TAM, toremifene, raloxifene (RAL) and SP500263 all have uterotrophic activity. At the human equivalent molar dose of TAM (0.33 or 1.0 mg/kg), SS5020 had much stronger antitumor potential than those same antiestrogens against 7,12-dimethylbenz(a)anthracene-induced mammary carcinoma in rats. The growth of human MCF-7 breast cancer xenograft implanted into athymic nude mice was also effectively suppressed by SS5020. SS5020, lacking genotoxic and estrogenic actions, could be a safer and stronger antiestrogen alternative to TAM and RAL for breast cancer therapy and prevention.

Equine estrogen-induced mammary tumors in rats.

Long-term hormone replacement therapy is associated with an increased risk of breast, ovarian and endometrial cancers in women. Equine estrogens are a principal component of hormone replacement therapy; however, their tumorigenic potential toward mammary tissue and reproductive organs has not been extensively explored. A pellet containing equilin was inserted under the skin of female ACI rats and the development of mammary tumors was monitored. Histological examination revealed premalignant lesions such as apocrine metaplasia in whole-mount preparations of mammary gland from the equilin-treated rats. ACI rats given 10mg equilin developed palpable mammary tumors at 13 weeks of treatment, and 37.5% of the rats developed mammary tumors within 15 weeks. For 2.5mg equilin, palpable tumors were observed in 8.3% of the rats after 8 weeks' treatment; the frequency was lower than that (42.9%) observed with 2.5mg E(2). No tumors were observed in the untreated rats. Evidently, equilin is a mammary carcinogen, and this potential may be associated with development of breast and reproductive cancers in women receiving hormone replacement therapy.

Anti-breast cancer potential of SS1020, a novel antiestrogen lacking estrogenic and genotoxic actions.

Long-term treatment with tamoxifen (TAM) increases the risk of developing endometrial cancer in women. Several antiestrogens developed in last decades have been discontinued from clinical testing because of their undesirable effects on the uterus. To avoid such serious side-effect while increasing the drug's anti-breast cancer potential, new triphenylethylene antiestrogens, 2E-3-{4-[(E)-4-chloro-1-(4-hydroxyphenyl)-2-phenylbut-1-enyl]-phenyl} acrylic acid (SS1020) and 2E-3-{4-[(Z)-4-chloro-1,2-diphenylbut-1-enyl]phenyl}acrylic acid (SS1010), were designed as safer alternatives. Unlike TAM, SS1020 does not present significant uterotrophic potential in rats; in contrast, SS1010, a compound removing a 4-OH moiety from SS1020, represented weak uterotrophic activity. The structurally related compounds 4-hydroxytamoxifen, toremifene, ospemifene, raloxifene (RAL) and GW5638 all have uterotrophic activity. In addition, SS1020 and SS1010 exhibit no genotoxic activity to damage hepatic DNA in rats. Therefore, SS1020 was selected as a safer antiestrogen candidate and used for evaluating the antitumor potential in animals. At the human equivalent doses of TAM, SS1020 had antitumor potential much higher than that of TAM, RAL and GW5638 against 7,12-dimethylbenz(a)anthracene-induced mammary carcinoma in rats. The growth of human MCF-7 breast cancer xenograft implanted into athymic nude mice was also effectively suppressed by SS1020. SS1020, lacking estrogenic and genotoxic actions and having strong antitumor potency superior to that of TAM and RAL, could be a safer alternative for breast cancer therapy and prevention.

Miscoding properties of 6alpha- and 6beta-diastereoisomers of the N(2)-(estradiol-6-yl)-2'-deoxyguanosine DNA adduct by Y-family human DNA polymerases.

Treatment with estrogen increases the risk of breast, ovary, and endometrial cancers in women. DNA damage induced by estrogen is thought to be involved in estrogen carcinogenesis. In fact, Y-family human DNA polymerases (pol) eta and kappa, which are highly expressed in the reproductive organs, miscode model estrogen-derived DNA adducts during DNA synthesis. Since the estrogen-DNA adducts are a mixture of 6alpha- and 6beta-diastereoisomers of dG-N(2)-6-estrogen or dA-N(6)-6-estrogen, the stereochemistry of each isomeric adduct on translesion synthesis catalyzed by DNA pols has not been investigated. We have recently established a phosphoramidite chemical procedure to insert 6alpha- or 6beta-isomeric N(2)-(estradiol-6-yl)-2'-deoxyguanosine (dG-N(2)-6-E(2)) into oligodeoxynucleotides. Using such site-specific modified oligomer as a template, the specificity and frequency of miscoding by dG-N(2)-6alpha-E(2) or dG-N(2)-6beta-E(2) were explored using pol eta and a truncated form of pol kappa (pol kappaDeltaC). Translesion synthesis catalyzed by pol eta bypassed both the 6alpha- and 6beta-isomers of dG-N(2)-6-E(2), with a weak blockage at the adduct site, while translesion synthesis catalyzed by pol kappaDeltaC readily bypassed both isomeric adducts. Quantitative analysis of base substitutions and deletions occurring at the adduct site showed that pol kappaDeltaC was more efficient than pol eta by incorporating dCMP opposite both 6alpha- and 6beta-isomeric dG-N(2)-6-E(2) adducts. The miscoding events occurred more frequently with pol eta, but not with pol kappaDeltaC. Pol eta promoted incorporation of dAMP and dTMP at both the 6alpha- and 6beta-isomeric adducts, generating G --> T transversions and G --> A transitions. One- and two-base deletions were also formed. The 6alpha-isomeric adduct promoted slightly lower frequency of dCMP incorporation and higher frequency of dTMP incorporation and one-base deletions, compared with the 6beta-isomeric adduct. These observations were supported by steady-state kinetic studies. Taken together, the miscoding property of the 6alpha-isomeric dG-N(2)-6-E(2) is likely to be similar to that of the 6beta-isomeric adduct.

Oxidative DNA damage in XPC-knockout and its wild mice treated with equine estrogen.

Long-term hormone replacement therapy with equine estrogens is associated with a higher risk of breast, ovarian, and endometrial cancers. Reactive oxygen species generated through redox cycling of equine estrogen metabolites may damage cellular DNA. Such oxidative stress may be linked to the development of cancers in reproductive organs. Xeroderma pigmentosa complementation group C-knockout ( Xpc-KO) and wild-type mice were treated with equilenin (EN), and the formation of 7,8-dihydro-8-oxodeoxyguanosine (8-oxodG) was determined as a marker of typical oxidative DNA damage, using liquid chromatography electrospray tandem mass spectrometry. The level of hepatic 8-oxodG in wild-type mice treated with EN (5 or 50 mg/kg/day) was significantly increased by approximately 220% after 1 week, as compared with mice treated with vehicle. In the uterus also, the level of 8-oxodG was significantly increased by more than 150% after 2 weeks. Similar results were observed with Xpc-KO mice, indicating that Xpc does not significantly contribute to the repair of oxidative damage. Oxidative DNA damage generated by equine estrogens may be involved in equine estrogen carcinogenesis.

Increased antitumor potential of the raloxifene prodrug, raloxifene diphosphate.

Raloxifene (RAL) significantly reduced the incidence of breast cancer in women at high risk of developing the disease. Unlike tamoxifen (TAM), an increased incidence of endometrial cancer was not observed in women treated with RAL. However, RAL, having two hydroxyl moieties, can be conjugated rapidly through phase II metabolism and excreted, making it difficult to achieve adequate bioavailability by oral administration in humans. As a result, higher doses must be administered to obtain an efficacy equivalent to that achieved with TAM. To improve oral bioavailability and antitumor potential, RAL diphosphate was prepared as a prodrug. RAL diphosphate showed several orders of magnitude lower binding potential to both ER alpha and ER beta and weak antiproliferative potency on cultured human MCF-7 and ZR-75-1 breast cancer cells, as compared to RAL. However, RAL diphosphate has a much higher bioavailability than RAL, endowing it with higher antitumor potential than RAL against both 7,12-dimethylbenz(a)anthracene-induced mammary carcinoma in rats and human MCF-7 breast cancer implanted in athymic nude mice. The RAL prodrug may provide greater clinical benefit for breast cancer therapy and prevention.

Mechanism of translesion synthesis past an equine estrogen-DNA adduct by Y-family DNA polymerases.

4-Hydroxyequilenin (4-OHEN)-dC is a major, potentially mutagenic DNA adduct induced by equine estrogens used for hormone replacement therapy. To study the miscoding property of 4-OHEN-dC and the involvement of Y-family human DNA polymerases (pols) eta, kappa and iota in that process, we incorporated 4-OHEN-dC into oligodeoxynucleotides and used them as templates in primer extension reactions catalyzed by pol eta, kappa and iota. Pol eta inserted dAMP opposite 4-OHEN-dC, accompanied by lesser amounts of dCMP and dTMP incorporation and base deletion. Pol kappa promoted base deletions as well as direct incorporation of dAMP and dCMP. Pol iota worked in conjunction with pol kappa, but not with pol eta, at a replication fork stalled by the adduct, resulting in increased dTMP incorporation. Our results provide a direct evidence that Y-family DNA pols can switch with one another during synthesis past the lesion. No direct incorporation of dGMP, the correct base, was observed with Y-family enzymes. The miscoding potency of 4-OHEN-dC may be associated with the development of reproductive cancers observed in women receiving hormone replacement therapy.

Translesion synthesis past tamoxifen-derived DNA adducts by human DNA polymerases eta and kappa.

The long-term treatment of tamoxifen (TAM), widely used for adjuvant chemotherapy and chemoprevention for breast cancer, increases a risk of developing endometrial cancer. A high frequency of K-ras mutations has been observed in the endometrium of women treated with TAM. Human DNA polymerase (pol) eta and pol kappa are highly expressed in the reproductive organs and are associated with translesion synthesis past bulky DNA adducts. To explore the miscoding properties of alpha-(N2-deoxyguanosinyl)tamoxifen (dG-N2-TAM), a major TAM-DNA adduct, site-specifically modified oligodeoxynucleotides containing a single diastereoisomer of trans or cis forms of dG-N2-TAM were prepared by phosphoramidite chemical procedure and used as templates. The primer extension reaction catalyzed by pol kappa deltaC, a truncated form of pol kappa, extended more efficiently past the adduct than that of pol eta by incorporating dCMP, a correct base, opposite the adduct. With pol eta, all diastereoisomers of dG-N2-TAM promoted small amounts of direct incorporation of dAMP and deletions. With pol kappa deltaC, dG-N2-TAM promoted small amounts of dTMP and/or dAMP incorporations and deletions. The miscoding properties varied depending on the diastereoisomer of dG-N2-TAM adducts and the DNA pol used. Steady-state kinetic studies were also performed using either the nonspecific sequence or the K-ras gene sequence containing a single dG-N2-TAM at the second base of codon 12. With pol eta, the bypass frequency past the dA x dG-N2-TAM pair positioned in the K-ras sequence was only 2.3 times lower than that for the dC x dG-N2-TAM pair, indicating that dG-N2-TAM in the K-ras sequence has higher miscoding potential than that in the nonspecific sequence. However, with pol kappa deltaC, the bypass frequency past the dC x dG-N2-TAM pair was higher than that of the dT x dG-N2-TAM pair in both sequences. The properties of pol eta and pol kappa are consistent with the mutagenic events attributed to TAM-DNA adducts.

Antiestrogens and the formation of DNA damage in rats: a comparison.

Tamoxifen (TAM) has been used as an agent for the treatment and prevention of breast cancer. However, long-term treatment of TAM in women increases the risk of developing endometrial cancer. The secondary cancer may be due to the genotoxicity of TAM. To find safer alternatives, four selective estrogen receptor modulators (SERMs), 4-hydroxytamoxifen (4-OHTAM), toremifene (TOR), raloxifene (RAL), and ICI 182,780, were administered to rats with an equimolar dose of TAM [54 micromol/kg (20 mg/kg)/day, p.o. for 7 days]. To evaluate the genotoxicity of each SERM, the presence of bulky DNA adducts was determined by (32)P-postlabeling/polyacrylamide gel electrophoresis and (32)P-postlabeling/high-performance liquid chromatography. The formation of 7,8-dihydro-8-oxodeoxyguanosine (8-oxodG) was analyzed as a marker of typical oxidative damage, using liquid chromatography electrospray tandem mass spectrometry. Among the SERMs, bulky DNA adducts were detected in the livers of rats treated with TAM; the total amount of TAM-DNA adducts was 26.1 adducts/10(7) nucleotides. However, with a detection limit of approximately 2 adducts/10(9) nucleotides, no bulky DNA adducts were observed with 4-OHTAM, TOR, RAL, or ICI 182,780. In addition, no significant increase of hepatic 8-oxodG lesions was detected in rats treated with any of the antiestrogens. Therefore, TOR, RAL, and ICI 182,780 are likely to be less genotoxic than TAM.

Translesion synthesis past equine estrogen-derived 2'-deoxyadenosine DNA adducts by human DNA polymerases eta and kappa.

Hormone replacement therapy (HRT) increases the risk of developing breast, ovarian, and endometrial cancers. Equilin and equilenin are the major components of the widely prescribed drug used for HRT. 4-Hydroxyequilenin (4-OHEN), a major metabolite of equilin and equilenin, promotes 4-OHEN-modified dC, dA, and dG DNA adducts. These DNA adducts were detected in breast tumor and adjacent normal tissues of several patients receiving HRT. We have recently found that the 4-OHEN-dC DNA adduct is a highly miscoding lesion generating C --> T transitions and C --> G transversions. To explore the mutagenic potential of another major 4-OHEN-dA adduct, site-specifically modified oligodeoxynucleotides containing a single diastereoisomer of 4-OHEN-dA (Pk-1, Pk-2, and Pk-3) were prepared by a postsynthetic method and used as DNA templates for primer extension reactions catalyzed by human DNA polymerase (pol) eta and kappa that are highly expressed in the reproductive organs. Primer extension catalyzed by pol eta or pol kappa occurred rapidly on the unmodified template to form fully extended products. With the major 4-OHEN-dA-modified templates (Pk-2 and Pk-3), primer extension was retarded prior to the lesion and opposite the lesion; a fraction of the primers was extended past the lesion. Steady-state kinetic studies with pol eta and pol kappa indicated that dTMP, the correct base, was preferentially incorporated opposite the 4-OHEN-dA lesion. In addition, pol eta and pol kappa bypassed the lesion by incorporating dAMP and dCMP, respectively, opposite the lesion and extended past the lesion. The relative bypass frequency past the 4-OHEN-dA lesion with pol eta was at least 2 orders of magnitude higher than that observed with pol kappa. The bypass frequency past Pk-2 was more efficient than that past Pk-3. Thus, 4-OHEN-dA is a miscoding lesion generating A --> T transversions and A --> G transitions. The miscoding frequency and specificity of 4-OHEN-dA varied depending on the stereoisomer of the 4-OHEN-dA adduct and DNA polymerase used.

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.

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.

Formation of tamoxifen-DNA adducts via O-sulfonation, not O-acetylation, of alpha-hydroxytamoxifen in rat and human livers.

Tamoxifen (TAM) is used as the standard endocrine therapy for breast cancer patients and as a chemopreventive agent for women at high risk for this disease. Unfortunately, treatment of TAM increases the incidence of endometrial cancer; this may be due to the genotoxic damage induced by TAM metabolites. Formation of TAM-DNA adducts in rat liver correlates with the development of hepatocarcinoma. TAM-DNA adducts are proposed to be formed through O-sulfonation and/or O-acetylation of alpha-hydroxylated TAM and its metabolites. However, the role of O-sulfonation and O-acetylation in the formation of TAM-DNA adducts has not been extensively investigated. Rat or human hydroxysteroid sulfotransferases (HST), acetyltransferases, and liver cytosol were incubated with calf thymus DNA, alpha-OHTAM, and either 3'-phosphoadenosine 5'-phosphosulfate (PAPS) or acetyl coenzyme A (acetyl-CoA) as a cofactor and analyzed for TAM-DNA adduct formation, using 32P postlableling/polyacrylamide gel electrophoresis analysis. TAM-DNA adduct was formed when PAPS, not acetyl-CoA, was used. No TAM-DNA adducts were produced using human N-acetyltransferase I and II. HST antibody inhibited approximately 90% of TAM-DNA adduct formation generated by the cytosol or HST, suggesting that HST is primarily involved in the formation of TAM-DNA adducts. The formation of TAM-DNA adducts with rat liver cytosol and HST was much higher than that of human liver cytosol and HST. Our results indicate that TAM-DNA adducts are formed via O-sulfonation, not O-acetylation, of alpha-hydroxylated TAM and its metabolites.

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.

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.

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.

Synthesis of oligodeoxynucleotides containing a single diastereoisomer of alpha-(N2-2'-deoxyguanosinyl)-N-desmethyltamoxifen.

A phosphoramidite chemical synthesis of oligodeoxynucleotides containing a diastereoisomer of (E)-alpha-(N(2)-deoxyguanosinyl)-N-desmethyltamoxifen, a major tamoxifen (TAM)-derived DNA adduct in animal and women treated with TAM, was described. The site-specifically modified oligodeoxynucleotide can be used for mutagenesis, DNA repair, and 3D structural studies and also as standard for quantitative analysis of TAM-DNA adducts in animal and human.

Asymmetric dearomatization of the furan ring promoted by conjugate organolithium addition to (menthyloxy)(3-furyl)carbene complexes of chromium.

The sequential low-temperature addition reaction of an organolithium compound and methyl triflate to (menthyloxy)(3-furyl)carbene complexes of chromium and tungsten proceeded with excellent regioselectivity (1,4-addition) and diastereoselectivity (2,3-trans disposition of the nucleophile and electrophile groups) to afford new 2,3-disubstituted (2,3-dihydro-3-furyl)carbene complexes. In addition, a high degree of diastereofacial selectivity was achieved by employing alkenyllithium compounds. After detachment of both the metal fragment and the chiral auxiliary group, trisubstituted 2,3-dihydrofuran derivatives containing a quaternary stereogenic center at the C3 position were obtained. The characterization, including X-ray crystallography, of a novel type of stable four-membered chelate (eta(2)-alkene)tetracarbonylcarbene complex of chromium is also reported.

Identification of tamoxifen-DNA adducts in monkeys treated with tamoxifen.

The risk of developing endometrial cancer is increased in breast cancer patients treated with tamoxifen (TAM) and in healthy women undergoing TAM chemoprevention. We have detected previously TAM-DNA adducts in the endometrium of women receiving TAM (Shibutani et al., Carcinogenesis, 21: 1461-1467, 2000). To investigate the genotoxic damage induced by TAM in the uterus and other tissues of primates, we gave adult female cynomolgus monkeys six times the human-equivalent dose of TAM (2 mg/kg body weight/day) for 30 days. DNA samples were prepared from the uterus, ovary, liver, kidney, and brain cortex of three TAM-exposed monkeys and one control monkey and were analyzed as coded specimens. To identify the TAM-DNA adducts, we established a new high-performance liquid chromatography gradient system for (32)P-postlabeling/high-performance liquid chromatography analysis, which can resolve the trans- and cis-diastereoisomers of alpha-(N(2)-deoxyguanosinyl)TAM (dG-N(2)-TAM), alpha-(N(2)-deoxyguanosinyl)-N-desmethylTAM, and alpha-(N(2)-deoxyguanosinyl)tamoxifen N-oxide. Trans-forms of dG-N(2)-TAM and dG-N(2)-N-desTAM adducts were detected in the livers of all three TAM-fed monkeys at levels of 2.7 adducts/10(8) nucleotides and 1.7 adducts/10(8) nucleotides, respectively. The levels of dG-N(2)-TAM adducts observed in the uterus of one monkey and in the ovaries of two monkeys were approximately 10-fold lower than those observed in the livers. TAM exposure also induced dG-N(2)-TAM adduct in the brain cortex of all three monkeys with a value of 1.5 adducts/10(8) nucleotides. No TAM-DNA adducts were detected in the kidneys or in any tissues obtained from the unexposed monkey. Our results suggest that women receiving TAM may form genotoxic damage in many organs, including the reproductive organs.

Mutagenic events induced by 4-hydroxyequilin in supF shuttle vector plasmid propagated in human cells.

Increased incidence of breast, ovarian and endometrial cancers are observed in women receiving estrogen replacement therapy (ERT). Equilin and equilenin are the major components of the widely prescribed drug used for ERT. These equine estrogens are metabolized primarily to 4-hydroxyequilin (4-OHEQ) and 4-hydroxyequilenin, respectively, which are autoxidized to react with DNA, resulting in the various DNA damages. To explore the mutagenic potential of equine estrogen metabolites, a double-stranded pMY189 shuttle vector carrying a bacteria suppressor tRNA gene, supF, was exposed to 4-OHEQ and transfected into human fibroblast. Plasmids containing mutations in the supF gene were detected with indicator bacteria and mutated colonies obtained were analyzed by automatic DNA sequencing. The proportion of plasmids with the mutated supF gene was increased dose-dependently. The majority of the 4-OHEQ-induced mutations were base substitutions (78%); another 22% were deletions and insertions. Among the base substitutions, 56% were single base substitutions and 19% were multiple base substitutions. The majority (86%) of the 4-OHEQ-induced single base substitutions occurred at the C:G site. C:G --> G:C and C:G --> A:T mutations were detected preferentially with lesser numbers of C:G --> T:A transitions. Sixty-two percent of base substitutions were observed particularly at C:G pairs in (5')-TC/AG-(5') sequences. Using (32)P-post-labeling/gel electrophoresis analysis, 4-OHEN-dC was a major adduct, followed by lesser amounts of 4-OHEN-dA adduct. Mutations observed at C:G pairs may result from 4-OHEN-dC adduct. These results indicated that 4-OHEQ is mutagenic, generating mutations primarily at C:G pairs in (5')-TC/AG-(5') sequences.