Nonsteroidal Antiestrogen Research Articles

Activation of Maxi Cl(-) channels by antiestrogens and phenothiazines in NIH3T3 fibroblasts.

The identification of alternative estrogen actions has been accumulating steadily over the past two decades. Typically, these novel actions are not directly related to nuclear transcriptional events but related to the interaction of estrogens with sites present at plasma membrane or cytosolic locations. These alternative effects, widely known as non-genomic effects, range from the modulation of plasma membrane ion channel activity to the regulation of different intracellular signalling cascades. In the present study we have investigated the modulation of a large conductance chloride channel (Maxi Cl(-)) by estrogens, non-steroidal triphenylethylene antiestrogens and phenothiazines in NIH3T3 fibroblasts and the dependence on guanosine triphosphate (GTP) of the Maxi Cl(-) activation. Our data identifies the non-steroidal antiestrogens toremifene and tamoxifen, and the phenothiazines chlorpromazine and triflupromazine as activators of Maxi Cl(-) channels. In contrast, 17 beta-estradiol and cAMP, added prior to the exposure to antiestrogens, prevent channel activation. The pure antiestrogen ICI 182780 did not activate the channel nor prevent its activation by non-steroidal antiestrogens. The activation of Maxi Cl(-) channels by toremifene and tamoxifen required the presence of intracellular nucleotides and was inhibited by the stable analog, GDP beta -S, suggesting the participation of a G-protein in the activation process. Little is known about the physiological relevance of Maxi Cl(-) channels. However, that fact that its regulation by estrogens and antiestrogens is shared by different cell types might imply a common role which needs to be identified.

Tamoxifen Decreases Fibroblast Function and Downregulates TGFβ2 in Dupuytren's Affected Palmar Fascia

Background. Dupuytren's contracture is a fibroproliferative disorder that is associated with increased collagen deposition. Isoforms of transforming growth factor beta (TGFβ), normally TGFβ1 and TGFβ2, are involved in the progressive fibrosis of Dupuytren's disease. It has been suggested that downregulation of TGFβ may be useful in the treatment of the condition. Tamoxifen, a synthetic nonsteroidal antiestrogen, is known to modulate the production of TGFβ. This study examined the role of tamoxifen in decreasing fibroblast function and downregulating TGFβ2.Methods. Primary cultures of fibroblasts were obtained from Dupuytren's affected fascia and carpal tunnel affected fascia as a control. Collagen lattices were prepared and populated with the fibroblasts. The fibroblast-populated collagen lattices (FPCL) were then measured for contraction every 24 h for 5 days. Supernatant was obtained from the culture medium following completion of the FPCL portion of the experiment and used for a TGFβ2 immunoassay.Results. Dupuytren's affected fibroblasts contracted the FPCLs significantly more than carpal tunnel control fibroblasts. Treating the fibroblasts with tamoxifen caused a decreased contraction rate in both Dupuytren's affected fibroblasts and carpal tunnel controls. There was increased TGFβ2 expression in the Dupuytren's affected fascia group compared to the carpal tunnel control group. Tamoxifen decreased TGFβ2 expression in Dupuytren's affected fascia group but not in the carpal tunnel control group.Conclusion. TGFβ appears to be the key cytokine in the fibrogenic nature of Dupuytren's disease. Tamoxifen treatment has been demonstrated to decrease the function of fibroblasts derived from Dupuytren's affected fascia and downregulated TGFβ2 production in these same fibroblasts. These data suggest a method to manipulate and control Dupuytren's contracture in the clinical setting.

Antagonists selective for estrogen receptor alpha.

To develop compounds that are antagonists on ER(alpha), but not ER(beta), we have added basic side-chains typically found in nonsteroidal antiestrogens to pyrazole compounds that bind with much higher affinity to ER(alpha) than to ER(beta). In this way we have developed basic side-chain pyrazoles (BSC-pyrazoles) that are high affinity, potent, selective antagonists on ER(alpha). These BSC-pyrazoles are themselves inactive on ER(alpha) and ER(beta), and they antagonize E2 stimulation by ER(alpha) only. We investigated seven basic side-chain substituents on various alkyl-triaryl-substituted pyrazoles, and the most ER(alpha)-selective compound was methyl-piperidino-pyrazole (MPP). ER(alpha)-selective antagonism was observed on diverse reporter-promoter gene constructs containing estrogen response elements that are consensus, nonconsensus (pS2), or comprised of multiple half-estrogen response elements (NHERF/EBP50) and on genes in which ER works indirectly by tethering to other DNA-bound proteins (TGF(beta)3). In contrast to these BSC-pyrazoles, the antiestrogens trans-hydroxytamoxifen, raloxifene, and ICI 182,780 suppress E2 activity via both ER(alpha) and ER(beta). The most effective BSC-pyrazole, MPP, fully antagonized E2 stimulation of pS2 mRNA in MCF-7 breast cancer cells, consistent with the fact that these cells contain almost exclusively ER(alpha). These compounds should be useful in studying the biological functions of ER(alpha) and ER(beta) and in selectively blocking responses that are mediated through ER(alpha).

Environmental estrogen-like chemicals and hydroxyl radicals induced by MPTP in the striatum: a review.

Oxygen free radical formation has been implicated in lesions caused by the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and iron. Although MPTP produces a parkinsonian syndrome after its conversion to 1-methyl-4-phenylpyridine (MPP+) by type B monoamine oxidase (MAO) in the brain, the etiology of this disease remains obscure. This review focuses on the role of an environmental neurotoxin chemically related to MPP+-induced free radical generation in the pathogenesis of Parkinson's disease. Environmental-like chemicals, such as para-nonylphenol or bisphenol A, significantly stimulated hydroxyl radical (*OH) formation in the striatum. Allopurinol, a xanthine oxidase inhibitor, prevents para-nonylphenol and MPP+-induced *OH generation. Tamoxifen, a synthetic nonsteroidal antiestrogen, suppressed the *OH generation via dopamine efflux induced by MPP+. These results confirm that free radical production might make a major contribution at certain stages in the progression of the injury. Such findings may be useful in elucidating the actual mechanism of free radical formation in the pathogenesis of neurodegenerative brain disorders, including Parkinson's disease and traumatic brain injuries.

Chemoprevention with antiestrogens: the beginning of the end for breast cancer. Daniel G. Miller Lecture.

In the 1960s, compounds known as nonsteroidal antiestrogens were identified as potential contraceptives, but the drugs caused the induction of ovulation in subfertile women. Tamoxifen and clomiphene were marketed for this indication. However, tamoxifen was advanced for the treatment of breast cancer in the 1970s through a close cooperation between the laboratory and the clinical trials community. The extensive use of long-term adjuvant tamoxifen has resulted in saving the lives of 400,000 women with breast cancer. Tamoxifen is a selective estrogen receptor modulator (SERM) that produces antiestrogenic actions in the breast but estrogen-like actions in bone and lowers serum cholesterol. These properties not only allowed the application of tamoxifen as the first chemopreventive in high-risk pre- and postmenopausal women but also the development of raloxifene to prevent osteoporosis with the potential to prevent breast cancer in postmenopausal women. The future development of SERMs holds the promise of preventing osteoporosis and coronary heart disease as well as breast and endometrial cancer.

Localization of tamoxifen-induced DNA adducts in rat liver

Tamoxifen is a non-steroidal anti-estrogen and is one of the most widely used and successful agents in the treatment of breast cancer. Tamoxifen competes with endogenous estrogens for estrogen receptors in mammary tissue, and thereby inhibits transcription of estrogen-responsive genes. The success of tamoxifen in the adjuvant therapy of breast cancer has resulted in its current widespread use as a prophylactic in healthy women who are at high risk of developing breast cancer. However, the prophylactic use of tamoxifen is controversial because of mixed results from clinical trials and, in particular, the increase in the incidence of endometrial cancer associated with tamoxifen therapy.The reason for this increase in endometrial cancer in humans is a matter of considerable debate. One theory is that tamoxifen is a genotoxic carcinogen – that is, it is metabolized to electrophilic, DNA-reactive intermediates that damage the genetic material and form DNA ‘adducts’. In support of this hypothesis, tamoxifen is a potent hepatocarcinogen in rats, and adducts are indeed found in the liver DNA of rats treated with tamoxifen. In humans, however, there are inconsistencies in the literature concerning the presence of DNA adducts in endometrial tissue from women undergoing tamoxifen therapy. It is therefore unclear whether tamoxifen is a genotoxic carcinogen in humans, or whether it exerts its carcinogenic effects by another mechanism.A recent report by Divi et al. 1xImmunohistochemical localization and semi-quantitation of hepatic tamoxifen-DNA adducts in rats exposed orally to tamoxifen. Divi, R.L. et al. Carcinogenesis. 2001; 10: 1693–1699CrossrefSee all References1 might help shed light on this contentious issue. The authors have developed a sensitive immunohistochemical assay to detect tamoxifen–DNA adducts in rat liver tissue sections. An advantage of this technique over conventional assays for adduct detection, in which DNA must first be isolated from tissue or cells, is that adducts can be localized to particular regions of the tissue in situ. In a beautiful series of immunohistochemical staining images, Divi and colleagues demonstrate nuclear localization of DNA adducts in liver sections from rats treated orally with tamoxifen at various doses. In subsequent experiments, the authors located regions within the liver tissue that expressed an altered form of glutathione-S-transferase, a phase II detoxification enzyme that reduces adduct formation. It was found that, in these regions, levels of tamoxifen–DNA adducts were much lower. Most striking of all is the presence of discrete foci of cells, positive for tamoxifen–DNA adducts, surrounded by areas of tissue that are relatively adduct free; this demonstrates localization of adducts at single-cell resolution within the tissue.The authors raise the possibility that this technique might be suitable for the analysis of human endometrial samples from women undergoing tamoxifen therapy, and might reveal specific regions or cells within the endometrial tissue that contain significant levels of tamoxifen–DNA adducts. This technique might well be a more suitable and sensitive method of analysis than other adduct-detection methods in which DNA must be isolated, and in which the presence of adducts in certain regions of the tissue are masked by the vast majority of surrounding tissue that contains no DNA adducts. If this is indeed the case, analysis of the localization of tamoxifen–DNA adducts in human tissues might help us understand the mechanism of tamoxifen-induced carcinogenesis in humans.

ChemInform Abstract: Synthesis and Biological Evaluation of Some Novel 4H‐Benzopyran‐4‐one Derivatives as Nonsteroidal Antiestrogens.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

The tamoxifen-responsive estrogen receptor alpha mutant D351Y shows reduced tamoxifen-dependent interaction with corepressor complexes.

The effects of estrogen and anti-estrogen are mediated through the estrogen receptors ERalpha and beta, which function as ligand-induced transcriptional factors. The nonsteroidal anti-estrogen tamoxifen is the most commonly used endocrine in the treatment of all stages of breast cancer in both pre- and postmenopausal women. Several lines of evidence have indicated that tamoxifen promotes association between ERalpha and corepressors N-CoR or silencing mediator for retinoid and thyroid hormone receptor (SMRT). Our results indicate that N-CoR/SMRT recognize and interact with helices H3 and H5 of the ERalpha ligand-binding domain in a 4-hydroxy tamoxifen-dependent manner. The mutant ERalpha(D351Y), derived from a tamoxifen-stimulated tumor and containing an amino acid substitution at position 351 within H3, showed reduced interaction with N-CoR/SMRT and high tamoxifen-induced activation function-1 (AF-1) activity. While the estradiol-dependent transcriptional activity of ERalpha(D351Y) was almost equal to that of wild-type ERalpha, the mutant exhibited higher levels of transcriptional activity in the presence of both E2 and 4-hydroxy tamoxifen compared with wild-type ERalpha. These results may explain the observation that the growth of tumor cells expressing ERalpha(D351Y) can be stimulated by tamoxifen, E2, or both.

DNA adduct formation and mutant induction in Sprague-Dawley rats treated with tamoxifen and its derivatives.

The non-steroidal anti-estrogen tamoxifen is used as an adjunct chemotherapeutic agent for the treatment of all stages of breast cancer and more recently as a chemoprotective agent in women with elevated risk of developing breast cancer. While beneficial for the treatment of breast cancer, tamoxifen increases the risk of endometrial cancer. In addition, it has been shown to induce liver and endometrial tumors in rats. Tamoxifen is genotoxic in rat liver, as indicated by the formation of DNA adducts, through a metabolic pathway involving the alpha-hydroxylation of tamoxifen and N-desmethyltamoxifen. Since the contribution of these alpha-hydroxy metabolites of tamoxifen to the induction of endometrial tumors is presently unknown, we compared the extent of DNA adduct formation in liver and selected non-hepatic tissues of female Sprague-Dawley rats treated by gavage with tamoxifen, alpha-hydroxytamoxifen, N-desmethyltamoxifen, alpha-hydroxy-N-desmethyltamoxifen and N,N-didesmethyltamoxifen, or intraperitoneal injection with tamoxifen, alpha-hydroxytamoxifen, 3-hydroxytamoxifen and 4-hydroxytamoxifen. In addition, spleen lymphocytes from rats treated by gavage with tamoxifen or alpha-hydroxytamoxifen were assayed for the induction of mutants in the hypoxanthine phosphoribosyl transferase (Hprt) gene. The relative levels of binding in rats treated by gavage were alpha-hydroxytamoxifen > tamoxifen approximately N-desmethyltamoxifen approximately alpha-hydroxy-N-desmethyltamoxifen > N,N-didesmethyltamoxifen. In rats dosed intraperitoneally, the relative order of binding was alpha-hydroxytamoxifen > tamoxifen > 3-hydroxytamoxifen approximately 4-hydroxytamoxifen. None of the compounds resulted in an increase in DNA adducts in uterus, spleen, thymus or bone marrow DNA from rats treated by gavage or in uterus DNA from rats injected intraperitoneally. Neither tamoxifen nor alpha-hydroxytamoxifen increased the Hprt mutant frequency in spleen T-lymphocytes. These results confirm previous observations that tamoxifen is activated to a genotoxic agent in rat liver through alpha-hydroxylation, and also suggest that endometrial tumors in rats do not arise from the formation of tamoxifen-DNA adducts.

Endometrial lesions after tamoxifen therapy in breast cancer women.

Tamoxifen, a nonsteroidal antiestrogen with a partial estrogen-antagonist activity, is widely used as a hormonal adjuvant therapy for breast cancer in women with positive receptors for estrogens. Its prolonged administration has been associated with a series of collateral effects, among which the endometrial carcinoma is the most important. The aim of our study was to investigate an eventual correlation between a therapy with tamoxifen and the onset of endometrial lesions. We recruited 228 postmenopausal patients who had been operated on for breast cancer. They were divided into two groups according to the presence of positive or negative estrogen receptors. The group with positive receptors was subjected to hormonal adjuvant therapy by tamoxifen (20 mg/day for 5 years), while the group with negative receptors was not treated. All the patients underwent a hysteroscopic evaluation of the uterine cavity before and after treatment. The follow-up carried out 5 years later showed the presence of a statistically higher risk (p < 0.00001) of endometrial lesions, such as low glandular hyperplasia and polyps, than in the treated patients compared with untreated patients. On the other hand, because there was no onset of endometrial carcinoma, the risk of this kind of lesion turned out to be practically absent. In all the treated patients who did not evidence any endometrial lesions (n = 90) and in all those with negative receptors (n = 104) the endometrium seemed to have an atrophic aspect. In conclusion, according to these data, we believe that hormonal adjuvant therapy by tamoxifen (20 mg/day), associated with a periodic hysteroscopic evaluation, and eventually a directed biopsy of the endometrium in order to keep under control the frequent onset of benign endometrial lesions, does not absolutely seem to increase the risk of endometrial carcinoma.

Effect of tamoxifen on sperm fertilising ability and preimplantation embryo development

Recent evidences point to a role of estrogens in males. We have earlier reported that tamoxifen, a synthetic non-steroidal antiestrogen, when administered to adult male rats, in the dose range of 0.04–0.4 mg/kg per day, reduced fertility. The reduced fertility was measured in terms of fertility index (a measure of the efficiency of the ovulated ovas to fertilise and implant), fecundity (siring ability) and litter size. The present study was done to investigate whether the reduction in fertility index was due to reduction in fertilising ability or increase in pre-implantation embryo loss. Also a dose related effect of tamoxifen from 0.02 mg to 2 mg/kg per day on the fertility of the male rats was studied. To study the fertilising ability, control and tamoxifen (0.4 mg/kg per day, the most effective dose) treated adult male rats were mated with normal cycling females and the females sacrificed at day 0–4 of gestation. Eggs fertilised/unfertilised were flushed from the oviduct/uterus and the number and types of eggs were noted. The index of fertilisation, a measure of the fertilising ability was determined. The studies demonstrate that the reduction in fertility is not due to decreased fertilising ability but because of the increased pre-implantation embryo loss as evident from an increase in number of abnormal eggs in the treated group with no change in index of fertilisation. A dose related decrease in fertility was observed. The present study suggests that tamoxifen at 0.02–2-mg dose is predominantly estrogenic in males and paternal factor/s sensitive to tamoxifen is involved in embryogenesis.

Tamoxifen inhibits nitrotyrosine formation after reversible middle cerebral artery occlusion in the rat.

Tamoxifen (TAM), a widely used non-steroidal anti-estrogen, has recently been shown to be neuroprotective in a rat model of reversible middle cerebral artery occlusion (rMCAo). Tamoxifen has several potential mechanisms of action including inhibition of the release of excitatory amino acids (EAA) and nitric oxide synthase (NOS) activity. The question addressed in this study was whether TAM reduces ischemia-induced production of nitrotyrosine, considered as a footprint of the product of nitric oxide and superoxide, peroxynitrite. In rat brain, 2 h rMCAo produced a time-dependent increase in nitrotyrosine content in the cerebral cortex, as measured by Western blot analysis. Compared with vehicle, TAM significantly reduced nitrotyrosine levels in the ischemic cortex at 24 h. The neuronal (n)NOS inhibitor, 7-nitroindazole also tended to reduce nitrotyrosine, but this reduction was not statistically significant. Immunostaining for nitrotyrosine was seen in cortical neurons in the MCA territory and this immunostaining was reduced by TAM. In vitro, TAM and the calmodulin inhibitor trifluoperazine inhibited, with similar EC(50) values, the activity of recombinant nNOS as well as NOS activity in brain homogenates, measured by conversion of [(3)H]arginine to [(3)H]citrulline. There was marginal inhibition of recombinant inducible (i)NOS activity up to 100 microM TAM. These data suggest that TAM is an effective inhibitor of Ca(2+)/calmodulin-dependent NOS and the derived peroxynitrite production in transient focal cerebral ischemia and this may be one mechanism for its neuroprotective effect following rMCAo.

Molecular and pharmacological aspects of antiestrogen resistance

Endocrine therapy is effective in approximately one-third of all breast cancers and up to 80% of tumors that express both estrogen and progesterone receptors. Despite the low toxicity, good overall response rates, and additional benefits associated with its partial agonist activity, most Tamoxifen-responsive breast cancers acquire resistance. The development of new antiestrogens, both steroidal and non-steroidal, provides the opportunity for the development of non-cross-resistant therapies and the identification of additional mechanisms of action and resistance. Drug-specific pharmacologic mechanisms may confer a resistance phenotype, reflecting the complexities of both tumor biology/pharmacology and the molecular endocrinology of steroid hormone action. However, since all antiestrogens will be effective only in cells that express estrogen receptors (ER), many mechanisms will likely be directly related to ER expression and signaling. For example, loss of ER expression/function is likely to confer a cross-resistance phenotype across all structural classes of antiestrogens. Altered expression of ERα and ERβ, and/or signaling from transcription complexes driven by these receptors, may produce drug-specific resistance phenotypes. We have begun to study the possible changes in gene expression that may occur as cells acquire resistance to steroidal and non-steroidal antiestrogens. Our preliminary studies implicate the altered expression of several estrogen-regulated genes. However, resistance to antiestrogens is likely to be a multigene phenomenon, involving a network of interrelated signaling pathways. The way in which this network is adapted by cells may vary among tumors, consistent with the existence of a highly plastic and adaptable genotype within breast cancer cells.

Synthesis and biological evaluation of some novel 4 H-benzopyran-4-one derivatives as nonsteroidal antiestrogens

The preparation and characterization of some novel 2- and 3-substituted-7-methoxy-4 H-1-benzopyran-4-one are presented. The synthesized compounds were evaluated for their uterotrophic, antiuterotrophic and antiimplantation activities in mature female albino rats. 3-Benzyl-7-methoxy-4 H-1-benzopyran-4-one ( 14) showed the highest uterotrophic activity (87%) based on dry uterine weight gain. The antifertility activity, as assessed by the post-coital antiimplantation activity test, was of weak potency for most compounds (14–29%). Among the products, the 2-(4′-methoxyphenyl)-7-methoxy-4 H-1-benzopyran-4-one ( 19) exhibited the highest antiestrogenic activity of 65%. It also elicited 31% of the uterotrophic activity of estradiol.

Glucuronidation of the nonsteroidal antiestrogen EM-652 (SCH 57068), by human and monkey steroid conjugating UDP-glucuronosyltransferase enzymes.

EM-652 (SCH 57068) is a new orally active antiestrogen that demonstrates pure antagonistic effects in the mammary gland and endometrium. In vivo studies have shown that EM-652 is primarily glucuronidated at the 7-hydroxy position in rats and that the metabolite is present in the plasma of female monkeys and human subjects after EM-800 (SCH 57050) or EM-652.HCl oral administration. Using hepatic microsomes from rat, monkey, and human, the formation of two EM-652 monoglucuronides at positions 4' and 7 was demonstrated by a liquid chromatographic tandem mass spectrometric method. Although no difference in EM-652 conjugation was observed between male and female monkey livers, an interindividual variation of hepatic EM-652 glucuronidation was shown with female human donors. Using microsome preparations from human embryonic kidney 293 cells stably expressing each of the 12 human and 11 monkey UGT enzymes cloned to date, the two EM-652-monoglucuronides were detected after incubation with microsomes containing human UGT1A1, UGT1A3, UGT1A8, UGT1A9, and monkey monUGT1A01, monUGT1A03, and monUGT1A09. Despite human UGT1A1 and monkey monUGT1A09 favored formation of EM-652-7-glucuronide, other active UGT1A enzymes formed both 4'- and 7-glucuronide derivatives in equal amounts. Kinetic analysis of EM-652 glucuronidation by these enzymes showed Michaelis constant (K(m)) values between 36 and 302 microM for EM-652-4'-glucuronide and 19 and 233 microM for EM-652-7-glucuronide. The present results demonstrate the importance of UGT1A isoforms, mainly UGT1A1, for EM-652 metabolism in humans.

Progress in the prevention of breast cancer: concept to reality

In 1936, Professor Antoine Lacassagne suggested that breast cancer could be prevented by developing drugs to block estrogen action in the breast. Jensen discovered the physiologic target, the estrogen receptor, that regulates estrogen action in its target tissues and Lerner discovered the first nonsteroidal antiestrogen MER25. However, the success of tamoxifen as a treatment of breast cancer opened the door for the testing of the worth of tamoxifen to reduce breast cancer incidence in high-risk women. In 1998, Fisher showed that tamoxifen could reduce breast cancer incidence by 50%. Nevertheless, only half the women who develop breast cancer have risk factors other than age, so what can be done for women without risk factors? The recognition that nonsteroidal antiestrogens have the ability to modulate estrogen action selectively has advanced the design and development of new drug for multiple diseases. Tamoxifen and raloxifene maintain bone density and raloxifene is now used to prevent osteoporosis and is being tested as a preventive for coronary heart disease and breast cancer. The drug group is now known as selective estrogen receptor modulators (SERMs) and the challenge is to design new agents for multiple applications. If the 20th century was the era of chemotherapy, the 21st century will be the era of chemoprevention.

Promising Results with Exemestane in the First-Line Treatment of Metastatic Breast Cancer: A Randomized Phase II EORTC Trial with a Tamoxifen Control

Because tamoxifen (TAM), a nonsteroidal antiestrogen, is routinely used in the adjuvant setting, other hormone therapies are needed as alternatives for first-line treatment of metastatic breast cancer (MBC). Currently, exemestane (EXE) and other antiaromatase agents are indicated for use in patients who experience failure of TAM. In this multicenter, randomized, open-label, TAM-controlled (20 mg/day), phase II trial, we examined the activity and tolerability of EXE 25 mg/day for the first-line treatment of MBC in postmenopausal women. Exemestane was well tolerated and demonstrated substantial first-line antitumor activity based on intent-to-treat analysis of peer-reviewed responses. In the EXE arm, values for complete, partial, and objective response, clinical benefit, and time to tumor progression (TTP) exceeded those reported for TAM although no statistical comparison was made. Based on these encouraging results, a phase III trial will compare EXE and TAM.

Accomodation of S-cis-tamoxifen-N2-guanine adduct within a bent and widened DNA minor groove

The non-steroidal anti-estrogen tamoxifen [TAM] has been in clinical use over the last two decades as a potent adjunct chemotherapeutic agent for treatment of breast cancer. It has also been given prophylactically to women with a strong family history of breast cancer. However, tamoxifen treatment has also been associated with increased endometrial cancer, possibly resulting from the reaction of metabolically activated tamoxifen derivatives with cellular DNA. Such DNA adducts can be mutagenic and the activities of isomeric adducts may be conformation-dependent. We therefore investigated the high resolution NMR solution conformation of one covalent adduct (cis-isomer, S-epimer of [TAM]G) formed from the reaction of tamoxifen [TAM] to N2-of guanine in the d(C-[TAM]G-C)·d(G-C-G) sequence context at the 11-mer oligonucleotide duplex level. Our NMR results establish that the S-cis [TAM]G lesion is accomodated within a widened minor groove without disruption of the Watson-Crick [TAM]G·C and flanking Watson-Crick G·C base-pairs. The helix axis of the bound DNA oligomer is bent by about 30° and is directed away from the minor groove adduct site. The presence of such a bulky [TAM]G adduct with components of the TAM residue on both the 5′- and the 3′-side of the modified base could compromise the fidelity of the minor groove polymerase scanning machinery.

The Predictive Value of Endometrial Thickness in Patients Receiving Tamoxifen

Tamoxifen is a nonsteroidal antiestrogen frequently given to patients with breast cancer as a long-term adjuvant therapy. It is known that it may also promote changes in the uterus that resemble endometrial neoplasia. This study was designed to investigate the value and cost-effectiveness of ultrasound measurement of endometrial thickness in predicting endometrial pathology. From a review of the medical charts of patients being treated with tamoxifen at Lehigh Valley Hospital from 1994 to 1997, 56 patients who had also received transvaginal ultrasonography with subsequent histological evaluation of endometrial samples were identified. The records were evaluated for patient age, duration of tamoxifen therapy, endometrial thickness, descriptive ultrasonographic findings, and the final histologic diagnosis. The 56 patients ranged in age from 31 to 82 years (mean = 62 years). They had been receiving tamoxifen therapy for an average of 18.2 months. In 49 women (87.5 percent), endometrial thickness was 5 mm or less, which is the accepted minimum for normal endometrium in asymptomatic postmenopausal women. Histological examination of the endometrial sample in these women showed proliferative changes in 13 (26.5 percent), atrophy in 8 (16.3 percent), polypoid changes in 9 (18.4 percent), adenomatous hyperplasia in 4 (8.2 percent), and adenocarcinoma in 1 (2.0 percent). The endometrium of this patient was more than 50 mm thick. Fourteen patients had insufficient tissue for evaluation. Of the seven women with a measured endometrial thickness greater than 5 mm, five had insufficient tissue for evaluation. The two remaining patients had atrophic or proliferative endometrium. The predictive values of different endometrial thicknesses are shown in Table 1. The sensitivity and negative predictive values decrease and specificity and positive predictive values increase as the threshold for endometrial thickness increases. In this study, maximum efficacy for ultrasound alone as a predictor for endometrial pathology was reached at a cutoff value of 9 mm for endometrial thickness. At this point, the sensitivity and specificity were 77 and 59 percent, respectively, with positive predictive value of 37 percent and a negative predictive value of 89 percent. Using 9 mm or less of endometrial thickness as a threshold for further study would have resulted in 15 fewer endometrial biopsies with no omission of significant pathology. Using conservative measures, a savings of $525 per patient or $7875 was estimated for the 9-mm threshold.Table 1: Sensitivity, specificity, and positive and negative predictive values of endometrial thickness for detection of pathologically significant histologyJ Pelvic Surg 2000;6:16–19

Analysis of Tamoxifen–DNA Adducts by High-Performance Liquid Chromatography Using Postcolumn Online Photochemical Activation

Tamoxifen, a widely used nonsteroidal antiestrogen in the treatment of breast cancer, forms several metabolites. 4-Hydroxytamoxifen (4-OHTam), a metabolite found in the bloodstream, has much higher affinity for the estrogen receptor than tamoxifen itself. Oxidative activation of 4-OHTam induces DNA damage. DNA isolated from HL-60 cells exposed to 10 μM 4-OHTam in the presence of 1 μM hydrogen peroxide was digested enzymatically to release both normal and modified nucleosides. The modified nucleosides were enriched by butanol extraction. Using UV detection, HPLC analysis of the butanol extract from 200 μg DNA digest detected ∼4 4-OHTam-dG adducts per 107 nucleotides (n = 3). Online postcolumn UV irradiation in HPLC and fluorescence detection improved the detection sensitivity by 3 × 102 times. Using 4-OHTam as an example, this report demonstrated for the first time the power of the technique to assay tamoxifen-DNA adducts directly in the DNA digest without relying on postlabeling.