Differing transcriptional responses to pulsed or continuous estradiol exposure in human umbilical vein endothelial cells.

This study used human umbilical vein endothelial cells (HUVECs) that were treated with 17beta-estradiol for 5 days as 1h pulse or 24h continuous treatment at concentrations such that the 24h exposure (concentration x time) was identical in both conditions. Cell proliferation was studied and gene expression profiling was carried out using the Affymetrix GeneChip microarray analysis. Changes in morphology and apoptosis in HUVECs were examined with electron microscopy. Time-course studies of expression of genes vascular endothelial growth factor (VEGF), inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) were performed by quantitative PCR. We observed that cell proliferation was significantly decreased over days 3-5 with pulsed estradiol treatment relative to constant exposure. Microarray results showed that after 5 days, 801 genes differed (P<0.05) between continuous versus pulsed estradiol treatment. Functional analysis showed a significant number of genes to be associated with apoptosis and cell cycle pathways. We did not find any evidence of apoptosis from flow cytometry or electron microscopy examination. Our study highlights a large number of significantly different molecular responses to estradiol depending upon the mode of administration of estradiol. Significant changes were observed in genes involved in apoptosis and proliferation including VEGF, IGF receptors, and tumor protein p53.

Absence of uterine tumours in CD-1 mice treated neonatally with subcutaneous tamoxifen or 4-hydroxyoestradiol.

The effects of subcutaneous dosing of neonatal CD-1 mice with tamoxifen on days 1-5 after birth at doses of 0, 5, 10, 25 or 50 microg/pup or with 4-hydroxyoestradiol at 2 microg/pup have been investigated. Animals were culled at 1.5, 3, 6, 12 and 18 months after dosing and changes in uterine and ovarian pathology examined. Results showed both compounds to result in uterine hypoplasia relative to controls. At 18 months after dosing in the uterus, there was a fairly marked atrophy of the muscle layer, mild to moderate glandular hyperplasia of the endometrium even though these irregularly shaped glands did not penetrate through the myometrium and no adenocarcinomas were detected. At 18 months after dosing, oviducts showed mild focal adenomatous changes characterized by penetration epithelial hyperplasia, changes similar to those previously reported as 'diverticulosis and salpingitis isthmica nodosa' following diethylstilbestrol treatment of mice. At this time, both tamoxifen and 4-hydroxyoestradiol also affected the ovaries which showed a paucity of follicles and no corpora lutea, suggesting that there had been disruption to the oestrus cycle, particularly with tamoxifen at the highest dose where the ovaries of mice contained no developing follicles. At 18 months, control mice were cycling normally. Results failed to substantiate that tamoxifen and 4-hydroxyoestradiol are uterine carcinogens in this neonatal mouse model.

Tamoxifen forms DNA adducts in human colon after administration of a single [14C]-labeled therapeutic dose.

Tamoxifen is widely prescribed for the treatment of breast cancer and is also licensed in the United States for the prevention of this disease. However, tamoxifen therapy is associated with an increased occurrence of endometrial cancer in women, and there is also evidence that it may elevate the risk of colorectal cancer. The underlying mechanisms responsible for tamoxifen-induced carcinogenesis in women have not yet been elucidated, but much interest has focused on the role of DNA adduct formation. We investigated the propensity of tamoxifen to bind irreversibly to colorectal DNA when given to 10 women as a single [(14)C]-labeled therapeutic (20 mg) dose, approximately 18 h before undergoing colon resections. Using the sensitive technique of accelerator mass spectrometry, coupled with high-performance liquid chromatography separation of enzymatically digested DNA, a peak corresponding to authentic dG-N(2)-tamoxifen adduct was detected in samples from three patients, at levels ranging from 1 to 7 adducts/10(9) nucleotides. No [(14)C]-radiolabel associated with tamoxifen or its major metabolites was detected. The presence of detectable CYP3A4 protein in all colon samples suggests that this tissue has the potential to activate tamoxifen to alpha-hydroxytamoxifen, in addition to that occurring in the systemic circulation, and direct interaction of this metabolite with DNA could account for the binding observed. Although the level of tamoxifen-induced damage displayed a degree of interindividual variability, when present, it was approximately 10 to 100 times higher than that reported for other suspect human colon carcinogens such as 2-amino-1-methyl-6-phenyimidazo[4,5-b]pyridine. These findings provide a mechanistic basis through which tamoxifen could increase the incidence of colon cancers in women.

Ishikawa cells exhibit differential gene expression profiles in response to oestradiol or 4-hydroxytamoxifen.

In this study, the oestrogen agonist/antagonist action of 4-hydroxytamoxifen (OHT; 1 x 10(-6) M) and 17beta-oestradiol (E(2); 1 x 10(-8) M) were assessed on the oestrogen receptor (ER)-positive epithelial cell line (Ishikawa) with respect to cell proliferation, and to gene and protein expression. qRT-PCR and western blotting confirmed that Ishikawa cells expressed both ER isoforms and that there was no change in transcript levels in response to either ligand. Gene expression profiles, using oligonucleotide arrays representing approxiamtely 19,000 human genes, showed that the expression of 716 and 534 genes were changed differentially by treatment with either OHT or E(2) respectively, at the 24-h time point, with modulation of 46 genes common to both ligands, whereas 335 (OHT) and 240 (E(2)) genes showed expression changes unique to ligand, with 13 common alterations at 48 h. Both OHT and E(2) had demonstrable oestrogen agonist actions on Ishikawa cells, exemplified by increased proliferation and expression of known oestrogen-responsive genes, such as creatine kinase B and by the induction of alkaline phosphatase activity. Additionally, the data indicate that the two oestrogen agonists generated not only common gene expression changes but also unique ligand-specific profiles, raising the intriguing possibility that tamoxifen has E(2)-independent effects on the uterine epithelium.

Experimental adenomyosis.

Adenomyosis has been reported in a number of different animal species, whereas endometriosis appears limited to humans and non-human primates. This suggests a different aetiology of the two conditions. Adenomyosis develops spontaneously in certain strains of laboratory mice. Its incidence in mice can be markedly enhanced by systemic exposure to various hormonal agents, including prolactin, progesterone, synthetic progestins, certain oestrogenic agents, as well as tamoxifen and toremifene. The precise hormonal changes necessary remain unclear, although the evidence suggests that adenomyosis in this model is not due to a simple oestrogenic effect. Study of the pathological and molecular alterations in this model indicates that disturbances to the uterine stroma, blood vessels and myometrium are also important factors in the development of adenomyosis.

Applications of accelerator mass spectrometry for pharmacological and toxicological research.

The technique of accelerator mass spectrometry (AMS), known for radiocarbon dating of archeological specimens, has revolutionized high-sensitivity isotope detection in pharmacology and toxicology by allowing the direct determination of the amount of isotope in a sample rather than measuring its decay. It can quantify many isotopes, including 26Al, 14C, 41Ca, and 3H with detection down to attomole (10(-18)) amounts. Pharmacokinetic data in humans have been achieved with ultra-low levels of radiolabel. One of the most exciting biomedical applications of AMS with 14C-labeled potential carcinogens is the detection of modified proteins or DNA in tissues. The relationship between low-level exposure and covalent binding of genotoxic chemicals has been compared in rodents and humans. Such compounds include heterocyclic amines, benzene, and tamoxifen. Other applications range from measuring the absorption of 26Al to monitoring 41Ca turnover in bone. In epoxy-embedded tissue sections, high-resolution imaging of 14C label in cells is possible. The uses of AMS are becoming more widespread with the availability of instrumentation dedicated to the analysis of biomedical samples.

Neonatal tamoxifen treatment of mice leads to adenomyosis but not uterine cancer.

Tamoxifen is contraindicated during pregnancy but many births have been reported in breast cancer patients taking this drug and numbers might be expected to increase with FDA approval of tamoxifen for risk reduction in women at high, risk of breast cancer. The neonatal mouse, exquisitely sensitive to xenobiotic estrogens, has been used to investigate the effects of short-term oral dosing with tamoxifen (1 mg/kg on days 2-5 after birth) on long-term changes in uterine pathology and gene expression. Increased adenomyosis incidence and severity was evident in the tamoxifen-treated mice with increasing age. Uterine weights in treated mice remained lower than the corresponding controls up until 9 months, after which they became greater but during life-time studies (up to 36 months), there was no development of uterine tumours. Pathological examination of uterine tissues showed there to be extensive down-growth of endometrial glands and stroma into thickened, abnormal myometrium that had disorganised fascicles of smooth muscle and increased interstitial collagen deposition. In advanced cases, the endometrial epithelium showed mild degrees of focal hyperplasia and squamous metaplasia but no atypical cytology suggestive of premalignant change. Microarray analysis of uterine RNA taken at 1.5, 3, 6, 9 and 12 months showed from 4500 ESTs, only 12 genes were continuously over-expressed by tamoxifen treatment over this time, while none was continuously down-regulated. Up-regulated genes include those for nerve growth factor (Ngfa), cathepsin B (Ctsb), transforming growth factor beta induced (Tqfbi) and collagens (Colla1, Colla2). Results provide a basis for understanding the mechanism for tamoxifen induced tissue remodelling and the development of adenomyosis.

Techniques: the application of accelerator mass spectrometry to pharmacology and toxicology.

The exquisite sensitivity of accelerator mass spectrometry (AMS) is being used in biomedical applications to quantitate many isotopes, including 14C, 3H, 41Ca and 27Al, at attomole (10(-18)) concentrations. This enables compounds and metabolites to be measured in human urine and plasma after administration of low pharmacologically or toxicologically relevant doses of labelled chemicals and drugs. The detection of modified proteins or DNA in target organs after dosing with potential carcinogens has also been achieved in many studies. Advances aimed at increasing sample throughput and expanding applications by coupling AMS instruments directly to chromatographic separation systems are currently underway.

Tamoxifen DNA damage detected in human endometrium using accelerator mass spectrometry.

This study was aimed to establish whether tamoxifen binds irreversibly to uterine DNA when given to women. Patients were given a single therapeutic dose of [(14)C]tamoxifen citrate orally (20 mg, 0.37 or 1.85 MBq) approximately 18 h prior to hysterectomy or breast surgery. Nonmalignant uterine tissue was separated into myometrium and endometrium. DNA and protein were isolated and bound radiolabel determined by the sensitive technique of accelerator mass spectrometry. Levels of irreversible DNA binding of tamoxifen in the endometrium of treated patients were 237 +/- 77 adducts/10(12) nucleotides (mean +/- SE, n = 10). In myometrial tissues, a similar extent of DNA binding was detected (492 +/- 112 adducts/10(12) nucleotides). Binding of tamoxifen to endometrial and myometrial proteins was 10 +/- 3 and 20 +/- 4 fmol/mg, respectively. In breast tissue, sufficient DNA could not be extracted but protein binding was an order of magnitude higher than that seen with endometrial proteins (358 +/- 81 fmol/mg). These results demonstrate that after oral administration, tamoxifen forms adducts in human uterine DNA but at low numbers relative to those previously reported in women after long-term tamoxifen treatment where levels, when detected, ranged from 15000 to 130000 adducts/10(12) nucleotides. Our findings support the hypothesis that the low level of DNA adducts in human uterus is unlikely to be involved with endometrial cancer development.

Identification of human CYP forms involved in the activation of tamoxifen and irreversible binding to DNA.

This study investigates which CYP forms are responsible for the conversion of tamoxifen to its putative active metabolite alpha-hydroxytamoxifen and irreversible binding to DNA. We have used eight different baculovirus expressed recombinant human CYP forms and liquid chromatography-mass spectrometry to show that only CYP3A4 is responsible for the NADPH-dependent alpha-hydroxylation of tamoxifen. Surprisingly, this CYP did not catalyse the formation of 4-hydroxytamoxifen. We demonstrate for the first time, by means of accelerator mass spectrometry, that CYP3A4 also catalysed the activation of [(14)C]tamoxifen to intermediates that irreversibly bind to exogenous DNA. Incubation of [(14)C]tamoxifen (20.6 kBq, 100 micro M) with CYP3A4, in the presence of NADPH for 60 min led to levels of DNA binding of 39.0+/-9.0 adducts/10(8) nucleotides (mean +/- SE, n = 6). While CYP3A4 converted tamoxifen to N-desmethyltamoxifen (38.3 +/- 7.20 pmol/20 min/pmol CYP, n = 4), the polymorphic CYP2D6 showed the highest activity for producing this metabolite (48.6+/-1.52pmol/20 min/pmol CYP). CYP2D6 was also the most active in catalysing 4-hydroxylation of tamoxifen, although an order of magnitude lower level was also detected with CYP2C19. With tamoxifen as substrate, no 3,4-dihydroxytamoxifen could be detected with any CYP form. CYP2B6 did not catalyse the metabolism or the binding of tamoxifen to DNA. It is concluded that CYP3A4 is the only P450 of those tested that converts tamoxifen to alpha-hydroxytamoxifen and the only one that results in appreciable levels of irreversible binding of tamoxifen to DNA.