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1.
Formation of estrogen metabolites that react with DNA is thought to be a mechanism of cancer initiation by estrogens. The estrogens estrone (E 1) and estradiol (E 2) can form catechol estrogen (CE) metabolites, catechol estrogen quinones [E 1(E 2)-3,4-Q], which react with DNA to form predominantly depurinating adducts. This may lead to mutations that initiate cancer. Catechol- O-methyltransferase (COMT) catalyzes an inactivation (protective) pathway for CE. This study investigated the effect of inhibiting COMT activity on the levels of depurinating 4-OHE 1(E 2)-1-N3Ade and 4-OHE 1(E 2)-1-N7Gua adducts in human breast epithelial cells. MCF-10F cells were treated with TCDD, a cytochrome P450 inducer, then with E 2 and Ro41-0960, a COMT inhibitor. Estrogen metabolites and depurinating DNA adducts in culture medium were analyzed by HPLC with electrochemical detection. Pre-treatment of cells with TCDD increased E 2 metabolism to 4-OHE 1(E 2) and 4-OCH 3E 1(E 2). Inclusion of Ro41-0960 and E 2 in the medium blocked formation of methoxy CE, and depurinating adducts were observed. With Ro41-0960, more adducts were detected in MCF-10F cells exposed to 1 μM E 2, whereas without the inhibitor, no increases in adducts were detected with E 2 ≤ 10 μM. We conclude that low COMT activity and increased formation of depurinating adducts can be critical factors leading to initiation of breast cancer. 相似文献
2.
The great majority of breast cancers are in their early stage hormone-dependent and it is well accepted that estradiol (E 2) plays an important role in the genesis and evolution of this tumor. Human breast cancer tissues contain all the enzymes: estrone sulfatase, 17β-hydroxysteroid dehydrogenase, aromatase involved in the last steps of E 2 bioformation. Sulfotransferases which convert estrogens into the biologically inactive estrogen sulfates are also present in this tissue. Quantitative data show that the ‘sulfatase pathway’, which transforms estrogen sulfates into the bioactive unconjugated E 2, is 100–500 times higher than the ‘aromatase pathway’, which converts androgens into estrogens. The treatment of breast cancer patients with anti-aromatases is largely developed with very positive results. However, the formation of E2 via the ‘sulfatase pathway’ is very important in the breast cancer tissue. In recent years it was found that antiestrogens (e.g. tamoxifen, 4-hydroxytamoxifen), various progestins (e.g. promegestone, nomegestrol acetate, medrogestone, dydrogesterone, norelgestromin), tibolone and its metabolites, as well as other steroidal (e.g. sulfamates) and non-steroidal compounds, are potent sulfatase inhibitors. In another series of studies, it was found that E2 itself has a strong anti-sulfatase action. This paradoxical effect of E2 adds a new biological response of this hormone and could be related to estrogen replacement therapy in which it was observed to have either no effect or to decrease breast cancer mortality in postmenopausal women. Interesting information is that high expression of steroid sulfatase mRNA predicts a poor prognosis in patients with +ER. These progestins, as well as tibolone, can also block the conversion of estrone to estradiol by the inhibition of the 17β-hydroxysteroid dehydrogenase type I (17β-HSD-1). High expressison of 17β-HSD-1 can be an indicator of adverse prognosis in ER-positive patients. It was shown that nomegestrol acetate, medrogestone, promegestone or tibolone, could stimulate the sulfotransferase activity for the local production of estrogen sulfates. This is an important point in the physiopathology of this disease, as it is well known that estrogen sulfates are biologically inactive. A possible correlation between this stimulatory effect on sulfotransferase activity and breast cancer cell proliferation is presented. In agreement with all this information, we have proposed the concept of selective estrogen enzyme modulators (SEEM). In conclusion, the blockage in the formation of estradiol via sulfatase, or the stimulatory effect on sulfotransferase activity in combination with anti-aromatases can open interesting and new possibilities in clinical applications in breast cancer. 相似文献
3.
In order to better understand the function of aromatase, we carried out kinetic analyses to asses the ability of natural estrogens, estrone (E 1), estradiol (E 2), 16-OHE 1, and estriol (E 3), to inhibit aromatization. Human placental microsomes (50 μg protein) were incubated for 5 min at 37°C with [1β- 3H]testosterone (1.24 × 10 3 dpm 3H/ng, 35–150 nM) or [1β- 3H,4- 14C]androstenedione (3.05 × 10 3 dpm 3H/ng, 3H/ 14C = 19.3, 7–65 nM) as substrate in the presence of NADPH, with and without natural estrogens as putative inhibitors. Aromatase activity was assessed by tritium released to water from the 1β-position of the substrates. Natural estrogens showed competitive product inhibition against androgen aromatization. The Ki of E 1, E 2, 16-OHE 1, and E 3 for testosterone aromatization was 1.5, 2.2, 95, and 162 μM, respectively, where the Km of aromatase was 61.8 ± 2.0 nM ( n = 5) for testosterone. The Ki of E 1, E 2, 16-OHE 1, and E 3 for androstenedione aromatization was 10.6, 5.5, 252, and 1182 μM, respectively, where the Km of aromatase was 35.4 ± 4.1 nM ( n = 4) for androstenedione. These results show that estrogens inhibit the process of andrigen aromatization and indicate that natural estrogens regulate their own synthesis by the product inhibition mechanism in vivo. Since natural estrogens bind to the active site of human placental aromatase P-450 complex as competitive inhibitors, natural estrogens might be further metabolized by aromatase. This suggests that human placental estrogen 2-hydroxylase activity is catalyzed by the active site of aromatase cytochrome P-450 and also agrees with the fact that the level of catecholestrogens in maternal plasma increases during pregnancy. The relative affinities and concentration of androgens and estrogens would control estrogen and catecholestrogen biosynthesis by aromatase. 相似文献
4.
The evaluation of estrogens (estrone, estradiol, and their sulfates) in the breast tissue of post-menopausal patients with breast cancer indicates high levels, particularly of estrone sulfate (E 1 S) which is 15–25 times higher than in the plasma. Breast cancer tissue contains the enzymes necessary for local synthesis of estradiol and it was demonstrated that, despite the presence of the sulfatase and its messenger in hormone-dependent and hormone-independent breast cancer cells, this enzyme operates particularly in hormone-dependent cells. Different progestins: Nomegestrol acetate, Promegestone, progesterone, as well as Danazol, can block the conversion of E 1 S to E 2 very strongly in hormone-dependent breast cancer cells. The last step in the formation of estradiol is the conversion of E 1 to this estrogen by the action of 17β-hydroxysteroid dehydrogenase. This activity is preferentially in the reductive direction (formation of E 2) in hormone-dependent cells, but oxidative (E 2 → E 1) in hormone-independent cells. Using intact hormone-dependent cells it was observed that Nomegestrol acetate can block the conversion of E 1 to E 2. It is concluded, firstly, that in addition to ER mutants other factors are involved in the transformation of hormone-dependent breast cancer to hormone-independent, this concerns the enzymatic activity in the formation of E 2; it is suggested that stimulatory or repressive factor(s) involved in the enzyme activity are implicated as the cancer evolves to hormone-independence; secondly, different drugs can block the conversion of E 1 S to E 2. Clinical trials of these “anti-enzyme” substances in breast cancer patients could be the next step to investigate new therapeutic possibilities for this disease. 相似文献
5.
Despite the dramatic fall in plasma estrogen levels at menopause, only minor differences in breast tissue estrogen levels have been reported comparing pre- and postmenopausal women. Thus, postmenopausal breast tissue has the ability to maintain concentrations of estrone (E 1) and estradiol (E 2) that are 2–10- and 10–20-fold higher than the corresponding plasma estrogen levels. This finding may be explained by uptake of estrogens from the circulation and/or local estrogen production. Local aromatase activity in breast tissue seems to be of crucial importance for the local estrogen production in some patients while uptake from the circulation may be more important in other patients. Beside aromatase, breast tissue expresses estrogen sulfotransferase and sulfatase as well as dehydrogenase activity, allowing estrogen storage and release in the cells as well as conversions between estrone and estradiol. The activity of the enzyme network in breast cancer tissue is modified by a variety of factors like growth factors and cytokines. Aromatase inhibitors have been used for more than two decades in the treatment of postmenopausal metastatic breast cancer and are currently investigated in the adjuvant treatment and even prevention of breast cancer. Novel aromatase inhibitors and inactivators have been shown to suppress plasma estrogen levels effectively in postmenopausal breast cancer patients. However, knowledge about the influence of these drugs on estrogen levels in breast cancer tissue is limited. Using a novel HPLC-RIA method developed for the determination of breast tissue estrogen concentrations, we measured tissue E 1, E 2 and estrone sulfate (E 1S) levels in postmenopausal breast cancer patients before and during treatment with anastrozole. Our findings revealed high breast tumor tissue estrogen concentrations that were effectively decreased by anastrozole. While E 1S was the dominating estrogen fraction in the plasma, estradiol was the estrogen fraction with the highest concentration in tumor tissue. Moreover, plasma estrogen levels did not correlate with tissue estrogen concentrations. The overall experience with aromatase inhibitors and inactivators concerning their influences on breast tissue estrogen concentrations is summarized. 相似文献
6.
Aromatase (estrogen synthase) is the cytochrome P450 enzyme complex that converts C 19 androgens to C 18 estrogens. Aromatase activity has been demonstrated in breast tissue in vitro, and expression of aromatase is highest in or near breast tumor sites. Thus, local regulation of aromatase by both endogenous factors as well as exogenous medicinal agents will influence the levels of estrogen available for breast cancer growth. The prostaglandin PGE 2 increases intracellular cAMP levels and stimulates estrogen biosynthesis, and previous studies in our laboratories have shown a strong linear association between aromatase ( CYP19) expression and expression of the cyclooxygenases ( COX-1 and COX-2) in breast cancer specimens. To further investigate the pathways regulating COX and CYP19 gene expression, studies were performed in normal breast stromal cells, in breast cancer cells from patients, and in breast cancer cell lines using selective pharmacological agents. Enhanced COX enzyme levels results in increased production of prostaglandins, such as PGE 2. This prostaglandin increased aromatase activity in breast stromal cells, and studies with selective agonists and antagonists showed that this regulation of signaling pathways occurs through the EP 1 and EP 2 receptor subtypes. COX-2 gene expression was enhanced in breast cancer cell lines by ligands for the various peroxisome proliferator-activated receptors (PPARs), and differential regulation was observed between hormone-dependent and -independent breast cancer cells. Thus, the regulation of both enzymes in breast cancer involves complex paracrine interactions, resulting in significant consequences on the pathogenesis of breast cancer. 相似文献
7.
The aromatase and estrone sulfatase enzymes are important sources of biologically active estrogens in postmenopausal women with breast cancer. Promising initial results in the treatment of endocrine-responsive breast cancer have been exhibited by 125-dihydroxyvitamin D 3 and the synthetic vitamin D analogues MC903 and EB1089. However, these compounds together with vitamin D 3 and vitamin D 3 sulfate did not inhibit the human placental aromatase enzyme when assayed up to 20 μm. Only vitamin D 3 sulfate and 125-dihydroxyvitamin D inhibited the estrone sulfatase activity in human placental microsomes, albeit at high concentration (32 and 37% inhibition, respectively with 50 μm each inhibitor). It is unlikely that inhibition of aromatase or estrone sulfatase enzymes contribute to the inhibitory effect of this group of compounds on breast cancer cells in vivo. 相似文献
8.
Silver nanoparticles (AgNPs) have antimicrobial properties, which have contributed to their widespread use in consumer products. A current issue regarding nanomaterials is the extent to which existing genotoxicity assays are useful for evaluating the risks associated with their use. In this study, the genotoxicity of 5 nm AgNPs was assessed using two standard genotoxicity assays, the Salmonella reverse mutation assay (Ames test) and the in vitro micronucleus assay. Using the preincubation version of the Ames assay, Salmonella strains TA102, TA100, TA1537, TA98, and TA1535 were treated with 0.15-76.8 μg/plate of the AgNPs. Toxicity limited the doses that could be assayed to 2.4-38.4 μg/plate; no increases in mutant frequency over the vehicle control were found for the concentrations that could be assayed. Human lymphoblastoid TK6 cells were treated with 10-30 μg/ml AgNPs, and additional cells were treated with water and 0.73 gy X-rays as vehicle and positive controls. Micronucleus frequency was increased by the AgNP treatment in a dose-dependent manner. At a concentration of 30 μg/ml (with 45.4% relative population doubling), AgNPs induced a significant, 3.17-fold increase with a net increase of 1.60% in micronucleus frequency over the vehicle control, a weak positive response by our criteria. These results demonstrate that the 5 nm AgNP are genotoxic in TK6 cells. Also, the data suggest that the in vitro micronucleus assay may be more appropriate than the Ames test for evaluating the genotoxicity of the AgNPs. 相似文献
9.
The 2-hydroxy and 4-hydroxyestradiols (2-/4-OHE 2) caused marked cytotoxic effects, including vacuolation and nuclear changes, in rat epididymal epithelia, after exposure to very low levels (40 ng/rat/week) for 20 weeks. The effects of the 2-/4-OHE 2 metabolites were more pronounced than that of estradiol-17β(E 2). 相似文献
10.
The implementation of aromatase inhibitors for treatment of early and metastatic breast cancer has been one of the major improvements in endocrine therapy of breast cancer. Measurement of endocrine effects of aromatase inhibition in vivo has been a major tool in the process of evaluating novel compounds. Biochemical efficacy of aromatase inhibitors in vivo may be determined from their effects on “total body aromatization” as well changes in plasma and tissue estrogen levels. Due to high sensitivity, tracer methods allowing calculation of whole body aromatase inhibition are still considered the gold standard. The method developed by our group in collaboration with the Royal Marsden Hospital and the results of this joint program are summarized and discussed. These studies allowed classification of the different aromatase inhibitors and their optimal dosage, selecting the best compounds for clinical evaluation. In vivo total body aromatase assessment is a work-consuming method, allowing such studies to be conducted in a limited number of patients only. In contrast, plasma estrogen measurement is a cruder but simpler method, allowing screening of larger groups of patients. As plasma estrogens arise through passive diffusion of estrogens synthesized in different body compartments, plasma estrogens, as well as total body aromatase assessment, present a rough estimate of total body tissue estrogen production, and changes associated with treatment with aromatase inhibitors reflect the effects on tissue estrogen production in general. However, plasma estrogen levels do not correlate to breast cancer tissue estrogen levels. This is due to the endocrine autonomy of breast cancer tissue with significant local estrogen production in some tumors. Thus, direct measurement of intratumor estrogens is demanded to evaluate the effects of aromatase inhibitors in malignant target tissues. Our group has developed a highly sensitive HPLC-RIA for the simultaneous measurement of estrone, estradiol, and estrone sulfate in malignant breast tissue samples, and we are currently using this method to assess alterations in intratumor estrogen levels during treatment with different aromatase inhibitors. 相似文献
11.
Estrogens are considered to play a major role in promoting the proliferation of both the normal and the neoplastic breast epithelium. Their role as breast carcinogens has long been suspected and recently confirmed by epidemiological studies. Three major mechanisms are postulated to be involved in their carcinogenic effects: stimulation of cellular proliferation through their receptor-mediated hormonal activity, direct genotoxic effects by increasing mutation rates through a cytochrome P450-mediated metabolic activation, and induction of aneuploidy. Recently it has been fully demonstrated that estrogens are carcinogenic in the human breast by testing in an experimental system the natural estrogen 17β-estradiol (E 2) by itself or its metabolites 2-hydroxy, 4-hydroxy, and 16- a-hydroxy-estradiol (2-OH-E 2, 4-OH-E 2, and 16--OH E 2), respectively, by inducing neoplastic transformation of human breast epithelial cells (HBEC) MCF-10F in vitro to a degree at least similar to that induced by the chemical carcinogen benz( a)pyrene (BP). Neither Tamoxyfen (TAM) nor ICI-182,780 abrogated the transforming efficiency of estrogen or its metabolites. The E 2 induced expression of anchorage independent growth, loss of ductulogenesis in collagen, invasiveness in Matrigel, is associated with the loss of 9p11-13 and only invasive cells that exhibited a 4p15.3-16 deletion were tumorigenic. Tumors were poorly differentiated ER- and progesterone receptor negative adenocarcinomas that expressed keratins, EMA and E-cadherin. The E 2 induced tumors and tumor-derived cell lines exhibited loss of chromosome 4, deletions in chromosomes 3p12.3-13, 8p11.1-21, 9p21-qter, and 18q, and gains in 1p, and 5q15-qter. The induction of complete transformation of the human breast epithelial cell MCF-10F in vitro confirms the carcinogenicity of E 2, supporting the concept that this hormone could act as an initiator of breast cancer in women. This model provides a unique system for understanding the genomic changes that intervene for leading normal cells to tumorigenesis and for testing the functional role of specific genomic events taking place during neoplastic transformation. 相似文献
12.
Aromatase (estrogen synthase) is the cytochrome P450 enzyme complex that converts C 19 androgens to C 18 estrogens. Aromatase activity has been demonstrated in breast tissue in vitro, and expression of aromatase is highest in or near breast tumor sites. Thus, local regulation of aromatase by both endogenous factors as well as exogenous medicinal agents will influence the levels of estrogen available for breast cancer growth. The prostaglandin E 2 (PGE 2) increases intracellular cAMP levels and stimulates estrogen biosynthesis, and our recent studies have shown a strong linear association between CYP19 expression and the sum of COX-1 and COX-2 expression in breast cancer specimens. PGE 2 can bind to four receptor subtypes, EP 1–EP 4, which are coupled to different intracellular signaling pathways. In primary human breast stromal cell cultures, aromatase activity was significantly induced by PGE 2, dexamethasone, and agonists for the EP 1 and EP 2 receptor subtypes. An EP 1 antagonist, SC-19220, inhibited the induction of enzyme activity by PGE 2 or 17-phenyltrinor-PGE 2, an EP 1 agonist. Sulprostone, an EP 3 agonist, did not alter aromatase activity levels. Investigations are also underway on the regulation of aromatase by exogenous medicinal agents. Selective steroidal and nonsteroidal agents are effective in inhibiting breast tissue aromatase. The benzopyranone ring system is a molecular scaffold of considerable interest, and this scaffold is found in certain flavonoid natural products that have weak aromatase inhibitory activity. Our novel synthetic route for benzopyranones utilizes readily available salicylic acids and terminal alkynes as starting materials. The synthesis of flavones with diversity on the benzopyranone moiety and at the C-2 position occurs with good to excellent yields using these reaction conditions, resulting in an initial benzopyranone library of thirty compounds exhibiting enhanced and differential aromatase inhibition. Current medicinal chemistry efforts focus on diversifying the benzopyranone scaffold and utilizing combinatorial chemistry approaches to construct small benzopyranone libraries as potential aromatase inhibitors. 相似文献
13.
Following the introduction of potent aromatase inhibitors for the treatment of breast cancer patients, highly sensitive methods have become mandatory to evaluate the influence of these drugs on plasma estrogen levels. Commercially available kits for estrogen measurements are not suitable for these kinds of evaluations due to their detection limits that are close to baseline estrogen levels in postmenopausal women. We describe here an optimised radioimmunoassay suitable for the simultaneous measurement of plasma estrone (E 1), estradiol (E 2) and estrone sulfate (E 1S) levels in the ultra-low range. Following incubation with [ 3H]-labelled estrogens as internal standards, crude estrogen fractions were separated by ether extraction. The E 1S fraction was hydrolysed with sulfatase followed by eluation on a Sephadex column. Free estrogens (E 1, E 2) were separated by chromatography (LH-20). Estrone and E 1S (following hydrolysis) were converted into E 2, and each estrogen fraction was measured by the same highly sensitive and specific radioimmunoassay using estradiol-6-(O-carboxymethyl)-oximino-2-(2-[ 125I]-iodo-histamine) as ligand. Although several purification steps were involved, the internal recovery values for tritiated estrogens were found to be 88%, 90%, and 49% for E 1, E 2 and E 1S, respectively. The intra-assay coefficient of variation was <5% for all recovery measurements. The detection limits were calculated following repeated blank measurements and found to be 1.14 pmol/L for E 1, 0.67 pmol/L for E 2, and 0.55 pmol/L for E 1S, respectively. The intra-assay coefficient of variation (CV) was found to be 3.4% for E 1, 5.1% for E 2 and 6.1% for E 1S, while the inter-assay CV was 13.6%, 7.6% and 7.5% for E 1, E 2, and E 1S, respectively. Considering normal plasma levels for E 2 (15 pmol/L), E 1 (80 pmol/L) and E 1S (400 pmol/L) in postmenopausal women, the method allows theoretically to detect suppression of plasma E 2, E 1 and E 1S levels by 95.5%, 98.6% and 99.9% when starting from average, normal postmenopausal levels. Thus, the method presented here is to our knowledge the currently most sensitive assay available for plasma estrogen measurements in the ultra-low range and, as such, a reliable tool for a proper evaluation of potent aromatase inhibitors and other potential drugs influencing on plasma estrogen levels. 相似文献
14.
Estrone sulfate (E1S) is an endogenous prodrug that delivers estrone and, subsequently, estradiol to the target cells following the hydrolysis by the enzyme estrone sulfatase which is active in various tissues including hormone dependent breast cancer cells. Blockade of this enzyme should reduce the estrogen level in breast cancer cells and prevent hormonal growth stimulation. Sulfamates of a variety of phenolic compounds have been shown to be inhibitors of estrone sulfatase. Our rational is based on findings that these inhibitors can undergo hydrolysis and the pharmacological effects of the free hydroxy compounds contribute to the bioactivity of the sulfamates. A desirable action of the metabolites would be an estrogen antagonism to block stimulatory effects of residual amounts of estrogens. Thus, we synthesized a number of sulfamoyloxy-substituted 2-phenylindoles with side chains at the indole nitrogen that guarantee antiestrogenic activity. All of the new sulfamates were studied for their inhibitory effects on the enzyme estrone sulfatase from human breast cancer cells and their (anti)hormonal activities in stably transfected human MCF-7/2a mammary carcinoma cells. The hormonal profile of the sulfamates was partly reflected by the properties of the corresponding hydroxy precursors. Some of the sulfamoylated antiestrogens strongly inhibited estrone sulfatase activity with IC 50 values in the submicromolar range. They were devoid of agonist activity and suppressed estrone sulfate-stimulated gene expression mainly by blocking the enzyme. Examples are the disulfamates of the indoles ZK 119, 010 and ZK 164, 015. Their IC 50s for sulfatase inhibition were 0.3 and 0.2 μM, respectively, and 50 and 80 nM, respectively, for the inhibition of E1S-stimulated luciferase expression in transfected MCF-7 cells. With some of the new sulfamates an additional direct antiestrogenic effect was noticed which might be due to a partial hydrolysis during incubation and would improve the growth inhibitory effect on estrogen-sensitive breast cancer cells. 相似文献
15.
The epoxy resin bisphenol F diglycidyl ether (BFDGE), was examined for its mutagenicity in prokaryotic assays (Salmonella typhimurium His(-) and Escherichia coli Trp(-) tests) and its genotoxicity in eukaryotic systems (sister chromatid exchange (SCE) and micronucleus tests in human lymphocytes), in the presence or absence of an exogenous metabolizing system (S9 from rat liver). In the prokaryotic tests, the concentrations of BFDGE ranged between 100 and 5000 micro g per plate, and in the eukaryotic assays from 12.5 to 62.5 micro g/ml. The compound is able to induce mutagenic effects in bacterial strains TA100, TA1535, WP2uvrA and IC3327, as revealed by the increase observed in the number of induced revertants. With respect to the genotoxicity assays, BFDGE induces an increase in the frequency of sister chromatid exchanges and micronuclei in human peripheral blood lymphocytes. 相似文献
16.
Steroid sulfatase (STS) hydrolyzes inactive estrone sulfate (E1-S) to estrone (E1), while estrogen sulfotransferase (EST; SULT 1E1 or STE gene) sulfonates estrogens to estrogen sulfates. They are considered to play important roles in the regulation of local estrogenic actions in various human tissues, however, their biological significance remains largely unknown. Therefore, we examined the expression of STS and EST in non-pathologic human tissues and breast carcinomas. STS expression was very weak except for the placenta, while EST expression was markedly detected in various tissues examined. In breast carcinoma tissues, STS and EST immunoreactivity was detected in carcinoma cells in 74 and 44% of cases, respectively, and was significantly associated with their mRNA levels and enzymatic activities. STS immunoreactivity was significantly correlated with the tumor size, and an increased risk of recurrence. EST immunoreactivity was inversely correlated with the tumor size or lymph node status. Moreover, EST immunoreactivity was significantly associated with a decreased risk of recurrence or improved prognosis. Our results suggest that EST is involved in protecting various peripheral tissues from excessive estrogenic effects. In the breast carcinoma, STS and EST are suggested to play important roles in the regulation of in situ estrogen production in the breast carcinomas. 相似文献
17.
Previous studies have shown that the selection of women who are at high breast cancer risk for treatment with chemoprevention agents leads to an enhanced benefit/risk ratio. However, further efforts to implement this strategy will require the development of new models to predict the breast cancer risk of particular individuals. Postmenopausal women with elevated plasma or serum estrogens are at increased risk for breast cancer. Therefore, the roles of various enzymes involved in the biosynthesis of estrogens in postmenopausal women have been reviewed in detail. In addition, the potential genotoxic and/or proliferative effects of the different estrogen metabolites as risk factors in the etiology of breast cancer have been examined. Unfortunately, much of the current bioanalytical methodology employed for the analysis of plasma and serum estrogens has proved to be problematic. Major advances in risk assessment would be possible if reliable methodology were available to quantify estradiol and its major metabolites in the plasma or serum of postmenopausal women. High performance liquid chromatography (HPLC) coupled with radioimmunoassay (RIA) currently provides the most sensitive and best validated immunoassay method for the analysis of estrone and estradiol in serum samples from postmenopausal women. However, inter-individual differences in specificity observed with many other immunoassays have caused significant problems when interpreting epidemiologic studies of breast cancer. It is almost impossible to overcome the inherent assay problems involved in using RIA-based methodology, particularly for multiple estrogens. For reliable measurements of multiple estrogens in plasma or serum, it will be necessary to employ stable isotope dilution methodology in combination with liquid chromatography–tandem mass spectrometry (LC–MS/MS). Extremely high sensitivity can be obtained with pre-ionized estrogen derivatives when employed in combination with a modern triple quadrupole mass spectrometer and nanoflow LC. Using [ 13C 6]-estrone as the internal standard it has proved possible to analyze estrone as its pre-ionized Girard T (GT) derivative in sub-fg (low amol) amounts on column. This suggests that in the future it will be possible to routinely conduct LC–MS assays of multiple estrogen metabolites in serum and plasma at even lower concentrations than the current lower limit of quantitation of 0.4 pg/mL (1.6 pmol/L). The ease with which the pre-ionization derivatization strategy can be implemented will make it possible to readily introduce high sensitivity stable isotope dilution methodology in laboratories that are currently employing LC–MS/MS methodology. This will help conserve important plasma and serum samples as it will be possible to conduct high sensitivity analyses using low sample volumes. 相似文献
18.
The contribution of local synthesis versus circulatory delivery of normal breast as well as breast cancer tissue estrogens has remained a controversial area for decades. Novel data on tissue estrogen levels confirm a positive normal breast tissue to plasma concentration gradient for estrone, and to a smaller extent estradiol. Remarkably, this gradient is similar for pre- and post-menopausal women. Together with pharmacokinetic data on estrogen disposition, these findings suggest plasma and breast tissue estrogens to rapidly equilibrate, with circulating estrogens being a major contributor to breast tissue estrone levels. A likely explanation to the concentration gradient could be the fact that non-polar estrogens easily dissolve into tissue fat compartments as compared to plasma. While intratumor estrone levels are low as compared to benign tissue concentrations, intratumor estradiol is elevated in ER+ tumors. The correlation between intratumor estradiol levels and expression levels of dehydrogenases reducing estrone into estradiol but also intratumor ER concentrations are consistent with intratumor estrogen activation but also a scavenger effect of the ER. 相似文献
19.
Two isothiocyanates (ITCs) commonly found in human diet, allyl isothiocyanate (AITC) and phenethyl isothiocyanate (PEITC), were tested for genotoxic effects in a battery of assays: Salmonella/microsome assay with TA 98 and TA 100, differential DNA repair assay with E. coli and micronucleus (MN) induction assay with human derived Hep G2 cells. Albeit to a different degree, both ITCs induced genotoxic effects in all test systems. AITC was more genotoxic in bacterial test systems than in Hep G2 cells; in contrast, the effect of PEITC was stronger in Hep G2 cells. In in vivo assays with E. coli indicators in which mice were exposed to relatively high doses of the compounds (90 and 270 mg/kg), AITC induced moderate but significant effects; PEITC failed to induce significant effects in any of the organs. To find out the reason for the weak genotoxicity of AITC and PEITC under in vivo test conditions, we exposed E. coli indicator cells to the test substances in the absence or presence of rat liver homogenate (with and without cofactors), bovine serum albumin (BSA) and human saliva. All of them markedly attenuated the genotoxicity of AITC and PEITC, implying that the test substances are detoxified by direct non-enzymatic binding to proteins. Additional experiments carried out on the mechanistic aspects of AITC and PEITC-induced genotoxicity showed that the compounds induce the formation of thiobarbituric acid reactive substances (TBARS) in Hep G2 cells. Furthermore, in in vitro assays with E. coli, radical scavengers reduced the differential DNA damage induced by AITC and PEITC. The latter two findings give a clue that reactive oxygen species might be involved in the genotoxic effect of the ITCs. Although ITCs have been repeatedly advocated as very promising anticancer agents, the data presented here indicate that the compounds are genotoxic, and probably carcinogenic, in their own right. 相似文献
20.
The study of DNA damage at the chromosome level is an essential part of genetic toxicology because chromosomal mutation is an important event in carcinogenesis. The micronucleus assays have emerged as one of the preferred methods for assessing chromosome damage because they enable both chromosome loss and chromosome breakage to be measured reliably. Because micronuclei can only be expressed in cells that complete nuclear division a special method was developed that identifies such cells by their binucleate appearance when blocked from performing cytokinesis by cytochalasin-B (Cyt-B), a microfilament-assembly inhibitor. The cytokinesis-block micronucleus (CBMN) assay allows better precision because the data obtained are not confounded by altered cell division kinetics caused by cytotoxicity of agents tested or sub-optimal cell culture conditions. The method is now applied to various cell types for population monitoring of genetic damage, screening of chemicals for genotoxic potential and for specific purposes such as the prediction of the radiosensitivity of tumours and the inter-individual variation in radiosensitivity. In its current basic form the CBMN assay can provide, using simple morphological criteria, the following measures of genotoxicity and cytotoxicity: chromosome breakage, chromosome loss, chromosome rearrangement (nucleoplasmic bridges), cell division inhibition, necrosis and apoptosis. The cytosine-arabinoside modification of the CBMN assay allows for measurement of excision repairable lesions. The use of molecular probes enables chromosome loss to be distinguished from chromosome breakage and importantly non-disjunction in non-micronucleated binucleated cells can be efficiently measured. The in vitro CBMN technique, therefore, provides multiple and complementary measures of genotoxicity and cytotoxicity which can be achieved with relative ease within one system. The basic principles and methods (including detailed scoring criteria for all the genotoxicity and cytotoxicity end-points) of the CBMN assay are described and areas for future development identified. 相似文献
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