Vitellogenin-inducing activities of natural, synthetic, and environmental estrogens in primary cultured Xenopus laevis hepatocytes

Vitellogenin (VTG)-inducing activities of natural estrogens (E1: estrone, E2:17beta-estradiol, E3: estriol, alpha-E2: 17alpha-estradiol), synthetic estrogens (EE2: 17alpha-ethynyl estradiol, DES: diethylstilbestrol,), phytoestrogen (GEN: genistein), and xeno-estrogens (BPA: bisphenol A, NP: nonylphenol, OP: octylphenol) were investigated by an assay system using primary-cultured hepatocytes of Xenopus laevis. An enzyme-linked immunoabsorbent assay (ELISA) was able to detect VTG at a minimum detection limit of 0.06 ng/mL. Relative estrogenic activities of the compounds were determined from their dose-response curves. The activities relative to E2 activity were 138% for DES, 121% for EE2, 6.1% for E3, 0.33% for E1, 0.29% for alpha-E2, 0.037% for GEN, 0.008% for BPA, 0.005% for NP, and 0.002% for OP. Comparison with data reported for other bioassay systems revealed that there were significant interspecies-and cell-type-differences in the activities of DES, E3, E1 and alpha-E2. BPA was found to have a substantial antagonistic activity (approximately 0.8% of tamoxifen activity) under the influence of physiological concentrations of E2. Complex-effects of endocrine disrupters on aquatic animals will be discussed.     

E2-3S在水平流人工湿地中的降解与转化

研究采用同时检测15种自由态雌激素(Free estrogens, FEs)及结合态雌激素(Conjugated estrogens, CEs; 包括雌激素手性分子、异构体、单位点及多位点结合的CEs)的方法, 探讨目标污染物17β-estradiol-3-sulfate (E2-3S)与其他类型雌激素(包括自由态雌激素与其他结合态雌激素)间的转化关系, 研究E2-3S在有/无植物水平流人工湿地(HFCWs)中的降解规律。结果显示: 在停留时间为1.5d的工况下, E2-3S在HFCWs进水端基质水平距离0 cm深度15 cm中转化率已达98%, E2-3S可转化为其他雌激素, 产物以FEs为最丰富(均占70%以上), 植物可以显著提高湿地DO浓度, 使FEs残留浓度比无植物CW(U-CW)更低, 有植物CW(P-CW)和U-CW对总雌激素去除效率分别为86%和58%; E2-3S的主要转化路径为经硫酯键断裂形成E2再氧化生成E1, 其次路径为直接氧化为E1-3S再水解生成E1, 少量路径为羟基化形成E3-3S再水解生成E3, 此外, E2-3S还可以产生痕量双位取代D-CEs(<总雌激素的5%), 且植物系统中存留量更低。     

Regulation of sex hormone-binding globulin secretion in human hepatoma G2 cells.

Our purpose was to examine the roles of natural (estradiol (E2) and estrone (E1)) and synthetic estrogens (ethinyl estradiol (EE), moxestrol (MOX), and tamoxifene (TAM)) in regulating production of sex hormone-binding globulin (SHBG) by human hepatoma G2 (Hep G2) cells, the rationale being that synthetic estrogens are less rapidly metabolized than natural estrogens and, thus, may alter SHBG levels more readily. In Hep G2 cells, E2, E1, and EE at 10(-7) M did not result in significantly greater SHBG secretion compared to control cells. The synthetic estrogens, MOX and TAM, caused significant, P < 0.001, increases of 30% and 51% in SHBG secretion at 10(-7) M compared to controls. However, when TAM and E2 were added together, each at 10(-7) M, no increase in SHBG secretion was noted. We conclude that natural estrogens at physiologic concentrations do not increase SHBG secretion by Hep G2 cells, but the increase of SHBG secretion caused by MOX and TAM suggests that the lack of effect of E2 and E1 may, in part, be due to their rapid metabolism. In addition, TAM stimulates SHBG secretion by interaction with the genome that is different, in certain respects, from that of E2.     

Sex steroids in serum of prepubertal male and female horses and correlation with bone characteristics

We used radioimmunoassay (RIA) to measure monthly serum levels of unconjugated and conjugated sex steroids (testosterone T, androstenedione A, estradiol E(2), and estrone E(1)) in 4 male and 4 female foals during their first year of life. Maximal production of sex steroids was detected from April to August with hormonal peaks, corresponding to the natural breeding season in adults. In males, only A levels were more steady. Total estrogens (unconjugated plus conjugated E(2) and E(1)) were the major steroids in immature males in contrast to adults. Estrogens generally peaked in young females before males; the major estrogen was E(1), and total estrogens overtook total androgens (unconjugated and conjugated T and unconjugated A). We also sampled 3 male and 3 female foals with bone alterations in adulthood. For all animals, serum levels of four bone formation markers were obtained: osteocalcin (O), hydroxyproline (HP), and alkaline phosphatase (AP), and a radiographic score was determined. Only male foals with normal skeletal frame (good radiographic score GRS) in adulthood showed a correlation (P < 0.01) between the distribution frequency of each bone formation marker and unconjugated E(2) or E(1) levels; this finding highlighted the role of unconjugated estrogens in bone maturation in horses, since this was not found in the groups with bone alterations. In females, the threshold of estrogen synthesis and sensitivity was probably sufficient to be a nonlimiting factor at this stage of development. Our results strongly suggest a differential regulation of the estrogen/androgen balance in horses according to sex, sexual maturation, and photoperiod. Moreover, estrogens appear to be crucial for skeletal development in male colts, and these steroids are good modulators of skeletal frame characteristics in adulthood.     

Behavior of natural estrogens in semicontinuous activated sludge biodegradation reactors

Biological degradation of natural estrogens was investigated by separately spiking 17 beta-estradiol (E2) and estrone (E1) into activated sludge reactors operated in a semi-continuous flow mode under aerobic conditions. By conducting runs for simultaneous addition of glucose over the designated initial concentrations of 0, 10, 30, 50 and 100 mg l(-1), respectively, the likely effect of the easily biodegradable organic constituent on the degradation rate of the natural estrogens was also studied. The spiked E2 disappeared in a manner much faster than E1; and its disappearance occurred through elimination of its metabolic byproduct of E1. The apparent first-order disappearance rate of E2 and E1 fell in the ranges of 0.84-4.31h(-1) and 0.15-0.84 h(-1), respectively when spiked separately into respective reactors. A general trend of decreases in the disappearance rate with increases of the initial glucose concentration was revealed. The presence of glucose was also found capable of lowering the conversion ratio from E2 to E1.     

Chemical kinetics and interactions involved in horseradish peroxidase-mediated oxidative polymerization of phenolic compounds

The primary objective of this research was to evaluate various factors that affect the reaction rate of oxidative coupling (OXC) reaction of phenolic estrogens catalyzed by horseradish peroxidase (HRP). Kinetic parameters were obtained for the conversion of phenol as well as natural and synthetic estrogens estrone (E(1)), 17β-estradiol (E(2)), estriol (E(3)), and 17α-ethinylestradiol (EE(2)). Molecular orbital theory and Autodock software were employed to analyze chemical properties and substrate binding characteristics. Reactions were first order with respect to phenolic concentration and reaction rate constants (k(r)) were determined for phenol, E(3), E(1), E(2) and EE(2) (in increasing order). Oxidative coupling was controlled by enzyme-substrate interactions, not collision frequency. Docking simulations show that higher binding energy and a shorter binding distance both promote more favorable kinetics. This research is the first to show that the OXC of phenolics is an entropy-driven and enthalpy-retarded process.     

The role of estrogens on the proliferation of human breast tumor cells (MCF-7)

The cloned human breast tumor cell line C7MCF7-173 behaved as an estrogen-dependent tumor in the nude mice. In contrast, E2 added to serum-less media did not increase the multiplication rate of these cells over the values obtained in the control cultures. Media supplemented with charcoal-dextran stripped (CD) human female serum (FHS) resulted in inhibition of cell proliferation in a concentration-dependent pattern (40% = 20% greater than 10% greater than 5% greater than 2.5%). E2 addition to all but the 2.5% CDFHS significantly increased the proliferation rate of these cells. The E2 concentration required to attain maximal proliferation rate increased as the serum concentration of the medium increased (e.g. 3 X 10(-11)M for 10% CDFHS, 3 X 10(-10)M for 40% CDFHS). E2 concentrations higher than the one needed to achieve maximal proliferation rate resulted in decreased cell yields (shut-off mechanism). Similar effects were obtained with synthetic and other natural estrogens. CD fetal bovine serum (FBS) also inhibited the proliferation of C7MCF7-173 cells; however, at similar concentration the inhibitory effect of CDFHS was more potent than the one obtained with CDFBS. The addition of "growth factors" (insulin, Epidermal Growth Factor and transferrin) and non-estrogenic steroids to 10% CDFHS failed to overcome the inhibitory effect of this serum. These results suggest that: (1) human and fetal bovine sera contain a specific inhibitor of the proliferation of E2-sensitive cells (estrocolyones), and (2) E2 promotes cell proliferation by neutralizing this inhibitor.     

Effects of thyroid hormones on human breast cancer cell proliferation

The involvement of estrogens in breast cancer development and growth has been well established. However, the effects of thyroid hormones and their combined effects with estrogens are not well studied. We investigated the response of human breast cancer cells to thyroid hormone, particularly the role of T3 in mediating cell proliferation and gene expression. We demonstrated that 17β-estradiol (E2) or triiodothyronine (T3) promoted cell proliferation in a dose-dependent manner in both MCF-7 and T47-D cell lines. The E2- or T3-dependent cell proliferation was suppressed by co-administration of the ER antagonist ICI. We also demonstrated that T3 could enhance the effect of E2 on cell proliferation in T47-D cells. Using an estrogen response element (ERE)-mediated luciferase assay, we determined that T3 was able to induce the activation of ERE-mediated gene expression in MCF-7 cells, although the effects were much weaker than that induced by E2. These results suggest that T3 can promote breast cancer cell proliferation and increase the effect of E2 on cell proliferation in some breast cancer cell lines and thus that T3 may play a role in breast cancer development and progression.     

Adrenal contribution to circulating estrogens in woman.

To study the contribution of adrenal glands to circulating estrogens in woman, the concentrations of estrone (E1), estradiol-17 beta (E2), and estriol (E3) in adrenal and peripheral venous blood were measured by radioimmunoassay and the grandular secretion of estrogens after ACTH stimulation was investigated by analyzing the adrenal vein levels of these steroids in patients with breast cancer who were undergoing a therapeutic adrenal operation. Furthermore, adrenal secretion rates of estrogens and cortisol were estimated. It was shown that there existed greater concentrations of estrogens in adrenal vein than in peripheral blood; about 3 times higher for E1 (p less than 0.001), and 2 times higher for E2 and E3 (p less than 0.05). Administration of ACTH caused a significant increase of E1 and E2 concentrations in adrenal venous blood to mean 150% of the basal levels that was comparable to the increase of cortisol. Apparent adrenal secretion rates of estrogens estimated under surgical situation were calculated to be 7.7 +/- 1.7 (M +/- SE) microgram/day for E1, 1.9 +/- 0.3 microgram/day for E2, and 0.3 +/- 0.2 microgram/day for E3, while the secretion rate of cortisol was 52.7 +/- 8.2 microgram/min. The present study demonstrates that the direct adrenal secretion of not only E1, but E2 and E3 contributes to the circulating estrogen levels, and it is suggested that the adrenal glands might be responsible for the relatively important source of estrogen production in the aged woman.     

Occurrence, fate, and biodegradation of estrogens in sewage and manure

The estrogens estrone (E1), 17α-estradiol (E2α), 17β-estradiol (E2β), and estriol (E3) are natural sex hormones produced by humans and animals. In addition, there are some synthetic estrogens, such as 17α-ethinylestradiol (EE2), used for contraception purposes. These compounds are able to produce endocrine disruption in living organisms at nanogram-per-liter levels. In both humans and animals, estrogens are excreted in urine and feces, reaching the natural environment through discharge from sewage treatment plants (STP) and manure disposal units. In STPs, hormone removal depends on the type of treatment process and on different parameters such as the hydraulic and sludge retention times. Thus, hormone elimination rates vary from 0% to 90% in different STPs. Animals are also an important source of estrogens in the environment. Indeed, animals produce high concentrations of hormones which will end up in manure which is typically spread on land. Hence, waste-borne animal hormones may transfer these pollutants to the soil. The purpose of this review is to highlight the significance for both health and the environment of pollution by estrogens and critically review the existing knowledge on their fate and removal in different treatment processes. Relevant information on the microbial degradation of hormones and metabolic pathways is also included.     

Estradiol and Estrogen Receptor Agonists Oppose Oncogenic Actions of Leptin in HepG2 Cells

Obesity is a significant risk factor for certain cancers, including hepatocellular carcinoma (HCC). Leptin, a hormone secreted by white adipose tissue, precipitates HCC development. Epidemiology data show that men have a much higher incidence of HCC than women, suggesting that estrogens and its receptors may inhibit HCC development and progression. Whether estrogens antagonize oncogenic action of leptin is uncertain. To investigate potential inhibitory effects of estrogens on leptin-induced HCC development, HCC cell line HepG2 cells were treated with leptin in combination with 17 β-estradiol (E2), estrogen receptor-α (ER-α) selective agonist PPT, ER-β selective agonist DPN, or G protein-coupled ER (GPER) selective agonist G-1. Cell number, proliferation, and apoptosis were determined, and leptin- and estrogen-related intracellular signaling pathways were analyzed. HepG2 cells expressed a low level of ER-β mRNA, and leptin treatment increased ER-β expression. E2 suppressed leptin-induced HepG2 cell proliferation and promoted cell apoptosis in a dose-dependent manner. Additionally E2 reversed leptin-induced STAT3 and leptin-suppressed SOCS3, which was mainly achieved by activation of ER-β. E2 also enhanced ERK via activating ER-α and GPER and activated p38/MAPK via activating ER-β. To conclude, E2 and its receptors antagonize the oncogenic actions of leptin in HepG2 cells by inhibiting cell proliferation and stimulating cell apoptosis, which was associated with reversing leptin-induced changes in SOCS3/STAT3 and increasing p38/MAPK by activating ER-β, and increasing ERK by activating ER-α and GPER. Identifying roles of different estrogen receptors would provide comprehensive understanding of estrogenic mechanisms in HCC development and shed light on potential treatment for HCC patients.     

Estrogen induction of progestophilins in rat estrogen-sensitive cells grown in media supplemented with sera from castrated rats and from rats bearing an alpha-fetoprotein-secreting hepatoma

The purpose of this work was to study the effect of alpha-fetoprotein (AFP) over cell multiplication and the induction of an estradiol-17 beta (E2)-dependent marker, i.e., progestophilins in E-sensitive cells C2(9)RAP derived from a W/Fu rat pituitary tumor. These cells proliferate in isogeneic hosts under the influence of E2, while they proliferate in culture regardless of the presence of E2. C2(9)RAP cells were grown in medium supplemented with 10% horse serum. Progestophilin levels were measured 48 h after adding serum (20% horse, or castrated rat, or AFP-secreting tumor-bearing rat) and estrogen to the 10% horse serum-supplemented medium in which the cells were growing. Maximal induction of progestophilins was obtained at 3 X 10(-10) M E2 in cells grown in medium containing horse or castrated rat serum. In contrast, maximal induction of progestophilins required 3 X 10(-8) M E2 in cells grown in medium supplemented with the serum of Morris hepatoma 7777-bearing rats. This serum contained AFP levels comparable to those present at birth in the rat. 11-Methoxy-17 beta ethynylestradiol (R2858), a synthetic estrogen with little affinity for AFP, was also tested for its ability to induce progestophilins. The degree of maximal induction of progestophilins expressed as percentage of the respective control, was similar for all experimental groups, both with E2 and with R2858. In addition, we compared the free E2 levels in the culture medium with the progestophilin levels and the cell proliferation rate. We found that the progestophilin levels were maximal at free E2 concentrations above 11 pg E2/ml, whereas there was no correlation between the free E2 levels and the proliferation rate. Moreover, the proliferation rate of cells in medium supplemented with horse or castrated rat serum was maximal at concentrations of free E2 below 0.4 pg/ml; whereas cell proliferation was inhibited with hepatoma serum even at concentrations of free E2 of 44 pg/ml. We conclude that the effect of hepatoma serum on the E2 induction of progestophilins seems to be mediated by the effect of AFP on the availability of free estrogen, since it is abolished by the addition of both natural and synthetic estrogens. The inhibitory effect of hepatoma serum upon cell proliferation is not reversed by estrogens and thus seems to be mediated by mechanisms other than E2 trapping by AFP.     

Effect of estrogens on boar sperm capacitation <Emphasis Type="Italic">in vitro</Emphasis>

Background  

Mammalian sperm must undergo a series of controlled molecular processes in the female reproductive tract called capacitation before they are capable of penetrating and fertilizing the egg. Capacitation, as a complex biological process, is influenced by many molecular factors, among which steroidal hormone estrogens play their role. Estrogens, present in a high concentration in the female reproductive tract are generally considered as primarily female hormones. However, there is increasing evidence of their important impact on male reproductive parameters. The purpose of this study is to investigate the effect of three natural estrogens such as estrone (E1), 17beta-estradiol (E2) and estriol (E3) as well as the synthetical one, 17alpha-ethynylestradiol (EE2) on boar sperm capacitation in vitro.     

Effects of selected xenoestrogens on liver peroxisomes, vitellogenin levels and spermatogenic cell proliferation in male zebrafish

Environmental estrogenic compounds or xenoestrogens can mimic natural estrogens and cause a variety of adverse effects on aquatic wildlife. The purpose of the present work was to investigate if xenoestrogens are able to cause proliferation of liver peroxisomes using zebrafish (Danio rerio) as a model. Adult male zebrafish were exposed for 15 days to 17beta-estradiol (E2) and the xenoestrogens dibutylphthalate (DBP), methoxychlor (MXC), 4-tert-octylphenol (OP) and 17alpha-ethynylestradiol (EE2). All five tested compounds caused significant proliferation of liver peroxisomes (p < 0.05) as indicated by increased peroxisomal surface and numerical densities and elevated activities of the peroxisomal beta-oxidation enzyme acyl-CoA oxidase (AOX). In the case of DBP, MXC and E2, positive significant correlations between peroxisomal density parameters and AOX were found. The treatments did not produce gross alterations in testis histology, but spermatogenic cell proliferation was disturbed in E2 and EE2-treated groups and vitellogenin levels increased significantly in fish exposed to MXC, OP, EE2 and E2 with respect to controls. Furthermore, a significant correlation between vitellogenin levels and AOX activity was found for MXC, OP and EE2 treatments, suggesting that for the latter xenoestrogens early estrogenic effects are associated with liver peroxisome proliferation. No such association occurred with typical peroxisome proliferators such as DBP.     

Genetic abolishment of hepatocyte proliferation activates hepatic stem cells

Quiescent hepatic stem cells (HSCs) can be activated when hepatocyte proliferation is compromised. Chemical injury rodent models have been widely used to study the localization, biomarkers, and signaling pathways in HSCs, but these models usually exhibit severe promiscuous toxicity and fail to distinguish damaged and non-damaged cells. Our goal is to establish new animal models to overcome these limitations, thereby providing new insights into HSC biology and application. We generated mutant mice with constitutive or inducible deletion of Damaged DNA Binding protein 1 (DDB1), an E3 ubiquitin ligase, in hepatocytes. We characterized the molecular mechanism underlying the compensatory activation and the properties of oval cells (OCs) by methods of mouse genetics, immuno-staining, cell transplantation and gene expression profiling. We show that deletion of DDB1 abolishes self-renewal capacity of mouse hepatocytes in vivo, leading to compensatory activation and proliferation of DDB1-expressing OCs. Partially restoring proliferation of DDB1-deficient hepatocytes by ablation of p21, a substrate of DDB1 E3 ligase, alleviates OC proliferation. Purified OCs express both hepatocyte and cholangiocyte markers, form colonies in vitro, and differentiate to hepatocytes after transplantation. Importantly, the DDB1 mutant mice exhibit very minor liver damage, compared to a chemical injury model. Microarray analysis reveals several previously unrecognized markers, including Reelin, enriched in oval cells. Here we report a genetic model in which irreversible inhibition of hepatocyte duplication results in HSC-driven liver regeneration. The DDB1 mutant mice can be broadly applied to studies of HSC differentiation, HSC niche and HSCs as origin of liver cancer.     

Effect of the activation of macrophages on the course of regeneration of rat liver following partial hepatectomy

Stimulation of the Kupffer cells with E. coli endotoxin (the purified lipopolysaccharide) or with prodigiosan (a polysaccharide from Serratia marcescens) 24 h before partial hepatectomy (resection of 65-70% of the liver) stimulated and intensified the onset of liver regenerative activity (evaluated from changes in liver DNA synthesis, the H5 labelling index and the mitotic activity of the hepatocytes). Liver DNA synthesis increased together with the dose of endotoxin (i.v., from 25 to 1000 micrograms/kg body weight). If E. coli endotoxin was injected during or 3 h after partial hepatectomy, partial inhibition of liver DNA synthesis was observed. In mice stimulated with zymosan (a polysaccharide isolated from yeast), administered 5 days before performing partial hepatectomy, proliferation of the hepatocytes (evaluated from changes in the 3H labelling index and in the mitotic activity of the hepatocytes) was evaluated. The results confirm that proliferation is correlated to the state of reactivity of the Kupffer cells.     

Chimeric molecule IL-6/soluble IL-6 receptor is a potent mitogen for fetal hepatocytes

A novel recombinant molecule, termed IL-6c and consisting of a chimera of interleukin 6 (IL-6) and its soluble receptor is extremely potent in stimulating proliferation of hematopoietic progenitors. We investigated the effect of the IL-6c on the proliferation and differentiation of E14 fetal hepatocytes. IL-6c, in a dose-dependent manner, stimulated proliferation of E14 fetal rat hepatocytes. Adult hepatocyte mitogens together with IL-6c showed no further effect on proliferation. Hematopoietic stem cells mitogens SCF and flt3 ligand (FL) were also mitogenic for fetal hepatocytes, but did not further enhance the effect of IL-6c on cell proliferation. IL-6c decreased expression of fetal markers alpha-fetoprotein (AFP) and gamma-glutamyltranspeptidase, and induced expression of adult enzyme glucose-6-phosphatase (Gluc-6-P) in E14 hepatocytes. On the other hand, IL-6c strongly reduced, in a dose-dependant manner, expression of albumin and tyrosine aminotransferase (TAT). However, when the cells were grown for 3 days with IL-6c, and IL-6c was removed for the next 5 days, expression of albumin and TAT returned to levels found in control cultures. In conclusion, IL-6c stimulated proliferation and affected gene expression in fetal hepatocytes in culture.     

Competitive product inhibition of aromatase by natural estrogens

In order to better understand the function of aromatase, we carried out kinetic analyses to asses the ability of natural estrogens, estrone (E₁), estradiol (E₂), 16-OHE₁, and estriol (E₃), to inhibit aromatization. Human placental microsomes (50 μg protein) were incubated for 5 min at 37°C with [1β-³H]testosterone (1.24 × 10³ dpm ³H/ng, 35–150 nM) or [1β-³H,4-¹⁴C]androstenedione (3.05 × 10³ 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 K_i of E₁, E₂, 16-OHE₁, and E₃ for testosterone aromatization was 1.5, 2.2, 95, and 162 μM, respectively, where the K_m of aromatase was 61.8 ± 2.0 nM (n = 5) for testosterone. The K_i of E₁, E₂, 16-OHE₁, and E₃ for androstenedione aromatization was 10.6, 5.5, 252, and 1182 μM, respectively, where the K_m 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.