Estrogen-like response to p-nonylphenol in human first trimester placenta and BeWo choriocarcinoma cells.

p-Nonylphenol (p-NP) is a metabolite of alkylphenol ethoxylates used as surfactants in the manufacturing industry. Although it is reported to have estrogenic activity and to be transferred from the mother to the embryo, no data are available on its effects on the development of the human placenta. In the present study, we investigated estrogen receptors' (ERs) expression in the first trimester human placenta. Using an in vitro model of chorionic villous explants, we then compared the effects of p-NP and 17beta-estradiol (17beta-E2). Finally, a trophoblast-derived choriocarcinoma cell line, BeWo, was used as a model of trophoblast cell differentiation. Our results showed that the first trimester placenta expresses three ER-alpha isoforms of 67, 46, and 39 kDa and one ER-beta isoform of 55 kDa. Immunohistochemistry revealed the expression of ER-alpha in the villous cytotrophoblast, whereas ER-beta was mainly expressed by the syncytiotrophoblast. Treatment of explant cultures with p-NP (10(-9)M) and 17beta-E2 (10(-9)M) significantly increased beta-hCG secretion and cell apoptosis but did not modify ER expression. After 72 h of exposure, hormone release was significantly higher in p-NP- than 17beta-E2-treated explant cultures. By this time, cleavage of caspase-3 was evident in cultures treated with 17beta-E2 and p-NP. In BeWo cells, a caspase-3 band of 20-16 kDa was evident after 1 h of treatment with p-NP and after 24 h of treatment with 17beta-E2 or forskolin. These findings suggest that the human trophoblast may be highly responsive to p-NP and raise concern about maternal exposure in early gestation.     

Effect of genistein as a selective estrogen receptor beta agonist on the expression of Calbindin-D9k in the uterus of immature rats.

Genistein, a phytoestrogen possessing a high affinity for estrogen receptor beta (ERbeta), is of increasing interest because of its possible influence on the physiology of mammalian reproductive tracts. Although estrogen has been demonstrated to regulate Calbindin-D9k (CaBP-9k) in the rat uterus as with other calcium binding proteins, the role of ERbeta on the modulation of CaBP-9k remains to be elucidated. To elucidate the effect of genistein as a selective ERbeta agonist on uterine expression of CaBP-9k mRNA and protein, immature female rats were injected with genistein daily for three consecutive days in a dose-dependent (0.4, 4, and 40 mg/kg/day) and time-dependent (40 mg/kg/day; 3, 6, 12, 24, 48, and 72 h) manner. Then, the expression of CaBP-9k mRNA and protein was analyzed by Northern hybridization and Western blot, respectively, in the absence or presence of ICI 182,780 (ICI), an estrogen antagonist. In addition, the protein levels of ERalpha and ERbeta and mRNA level of progesterone receptor (PR) were further measured following genistein treatment to elucidate which of ERs is involved in CaBP-9k modulation. In a dose-dependent experiment, the highest dose of genistein (40 mg/kg/day) for 3 days significantly induced uterine CaBP-9k protein as 17beta-estradiol (E2) did. In addition, its maximal mRNA expression was observed at 3 and 6 h, and it returned to control level at 24 h in a time-dependent experiment. In parallel with its mRNA level, the protein level of CaBP-9k was significantly induced by genistein at 3 h and sustained up to 48 h. The pretreatment with ICI, followed by genistein or E2, completely blocked genistein- and E2-induced CaBP-9k protein in the uterus of immature rats. Interestingly, genistein was demonstrated to induce ERalpha protein, but not ERbeta and PR mRNA, an E2-responsive gene, in this tissue. These results imply that genistein, an ERbeta ligand, may regulate CaBP-9k gene through ERalpha pathway. Taken together, the present study demonstrated that genistein enhanced CaBP-9k gene via ERalpha in the uterus of immature rats, suggesting that ERalpha may be a key mediator in uterine CaBP-9k gene induction in immature rats.     

A rapid and sensitive reporter gene that uses green fluorescent protein expression to detect chemicals with estrogenic activity.

A reporter gene sequence was constructed within a eukaryotic expression vector. The altered plasmid contained 2 sequential estrogen response elements (ERE) coupled to a human phosphoglycerate kinase (PGK) promoter inserted upstream from a cDNA sequence encoding enhanced green fluorescent protein (GFP) with a 3'-polyadenylation signal. The plasmid was linearized and transfected into MCF-7 cells, a human breast cancer-derived line that expresses the estrogen receptor (ER). No selectable marker was present in the plasmid, requiring stably transfected cells to be selected by fluorescence-activated cell sorting based on GFP expression after the cells were treated with 10(-9) M 17beta-estradiol (E2). Stably transfected MCF-7 cells (MCF7-ERE) exhibited 2000-3000 times more fluorescence at 488 nm excitation and 512 nm emission than non-transfected cells. MCF7-ERE cells exhibited a linear increase in GFP expression induced over a range of 10(-12) M E2, a concentration giving 2 times the background expression, to maximal expression at 3 x 0(-10) M E2. From the maximal level, GFP expression plateaued, and then declined when E2 was increased to the highest concentration tested, 10(-7) M. 4-Hydroxytamoxifen (TFN-OH) treatment of cells produced a dose-dependent inhibition of E2-induced GFP expression, indicating the interaction of ER in the regulation of GFP gene expression. A series of estrogenic chemicals were evaluated for their capacity to induce GFP expression in MCF7-ERE cells, showing induced expression of GFP at concentrations 2-4 log units higher than the E2 concentration giving maximal GFP expression. The ERE-PGK-GFP reporter gene system is capable of rapid GFP expression in the presence of low concentrations of E2, and of quantifying estrogenicity of chemicals compared with a standard curve of the natural ligand, 17beta-estradiol.     

Genistein accumulates in body depots and is mobilized during fasting, reaching estrogenic levels in serum that counter the hormonal actions of estradiol and organochlorines.

Isoflavones are important dietary compounds that are consumed with the daily diet and elicit important biological actions. Here we report on the ability of genistein to partially accumulate in body depots of male mice, be released following fasting, and modulate the actions of estradiol and environmental estrogens in reproductive and nonreproductive target organs of estrogen-reporter mice (ERE-tK-luciferase). After the consumption of 50 mg/kg/day for 3 days, genistein accumulates in body compartments where it remains at functionally active levels for at least 15 days. Following 48 h of fasting, its concentration increased in serum from 99 +/- 13 to 163 +/- 17 nM. These levels are sufficient to exert an estrogenic effect in the testis and liver, as revealed by a twofold increase in luciferase gene expression. beta-Benzene-hexachloride (betaBHC) given at the concentration of 100 mg/kg/day for 3 days also accumulates in the body and is released by fasting, reaching serum levels of 176 +/- 33 nM, upregulating the luciferase gene in the liver and inhibiting its expression in the testis. When genistein was given in combination with betaBHC at doses sufficient to induce accumulation of both in body depots, the genistein mobilized by fasting reversed the action of the mobilized betaBHC in the testis. Acute administration of nutritional doses of genistein inhibited the action of estradiol and reversed the antiestrogenic action of o,p'-DDT: 1,1,1,-trichloro-2(p-chlorophenyl)-2-(o-chlorophenyl)ethane in the liver and the antiestrogenic action of betaBHC in the testis. Genistein had an additive effect with the ER agonist p,p'-DDT: 1,1,1,-trichloro-2,2-bis(p-chlorophenyl)ethane in the liver. The observed effects may be relevant to a protective action of phytoestrogens against estrogen receptor-interacting pollutants as well as the dietary modulation of estradiol action.     

Phytoestrogens and their human metabolites show distinct agonistic and antagonistic properties on estrogen receptor alpha (ERalpha) and ERbeta in human cells.

Phytoestrogens exert pleiotropic effects on cellular signaling and show some beneficial effects on estrogen-dependent diseases. However, due to activation/inhibition of the estrogen receptors ERalpha or ERbeta, these compounds may induce or inhibit estrogen action and, therefore, have the potential to disrupt estrogen signaling. We performed a comprehensive analysis and potency comparison of phytoestrogens and their human metabolites for ER binding, induction/suppression of ERalpha and ERbeta transactivation, and coactivator recruitment in human cells. The soy-derived genistein, coumestrol, and equol displayed a preference for transactivation of ERbeta compared to ERalpha and were 10- to 100-fold less potent than diethylstilbestrol. In contrast, zearalenone was the most potent phytoestrogen tested and activated preferentially ERalpha. All other phytoestrogens tested, including resveratrol and human metabolites of daidzein and enterolactone, were weak ER agonists. Interestingly, the daidzein metabolites 3',4',7-isoflavone and 4',6,7-isoflavone were superagonists on ERalpha and ERbeta. All phytoestrogens tested showed reduced potencies to activate ERalpha and ERbeta compared to diethylstilbestrol on the estrogen-responsive C3 promoter compared to a consensus estrogen response element indicating a degree of promoter dependency. Zearalenone and resveratrol were antagonistic on both ERalpha and ERbeta at high doses. The phytoestrogens enhanced preferentially recruitment of GRIP1 to ERalpha similar to 17beta-estradiol. In contrast, for ERbeta no distinct preference for one coactivator (GRIP1 or SRC-1) was apparent and the overall coactivator association was less pronounced than for ERalpha. Due to their abundance and (anti)-estrogenic potencies, the soy-derived isoflavones, coumestrol, resveratrol, and zearalenone would appear to have the potential for effectively functioning as endocrine disruptors.     

The colloidal thyroxine (T4) ring as a novel biomarker of perchlorate exposure in the African clawed frog Xenopus laevis.

The purpose of this study was to determine if changes in colloidal thyroxine (T(4)) immunoreactivity can be used as a biomarker of perchlorate exposure in amphibian thyroid tissue. Larval African clawed frogs (Xenopus laevis) were exposed to 0, 1, 8, 93, and 1131 microg perchlorate/l for 38 and 69 days to cover the normal period of larval development and metamorphosis. The results of this study confirmed the presence of an immunoreactive colloidal T(4) ring in thyroid follicles of X. laevis and demonstrated that the intensity of this ring is reduced in a concentration-dependent manner by perchlorate exposure. The smallest effective concentration of perchlorate capable of significantly reducing colloidal T(4) ring intensity was 8 microg perchlorate/l. The intensity of the immunoreactive colloidal T(4) ring is a more sensitive biomarker of perchlorate exposure than changes in hind limb length, forelimb emergence, tail resorption, thyrocyte hypertrophy, or colloid depletion. We conclude that the colloidal T(4) ring can be used as a sensitive biomarker of perchlorate-induced thyroid disruption in amphibians.     

Oestrogenic activity of p-hydroxybenzoic acid (common metabolite of paraben esters) and methylparaben in human breast cancer cell lines

This paper addresses the question of whether p-hydroxybenzoic acid, the common metabolite of parabens, possesses oestrogenic activity in human breast cancer cell lines. The alkyl esters of p-hydroxybenzoic acid (parabens) are used widely as preservatives in consumer products to which the human population is exposed and have been shown previously to possess oestrogenic activity and to be present in human breast tumour tissue, which is an oestrogen-responsive tissue. Recent work has shown p-hydroxybenzoic acid to give an oestrogenic response in the rodent uterotrophic assay. We report here that p-hydroxybenzoic acid possesses oestrogenic activity in a panel of assays in human breast cancer cell lines. p-Hydroxybenzoic acid was able to displace [(3)H]oestradiol from cytosolic oestrogen receptor of MCF7 human breast cancer cells by 54% at 5 x 10(6)-fold molar excess and by 99% at 10(7)-fold molar excess. It was able to increase the expression of a stably integrated oestrogen responsive reporter gene (ERE-CAT) at a concentration of 5 x 10(-4) M in MCF7 cells after 24 h and 7 days, which could be inhibited by the anti-oestrogen ICI 182 780 (Faslodex, fulvestrant). Proliferation of two human breast cancer cell lines (MCF7, ZR-75-1) could be increased by 10(-5) M p-hydroxybenzoic acid. Following on from previous studies showing a decrease in oestrogenic activity of parabens with shortening of the linear alkyl chain length, this study has compared the oestrogenic activity of p-hydroxybenzoic acid where the alkyl grouping is no longer present with methylparaben, which has the shortest alkyl group. Intrinsic oestrogenic activity of p-hydroxybenzoic acid was similar to that of methylparaben in terms of relative binding to the oestrogen receptor but its oestrogenic activity on gene expression and cell proliferation was lower than that of methylparaben. It can be concluded that removal of the ester group from parabens does not abrogate its oestrogenic activity and that p-hydroxybenzoic acid can give oestrogenic responses in human breast cancer cells.     

木豆叶提取物对人的类成骨细胞TE85成骨功能和体外破骨细胞分化的影响

木豆素是从天然植物木豆叶中提取的单体化合物，结构类似乙烯雌酚。本文观察木豆素及4种木豆叶提取物总成分(32-1，35-1，35-2和35-3)对人的类成骨细胞HOS TE85成骨功能、间质矿化及体外破骨细胞分化的影响。以木豆叶提取物作用于细胞48 h，观察细胞增殖、³H-脯氨酸掺入量及碱性磷酸酶活性；用抗酒石酸酸性磷酸酶(TRAP)染色法观察体外破骨细胞形成；作用于细胞18 d，用茜素红染色法观察细胞间质矿化。结果表明，作用于细胞48 h后，木豆素可促进细胞增殖，在1×10^-8 g·mL^-1时细胞数增加57.7%，³H-脯氨酸掺入量增加98.5%。35-1和35-3还可明显增加碱性磷酸酶活性。长时程作用后，32-1和35-3可明显增加成骨细胞间质矿化程度。木豆素(1×10^-7 g·mL^-1)对破骨细胞形成有明显的抑制作用，抑制率为22.8%，32-1(1×10^-7 g·mL^-1)的抑制率为37.9%。木豆素及木豆叶提取物都具有刺激成骨细胞骨形成、促进细胞间质矿化及抑制破骨细胞形成的活性，提示木豆素在骨细胞上有类雌激素样作用，具有抗骨质疏松新药的研究前景。     

Development of an in vivo anti‐androgenic activity detection assay using fenitrothion in Japanese medaka (Oryzias latipes)

The effects of endocrine disruptors, including anti‐androgenic chemicals, on aquatic environments have received increased attention in recent years. Currently, the method used to screen chemicals for anti‐androgenic activity is called the androgenized female stickleback screen, and it was established by the Organization of Economic Cooperation and Development in 2011 using the three‐spined stickleback. However, screening chemicals for anti‐androgenic activity has yet to be established using Japanese medaka. Thus, the purpose of this study was to establish a screening method for anti‐androgenic activity utilizing the number of papillary processes in Japanese medaka (Oryzias latipes ) as an indicator of the chemical's anti‐androgenic activity. Thus, at 35 days post‐fertilization, medaka were exposed to fenitrothion, an anti‐androgenic compound, for 28 days. In the control group, the formation of papillary processes was observed in XY medaka, but not in XX medaka. However, after fenitrothion exposure, the number of papillary processes was significantly decreased in a dose‐dependent manner in XY medaka; in the 300 μg l⁻¹ concentration group, four of 11 XY medaka showed no papillary processes even if there were no significant effects on total length and wet body weight compared with the control group. Our results indicate that the number of papillary processes in Japanese medaka can be used as an indicator of anti‐androgenic activity and that this model may prove useful as a chemical screening method. Copyright © 2016 John Wiley & Sons, Ltd.     

Differential binding affinities of PCBs, HO-PCBs, and aroclors with recombinant human, rainbow trout (Onchorhynkiss mykiss), and green anole (Anolis carolinensis) estrogen receptors, using a semi-high throughput competitive binding assay.

A comparative study was undertaken to assess the ability of 44 polychlorinated biphenyls (PCBs), 9 hydroxylated PCBs (HO-PCBs), and 8 aroclors at concentrations ranging from 1 nM to 10 microM to compete with [3H]17beta-estradiol (E2) for binding to bacterially expressed fusion proteins using a semi-high throughput competitive-binding assay. The fusion proteins consisted of the D, E, and F domains of human (alpha), cloned reptilian (Anolis carolinensis) and recloned rainbow trout (Onchorhynkiss mykiss) estrogen receptors (ER) linked to the glutathione S-transferase (GST) protein. GST-hERalphadef (human), GST-aERdef (reptile) and GST-rtERdef (rainbow trout) fusion proteins exhibited high affinity for E2 with dissociation constants (Kd) of 0.4+/-0.1 nM, 0.7+/-0.2 nM, and 0.6+/-0.1 nM, respectively. Of the 44 PCBs examined, only PCBs 104, 184, and 188 effectively competed with [3H]E2 for binding to the GST-rtERdef protein with IC50 values ranging from 0.4-1.3 microM. In contrast, these same congeners only caused a 30% displacement of [3H]E2 in GST-hERalphadef and GST-aERdef proteins. Several additional congeners were found to bind to the GST-rtERdef fusion protein, although the degree of interaction varied among congeners. Among the HO-PCBs, 2',3',4',5'-tetrachloro-4-biphenylol and 2,6,2',6'-tetrachloro-4-biphenylol bound to all three fusion proteins with IC50 values ranging from 0.1-0.3 microM. Dimethyl sulphoxide (DMSO) concentrations of 20% significantly increased the ability of PCBs 104, 184, and 188 to compete with [3H]E2 for binding to the GST-ERdef fusion proteins, whereas at 20% DMSO, a significant reduction in saturable binding was observed. These results demonstrate that ERs from different species exhibit differential ligand preferences and relative binding affinities for PCBs, which can be dramatically affected by DMSO concentration.     

Quantitative analysis of expression level of estrogen and progesterone receptors and VEGF genes in human endometrial stromal cells after treatment with nicotine

Cigarette smoke is a complex mixture of toxic chemicals, including nicotine, carbon monoxide, and several recognized carcinogens and mutagens. Nicotine has a direct disturbing influence on steroid hormones (estrogen and progesterone), which are essential components of the female reproductive system, but the effect of nicotine on the hormone receptors is not yet clear. The aim of this study was to elucidate the effect of nicotine on the expression of estrogen receptor (ER), progesterone receptor (PR), and vascular endothelial growth factor (VEGF) in endometrial stromal cells. Expression levels of PR, ER, and VEGF in human endometrial stromal primary cells treated with nicotine (0, 10^?11, 10^?8, and 10^?6?μM) for 24?h were measured by quantitative real-time PCR. MTT assay demonstrated that nicotine decreased cell viability in a dose-dependent manner. Real-time PCR data showed that despite decrease in ER expression in the nicotine-treated groups compared with the control, nicotine exerted an increased inhibitory effect on PR expression compared to that on ER expression. VEGF mRNA expression in nicotine-treated endometrial stromal cells was increased. The results from this study provide novel evidence for inhibitory effects of nicotine on steroid hormones receptor expression in human primary endometrial cells. Also, our data suggest that nicotine might have angiogenesis effects on these cells.     

Biodegradability of para-aramid respirable-sized fiber-shaped particulates (RFP) in human lung cells.

Using both in vivo (inhalation) and in vitro (cell culture) studies, we previously reported that p-aramid respirable fibers (RFP--defined as respirable-sized fiber-shaped particulates) are biodegraded in lungs and lung cells of rats following exposures. The current studies were undertaken to determine whether shortening mechanisms of p-aramid RFP biodegradability are also operative in human lung cells. Cultures of human A549 lung epithelial cells (A549), primary alveolar macrophages (HBAL) (collected via bronchoalveolar lavage [BAL]) from volunteers), and co-cultures (Co) of the A549 and HBAL were incubated with p-aramid RFP for either 1 h, 1 day, or 1 week to assess RFP shortening. Lengths of RFP were measured using scanning electron microscopy (SEM) following fixation, digestion of culture tissue components, and processing. Similar to findings using rat lung cells, only slight RFP shortening was measured in A549 cultures at 1-day and 1-week post-incubation. More importantly, in HBAL and Co groups, greater transverse cleavage of p-aramid RFP was measured at 1-day and 1-week postexposure compared to 1-h HBAL or Co groups, or in any A549 groups. In contrast, cellulose RFP, a biopersistent reference control fiber, were not measurably shortened under similar circumstances. Second, p-aramid RFP were incubated either with phosphate-buffered saline (PBS), or acellular BAL fluids from human volunteers or rats and processed for SEM analysis of RFP lengths. Mean lengths of p-aramid RFP incubated with human or rat BAL fluids were substantially decreased compared to PBS. Similar to our findings with rat lung cells, components of human lung fluids coat the p-aramid RFP as a prerequisite for subsequent enzymatic cleavage by human phagocytic lung cells and this finding reinforces the concept that inhaled p-aramid RFP are likely to be biodegradable in the lungs of humans.     

Effects of 2,4-dichlorophenol on the expression of vitellogenin and estrogen receptor genes and physiology impairments in Chinese rare minnow (Gobiocypris rarus)

2,4-Dichlorophenol (2,4-DCP) is known as a toxicant, but unknown as an environmental endocrine disruptor. In the present work, Chinese rare minnow (Gobiocypris rarus) was exposed to 0.03, 0.3, and 3 mg/L 2,4-DCP for 3 d and 0.1, 0.3, and 1 mg/L 2,4-DCP for 21 d, respectively. Endpoints including somatic index, vitellogenin (VTG) mRNA and protein level, estrogen receptor (ER) mRNA and histopathology were measured. In the 3-d exposure experiment, the effect concentrations were 0.3 mg/L and above, and in 21-d exposure, the effect concentrations were 0.1 mg/L and above. When exposed to the effect concentrations, GSI was significantly reduced for both male and female, ER mRNA was upregulated in male and downregulated in female. There were no significant variations of VTG mRNA in both male and female in 3-d exposure and in male in 21-d exposure. However, VTG mRNA in female in 21-d exposure was upregulated, corresponding to an increase of VTG protein in serum. Histopathological observation showed that ovaries were degenerated in the effect concentrations, where follicular atresias were more frequently observed. Because the sex hormones related genes and toxicological endpoints were affected in the dose-dependent manner, the results suggest that 2,4-DCP could be a potential endocrine disruptor and might cause adverse effects in female sex organs through interruption of ER-mediated processes.     

Stimulatory effect of naturally occurring flavonols quercetin and kaempferol on alkaline phosphatase activity in MG-63 human osteoblasts through ERK and estrogen receptor pathway

Many plant-derived substances have estrogenic activities. Due to their ability to bind the estrogen receptor (ER), these compounds have the potential to counteract the deleterious effects of estrogen deficiency on bone. In this study, we investigated the in vitro effect of two widespread flavonols, quercetin and kaempferol, on alkaline phosphatase (ALP) activity in MG-63 cultured human osteoblasts. We found that both flavonols significantly increased ALP activity. This effect was markedly reduced by PD 98059, an inhibitor of the extracellular regulated kinase (ERK) pathway, and by ICI 182780, an antagonist of ERs. Western blot studies confirmed that ERK is rapidly activated in cells treated by both flavonols. Finally, ICI 182780 markedly inhibits the flavonol-induced ERK activation. The data presented in this study support the conclusion that, in MG-63 osteoblasts (i) the increase in ALP activity by flavonols involves a rapid stimulation of ERK activation but also involves the ER, and that (ii) the activation of ERK by flavonols occurs most likely downstream of the ERs activation. Taken together, these results suggest that flavonols derivatives as quercetin and kaempferol can stimulate osteoblastic activity. Such compounds may represent new pharmacological tools for the treatment of osteoporosis.     

Brain cytochrome P450 aromatase gene isoforms and activity levels in atlantic salmon after waterborne exposure to nominal environmental concentrations of the pharmaceutical ethynylestradiol and antifoulant tributyltin.

In this study, the effects of two environmental endocrine disruptors, the synthetic pharmaceutical estrogen (ethynylestradiol, EE2) and antifoulant (tributyltin, TBT) representing two different modes of action on the endocrine system, were studied on brain steroidogenic pathway of juvenile Atlantic salmon (Salmo salar). Neurosteroidogenesis was studied using brain aromatase gene isoforms and enzyme activity, in parallel with typical xenoestrogen responses, such as brain estrogen receptor (ERalpha) and plasma vitellogenin (Vtg) levels. Fish were exposed to nominal waterborne EE2 (5 and 50 ng/l) and TBT (50 and 250 ng/l) concentrations dissolved in dimethyl sulfoxide (DMSO), singly and in combination. Gene expressions were quantified using real-time PCR with gene-specific primers, aromatase activity was analyzed using the tritiated water-release assay, and plasma Vtg was analyzed using competitive ELISA. Our data show that EE2 induced a concentration-specific modulation of P450aromA, P450aromB, and aromatase activity in addition to ERalpha and plasma Vtg levels in juvenile salmon at day 3 postexposure. TBT exposure caused both the elevation and inhibition of P450aromA, P450aromB, and aromatase activity levels, depending on concentration, at day 7 postexposure. TBT elevated and inhibited ERalpha and plasma Vtg and also antagonized EE2-induced expression of the studied variables at day 7 postexposure. Interestingly, the carrier vehicle DMSO modulated the receptor-mediated and non-receptor-mediated estrogenic responses at day 7 postexposure, compared to day 3. In general, these findings suggest that the exposed animals are experiencing impaired steroidogenesis and modulations of receptor-mediated endocrine responses. Given the integral role of neurosteroids in homeostatic process, growth, metabolism, reproduction, and development of central nervous system and function, these effects may have serious impact on this endocrine pathway and potentially affect organismal reproductive performance and health. In conclusion, the regulation of steroidogenesis is a fundamental mechanism involved in the biosynthesis of important biological compounds, irrespective of organ; therefore, the search for the molecular targets of xenoestrogens, given singly and also in combination, in these pathways will increase our understanding of organismal endocrine disruption and potential consequences.     

乌骨藤提取物对人结肠腺癌细胞增殖的实验研究

目的:研究乌骨藤提取物对体外培养的人肠癌细胞(Lovo)增殖的影响。方法:用两种方法(A、B)制备乌骨藤提取物,分别采用不同浓度作用Lovo细胞,通过细胞形态学和MTT法检测乌骨藤提取物对Lovo细胞增殖的影响。结果:乌骨藤提取物A对细胞的最大无毒浓度约为0.648 mg/mL;药物浓度在0.389、0.648、1.08、1.8、3 mg/mL时的抑制率分别为6.58%、8.24%、13.02%、59.69%、66.23%,用回归法求出乌骨藤提取物A对Lovo细胞增殖的半数抑制浓度(IC_(50))为1.625 mg/mL。乌骨藤提取物B对细胞的最大无毒浓度约为0.648 mg/mL,药物浓度在0.389、0.648、1.08、1.8、3 mg/mL时的抑制率分别为14.49%、15.48%、29.69%、63.26%、64.05%,用回归法求出乌骨藤提取物B对Lovo细胞增殖的半数,IC_(50)为1.84 mg/mL。结论:乌骨藤提取物对体外培养的Lovo细胞的增殖有明显抑制作用。     

Antiproliferative activity of monastrol in human adenocarcinoma (MCF-7) and non-tumor (HB4a) breast cells

Monastrol is an allosteric inhibitor of the mitotic kinesin Eg5 that exhibits an antiproliferative effect against several cell lines. We investigated the antiproliferative effect of monastrol on human breast adenocarcinoma cells (MCF-7) and mammary epithelial cells (HB4a, non-tumoral). Monastrol treatment decreased cell viability only in MCF-7 tumor cells. Real-time cell growth kinetic analysis showed a decrease in the proliferation of MCF-7 cells exposed to monastrol, while in the HB4a cells, only a concentration of 100 μM was able to induce this effect. In a cell cycle analysis, exposure of MCF-7 cells to monastrol led to an increased population of cells in both the G1 and G2/M phases. In HB4a cells, the proportion of cells in the G2/M phase was increased. Monastrol led to an increased mitotic index in both cell lines. Monastrol was not able to induce cell death by apoptosis in any of the cell lines studied. Gene expression analysis was performed to measure the mRNA levels of cell cycle genes, DNA damage indicator gene, and apoptotic related genes. Treatment with monastrol induced in MCF-7 cells a 5-fold increase in the mRNA levels of the CDKN1A gene, an inhibitor of CDKs related with cell cycle arrest in response a stress stimulus, and a 2-fold decrease in CDKN1C mRNA levels in HB4a cells. These results provide evidence that monastrol has a greater antiproliferative effect on MCF-7 tumor cells compared with non-tumor HB4a cells; however, no selective is observed.