Hepatic transcriptome analysis of male and female Eastern Mosquitofish (Gambusia holbrooki) exposed to a progestin and anti-progesterone reveals the mode of action of endocrine disruption of synthetic steroids in an aquatic organism
ABSTRACT: Major classes of hormone mimics that have been studied include environmental estrogens and androgens, but recent studies have also demonstrated the significant impacts of natural and synthetic progesterones in the environment. The objective of this study was to evaluate the molecular and physiological impacts of progestin, anti-progestin, and mixture exposures in the Eastern Mosquitofish (G. holbrooki). By comparison of gene expression profiles and modulated biological processes in the three groups, it was determined that mifepristone acts more as a progestin than as an anti-progestin, as has also been demonstrated in other species of fish. This work contributes to the overall knowledge of the impacts of this class of chemical contaminats on aquatic organisms, which are a sentinel species for pollutants as aquatic ecosystems often become a reservoir for anthropogenic contaminants. G. holbrooki adult males and females were exposed to one of the following conditions: vehicle control (ethanol), 100 ng/L of levonorgestrel, 100 ng/L of mifepristone, or a mixture of both levonorgestrel and mifepristone. All exposures were conducted for 48 hours with water changed and chemicals renewed daily. Fish were anesthetized using 100 mg/L Benzocaine (Ethyl 4-aminobenzoate). Livers were removed and stored in RNAlater (Qiagen, Hilden, Germany) overnight at 4 C before storage at -80 C. RNA was isolated from the livers using TRIzol (Invitrogen, Grand Island, USA), hydrated using RNAsecure (Ambion, Grand Island, USA), and DNase treated using the Turbo DNA-free kit (Ambion, Grand Island, USA). Four oocyte-development stage-matched RNA samples per treatment were evaluated for RNA integrity using the 2100 BioAnalyzer (Agilent, Santa Clara, USA). The range of RIN values was 8.2-9.6
Project description:Masculinized female Eastern Mosquitofish (Gambusia holbrooki) have resided downstream of paper mills in Florida since the 1980's. The potential impacts of this effluent on the mosquitofish endocrine system are unknown. The objective of this study was to evaluate gene expression patterns of endocrine system genes and global gene expression patterns in female G. holbrooki from a paper mill-impacted site. Masculinized female G. holbrooki were collected from a paper mill-impacted site (Fenholloway River) and from a reference site (Econfina River) and microarray analysis in livers was conducted. Hepatic microarray analysis revealed an increase in the expression of metabolic genes at the Fenholloway, with similarities in individual genes and biological processes compared to G. holbrooki exposed to androgens. These data indicate G. holbrooki from the Fenholloway may be impacted by a mixture of endocrine-active chemicals, including androgens. During the summer of 2012, G. holbrooki were captured from one site downstream of the Buckeye Pulp and Paper Mill (Taylor County, Perry, FL, USA) on the Fenholloway River (GPS coordinates: N 30 058.341’, W 83 588.569’) and one site in the Econfina conservation area (GPS coordinates: N 30 08.549', W 83 51.962') . Only sexually mature G. holbrooki (females > 15cm standard length and with the presence of the gravid spot near the vent) were collected. A 1/8 mesh seine was used for sample collection. Female G. holbrooki were transferred to 5 gallon aerated buckets filled with site water and were processed at the site immediately after collection. Fish were anesthetized using Tricaine-S (Western Chemical, Ferndale, USA) and sacrificed via spinal transection. Oocyte development was assessed upon dissection and livers were removed and stored in RNAlater (Qiagen, Hilden, Germany) overnight at 4 C before storage at -80 C. RNA was isolated from the livers using TRIzol (Invitrogen, Grand Island, USA), hydrated using RNAsecure (Ambion, Grand Island, USA), and DNase treated using the Turbo DNA-free kit (Ambion, Grand Island, USA). Four oocyte-development stage-matched RNA samples per treatment were evaluated for RNA integrity using the 2100 BioAnalyzer (Agilent, Santa Clara, USA). The range of RIN values was 7.8-8.9.
Project description:The primary aim of this project was to identify novel factors, in particular the cell-surface protein CD109, which regulate osteoclastogenesis. Microarray analysis was performed comparing two pre-osteoclast cell lines generated from the RAW 264.7 osteoclast cell line: one that has the capacity to fuse forming large multinucleated cells and one that does not fuse. It was found that CD109 was up-regulated by > 17-fold in the osteoclast forming cell line when compared to the cell line that does not fuse. H10 (osteoclastogenic cell line) and C8 (non osteoclastogenic cell line) cells were plated for two days in 60 mm tissue culture dishes at a density of 0.5 x 10^6 in a culture medium consisting of 8 ml of Dulbecco's Modified Eagle Medium (DMEM, Life Technologies, Grand Island, NY, USA) supplemented with 10% Fetal Bovine Serum (FBS) and 10% antibiotics (164 IU/mL of penicillin G, 50 mg/ml of gentamicin, and 0.25 mg/ml of fungizone) as well as purified recombinant RANKL (60ng/ml). Following two days of culture, total RNA was extracted from the H10 and C8 cells (Qiagen RNeasy Minikit, Germantown, MD, USA) and the concentration of extracted RNA was measured (using a nanodrop method). Experiment was done in triplicates
Project description:The Eastern Mosquitofish (Gambusia holbrooki) is a non-model species with the potential for development of biomarkers of endocrine disrutpting chemical (EDC) exposure due to its androgen-driven secondary sexual characteristics. While G. holbrooki have been utilized in the field as bioindicator organisms EDC exposure in areas impacted by pulp and paper mills, the lack of molecular tools and general understanding of how G. holbrooki are impacted by androgen exposure hinder the use of this organism as a widespread tool for the evaluation of these chemicals in the environment. While traditional gene-by-gene approaches have provided a list of genes that may be appropriate for developing into biomarkers of androgen exposure, a more inclusive method could provide more rapid and expansive determination of genes that can be developed into biomarkers of androgen exposure. The objective of this study is toutilize a mosquitofish microarray to determine potential biomarkers of chronic androgen exposure. The specific aim was to determine genes that may be developed into biomarkers of chronic androgen exposure in hepatic tissues using 17β-trenbolone (TB) exposed adult female G. holbrooki by microarray analysis. Liver tissues from a 14-day exposure of female G. holbrooki to 1 µg/L of the potent androgen receptor agonist 17β-trenbolone (17β-hydroxyestra-4,9,11-trien-3-one; abbreviated as TB) [Brockmeier 2013] were used as the first sample for microarray analysis using the custom G. holbrooki microarray to determine genes that were significantly up or down-regulated during a chronic androgen agonist exposure. RNA was isolated from the livers as previously described using TRIzol (Invitrogen, Grand Island, USA), hydrated using RNAsecure (Ambion, Grand Island, USA), and DNase treated using the Turbo DNA-free kit (Ambion, Grand Island, USA). Four oocyte-development stage-matched RNA samples per treatment were evaluated for RNA integrity using the 2100 BioAnalyzer (Agilent, Santa Clara, USA). The range of RIN values was 8.3-8.9.
Project description:Aim of the study was to characterize at a molecular level (changes in transcriptomes) the effect of monosodium urate crystal (MSU) on HaCaT keratinocyte cell line. This was adressed by using a culture model. The HaCaT cell line (human keratinocytes) was stimulated by MSU (1mg/mL) vs control for 12 hrs. By using genome-wide expression profiling, we identified deregulation of functionally relevant gene networks. HaCaT were obtained from Cell Lines Service (Eppelheim, Germany) and grown in DMEM medium (PAN biotech, Aidenbach, Germany) supplemented with 10% FBS (Life Technology, Grand Island, NY, USA), L-glutamine and non-essential amino acid. Before the treatment HaCaT cells were cultured in serum-free medium for 12hrs. HaCaT were treated with MSU (1mg/ml) vs DMEM control for 12hrs then submitted to RNA extration and gene expression profiling. Triplicate experiments were performed: HaCaT control (n=3), MSU-treated (n=3).
Project description:Patients with endometrial hyperplasia representing preliminary stages of endometrial cancer have shown to respond to therapy in 100% of the cases when treated with levonorgestrel -impregnated intrauterine device. Antiproliferative effect has also been reported after application of an anti-progestin impregnated intrauterine device which showed to induce endometrial atrophy. The intention of the present study was to obtain more information of novel therapeutic targets for hormonal treatment in endometrial hyperplasia and endometrial cancers. Gene expression of signaling pathways after stimulation of Ishikawa cells with high doses of progesterone (10 μM) or Mifepristone (10 μM) was performed. After using an oligo microarrays representing 24,650 human genes and 37,580 gene transcripts, 6,154 genes remained after pre-processing and filtering. This resulted in a total of 993 up-regulated genes with 189 genes for progesterone and 255 genes for Mifepristone. The 550 down regulated genes were distributed with 256 genes for progesterone, 127 genes for RU 486. The results showed that genes presenting the epidermal growth factor (EGF)/MAP-kinase pathway were significantly over-represented by progesterone treatment, whereas, by Mifepristone treatment genes involved in the p53 pathway were also up-regulated (data not shown). These genes may be interesting as potential new therapeutic targets in endometrial hyperplasia and endometrial cancer, as candidate genes for therapy response or as candidate markers for tumor progression. 20 samples, 4 different condition groups with 5 replicates for each group, linear factorial experimental design. Short time (4hrs) dose response to progesterone and Mifepristone in combination and alone. Only 18 samples are represented in this Series.
Project description:Host factors governing mild disease in adults who have developed clinical immunity to Plasmodium falciparum may provide insights for disease altering vaccine or interventions, to prevent severe malaria We used microarrays of whole blood samples during mild malaria and compared them in a paired analysis to whole blood microarray 30 days later. (n=19 pairs) 3mL of whole blood was immediately placed in Tri-Reagent BD, frozen; high quality pairs were selected, were randomly selected for hybridization and analysis. Sense strand cDNA was generated from total RNA using the Ambion® WT Expression Kit (Life Technologies, Grand Island, NY) and labeling was done with the GeneChip® WT Terminal Labeling Kit (Affymetrix, Santa Clara, CA) for use with the GeneChip® Human Gene 1.0 ST Arrays (Affymetrix). The array image was generated by a high-resolution GeneArray Scanner 3000 7G (Affymetrix).
Project description:The goal of this experiment was to identify genes that were expressed at higher levels in benign human mammary epithelial cells than in breast cancer cell lines and that were induced by 5AZA treatment in breast cancer cell lines. Six breast cancer cell lines were selected for demethylation studies based on known tumor suppressor gene expression regulation by promoter region hypermethylation: HCC1569 (CCND2), HCC1954 (SCGB3A1, APC, RASSF1A), MCF-7 (RAR-beta2), MDA-MB-231 (ESR1), UACC3199 (BRCA1), and BT-549 (hypermethylator phenotype). Other than MCF10A we specifically avoided immortalized benign human mammary epithelial cell lines for this experiment as these cells frequently show tumor suppressor gene methylation (e.g. p16) and gene expression profiles that are intermediate between normal breast epithelial cells and breast cancer. Instead, we opted to test six first-passage benign human mammary epithelial cell cultures (HME) generated in serum-free media from small fragments of normal breast tissue obtained from young women undergoing fibroadenoma excision. The 5AZA dose (0.5 microM) was selected based on evaluation of growth curves and induction of BNC1, SERPINB, and TKTL1 gene expression measured by RT-PCR in benign and malignant cells. The breast cancer cell lines, HME cultures, and MC10A cells were treated with 0.5 microM 5AZA (Sigma-Aldrich, St. Louis, MO) in DMSO or DMSO alone for six days after which the cells were harvested, and RNA prepared using the Illumina TotalPrep kit (AMIL1791, Life Technologies, Grand Island, NY). Whole genome expression was assessed using the Illumina HumanWG-6-v3 chip Gene expression was evaluated in 6 breast cancer cell lines, 6 primary breast epithelial cell cultures, and MCF10A cells after 6 days in DMSO or DMSO plus 0.5 microM 5AZA.
Project description:Tumor cell invasion and metastasis are hallmarks of malignancy. Despite recent advances in the understanding of lymphatic spread, the mechanisms by which tumors metastasize to sentinel/distant lymph nodes and beyond are poorly understood. To gain new insights into this complex process, we established a highly metastatic melanoma cell line (B16F1-variant) by in vivo passaging the B16 parental cell line through the lymphatic system. Here, we characterized morphology, rate of cell proliferation, colony formation, migration, tumorogenicity, lymph flow, and capacities to induce tumor- and sentinel lymph node- lymphangiogenesis. Furthermore, microarray-based comparative analysis bewteen parental and passaged cell lines was performed to identify specific gene expression profiles. The most differentially expressed gene was SPP (osteopontin), a secreted glycophosphoprotein which is known to be involved in cancer metastasis. Overexpression of osteopontin in B16 F1-variant was confirmed by Western blot and quantitative RT-PCR. Treatment of cultured lymphatic endothelial cells (LEC) with osteopontin promoted cell migration mediated by the integrin α9 pathway. Our results identify osteopontin as a novel lymphangiogenic factor. B16 and B16 variants were cultured in DMEM supplemented with 20% fetal bovine serum (FBS; Invitrogen, Grand Island, NY) supplemented with antibiotic-antimycotic solution. Every B16 variant was cultured in duplicates. Total cellular RNA was isolated using the Trizol reagent (Invitrogen, Carlsbad, CA) extracted with chloroform, precipitated with isopropanol, washed with 70% ethanol, and dissolved in DNase-free/RNase-free distilled water. The concentration of RNA was measured using NanoDrop ND-1000 spectrophotometer (Witec AG, Littau, Switzerland) and RNA quality was assessed using 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). Digoxigenin-UTP–labeled cRNA was generated and amplified from 500ng of total RNA using the NanoAmp RT-IVT Labeling Kit (Applied Biosystems, Foster City, CA) following the manufacturer's protocol, and was hybridized to Applied Biosystems Mouse Genome Survey Microarrays V2.0. Chemiluminescence detection, image acquisition, and analysis were performed using the Chemiluminescence Detection Kit (Applied Biosystems) and the Applied Biosystems 1700 Chemiluminescent Microarray Analyzer following the manufacturer's protocol. A total of two biological replicates were generated for each B16 variant and each cell line data consisted of pooled RNA of two diffferent passage numbers.
Project description:To investigate the effect of the dexamethasone-eluting electrode in the guinea pig cochlea, and compared the gene expression after 7 days insertion with that of a normal electrode or non-treated control by microarray. Male Hartley guinea pigs (SLC, Shizuoka, Japan) with an age of seven weeks were used for the study. Three were implanted with normal electrodes while three others received a dexamethasone-eluting electrode. The cochleae from two animals, which did not undergo surgery. Seven days after electrode implantation the whole temporal bone was removed and placed into RNAlater solution (Ambion, Life Technologies Co., Grand Island, NY) to stabilize and protect cellular RNA. The whole cochlea was dissected out under a microscope and total RNA were extracted.
Project description:Epidemiological studies indicate that progestin-containing contraceptives may increase susceptibility to HIV and other infections; however, underlying mechanisms involving the upper female reproductive tract are undefined. To determine the effects of depot medroxyprogesterone acetate (DMPA) and the levonorgestrel intrauterine system (LNG-IUS) on gene expression and physiology of the human endometrial and cervical transformation zone (TZ), microarray analyses were performed on whole tissue biopsies. In endometrium, activated pathways included leukocyte chemotaxis, attachment, and inflammation in DMPA (z>2.5) and LNG-IUS (z>3.5) users, and regulation of pattern recognition receptors and other immune mediators. In cervical TZ, progestin treatment altered expression of tissue remodeling and viability genes, but not those of immune functions. Together, these results indicate that progestins influence expression of immune-related genes in endometrium that would be expected to result in the local recruitment of HIV target cells, and thus may increase HIV susceptibility. It is important to consider the upper reproductive tract in the assessment of effects of contraceptives that may influence susceptibility to pathogens, such as HIV. Cross-sectional study conducted at an academic medical center. Cervical transformation zone and endometrial biopsies were obtained from 3 groups of volunteers: those using no hormonal contraceptives (controls, mid-secretory phase, n=20 cervix, 11 endometrium), DMPA users (150mg, n=15, 8), or LNG-IUS users (n=17, 13). DMPA and LNG-IUS groups had used these contraceptives for at least 6 months.