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  • 1.
    Asnake, Solomon
    et al.
    Örebro University, School of Science and Technology.
    Pradhan, Ajay
    Örebro University, School of Science and Technology.
    Banjop-Kharlyngdoh, Joubert
    Örebro University, School of Science and Technology.
    Modig, Carina
    Örebro University, School of Science and Technology.
    Olsson, Per-Erik
    Örebro University, School of Science and Technology.
    1,2-dibromo-4-(1,2 dibromoethyl) cyclohexane (TBECH)-mediated steroid hormone receptor activation and gene regulation in chicken LMH cells2014In: Environmental Toxicology and Chemistry, ISSN 0730-7268, E-ISSN 1552-8618, Vol. 33, no 4, p. 891-899Article in journal (Refereed)
    Abstract [en]

    The incorporation of brominated flame retardants into industrial and household appliances has increased their occurrence in the environment, resulting in deleterious effects on wildlife. With the increasing restraints on available compounds, there has been a shift to using brominated flame retardants that has seen the production of alternative brominated flame retardants such as 1,2-dibromo-4-(1,2 dibromoethyl) cyclohexane (TBECH), which has been detected in the environment. In previous in silico and in vitro studies the authors have shown that TBECH can activate both the human androgen receptor (hAR) and the zebrafish AR (zAR) suggesting that it is a potential endocrine disruptor. The present study was aimed at determining the interaction of TBECH with the chicken AR (cAR). In the present study, TBECH bound to cAR, but in vitro activation assay studies using the chicken LMH cell line showed it had a potency of only 15% compared with testosterone. Sequence difference between ARs from different species may contribute to the different responses to TBECH. Further quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) analysis showed that TBECH interacted with and altered the expression of both thyroid receptors and estrogen receptors. In addition, the qRT-PCR analysis showed that TBECH altered the transcription pattern of genes involved in inflammatory, apoptotic, proliferative, DNA methylation, and drug-metabolizing pathways. This demonstrates that TBECH, apart from activating cAR, can also influence multiple biological pathways in the chicken.

  • 2.
    Asnake, Solomon
    et al.
    Örebro University, School of Science and Technology.
    Pradhan, Ajay
    Biology, The Life Science Center, School of Science and Technology, Örebro University, Örebro, Sweden.
    Kharlyngdoh, Joubert Banjop
    Örebro University, School of Science and Technology.
    Modig, Carina
    Örebro University, School of Science and Technology.
    Olsson, Per-Erik
    Örebro University, School of Science and Technology.
    The brominated flame retardants TBP-AE and TBP-DBPE antagonize the chicken androgen receptor and act as potential endocrine disrupters in chicken LMH cells2015In: Toxicology in Vitro, ISSN 0887-2333, E-ISSN 1879-3177, Vol. 29, no 8, p. 1993-2000Article in journal (Refereed)
    Abstract [en]

    Increased exposure of birds to endocrine disrupting compounds has resulted in developmental and reproductive dysfunctions. We have recently identified the flame retardants, ally1-2,4,6-tribromophenyl ether (TBP-AE), 2-3-dibromopropy1-2,4,6-tribromophenyl ether (TBP-DBPE) and the TBP-DBPE metabolite 2-bromoallyI-2,4,6-tribromophenyl ether (TBP-BAE) as antagonists to both the human androgen receptor (AR) and the zebrafish AR. In the present study, we aimed at determining whether these compounds also interact with the chicken AR. In silico modeling studies showed that TBP-AE, TBP-BAE and TBP-DBPE were able to dock into to the chicken AR ligand-binding pocket. In vitro transfection assays revealed that all three brominated compounds acted as chicken AR antagonists, inhibiting testosterone induced AR activation. In addition, qRT-PCR studies confirmed that they act as AR antagonists and demonstrated that they also alter gene expression patterns of apoptotic, anti-apoptotic, drug metabolizing and amino acid transporter genes. These studies, using chicken LMH cells, suggest that TBP-AE, TBP-BAE and TBP-DBPE are potential endocrine disrupters in chicken.

  • 3.
    Asnake, Solomon
    et al.
    Örebro University, School of Science and Technology.
    Pradhan, Ajay
    Örebro University, School of Science and Technology.
    Kharlyngdoh, Joubert Banjop
    Örebro University, School of Science and Technology.
    Olsson, Per-Erik
    Örebro University, School of Science and Technology.
    The brominated flame retardants TBP-AE and TBP-DBPE antagonize the chicken androgen receptor and alter gene expression in chicken LMH cellsManuscript (preprint) (Other academic)
  • 4.
    Banjop Kharlyngdoh, Joubert
    et al.
    Örebro University, School of Science and Technology.
    Pradhan, Ajay
    Örebro University, School of Science and Technology.
    Asnake, Solomon
    Örebro University, School of Science and Technology.
    Olsson, Per-Erik
    Örebro University, School of Science and Technology.
    Androgen receptor mutations associated with prostate cancer lead to differential activation by DBE-DBCH diastereomersManuscript (preprint) (Other academic)
  • 5.
    Banjop-Kharlygdoh, Joubert
    et al.
    Örebro University, School of Science and Technology.
    Pradhan, Ajay
    Örebro University, School of Science and Technology.
    Asnake, Solomon
    Örebro University, School of Science and Technology.
    Walstad, Anders
    Örebro University, School of Science and Technology.
    Ivarsson, Per
    ALS Laboratory Group, Analytical Chemistry & Testing Services, Stockholm, Sweden.
    Olsson, Per-Erik
    Örebro University, School of Science and Technology.
    Identification of a group of brominated flame retardants as novel androgen receptor antagonists and potential neuronal and endocrine disrupters2015In: Environment International, ISSN 0160-4120, E-ISSN 1873-6750, Vol. 74, p. 60-70Article in journal (Refereed)
    Abstract [en]

    Brominated flame-retardants (BFRs) are used in industrial products to reduce the risk of fire. However, their continuous release into the environment is a concern as they are often persistent, bioaccumulating and toxic. Information on the impact these compounds have on human health and wildlife is limited and only a few of them have been identified to disrupt hormone receptor functions. In the present study we used in silico modeling to determine the interactions of selected BFRs with the human androgen receptor (AR). Three compounds were found to dock into the ligand-binding domain of the human AR and these were further tested using in vitro analysis. Allyl 2,4,6-tribromophenyl ether (ATE), 2-bromoallyl 2,4,6-tribromophenyl ether (BATE) and 2,3-dibromopropyl-2,4,6-tribromophenyl ether (DPTE) were observed to act as AR antagonists. These BFRs have recently been detected in the environment, in house dust and in aquatic animals. The compounds have been detected at high concentrations in both blubber and brain of seals and we therefore also assessed their impact on the expression of L-type amino acid transporter system (LAT) genes, that are needed for amino acid uptake across the blood-brain barrier, as disruption of LAT gene function has been implicated in several brain disorders. The three BFRs down-regulated the expression of AR target genes that encode for prostate specific antigen (PSA), 5. α-reductases and β-microseminoprotein. The potency of PSA inhibition was of the same magnitude as the common prostate cancer drugs, demonstrating that these compounds are strong AR antagonists. Western blot analysis of AR protein showed that ATE, BATE and DPTE decreased the 5. α-dihydrotestosterone-induced AR protein levels, further confirming that these BFRs act as AR antagonists. The transcription of the LAT genes was altered by the three BFRs, indicating an effect on amino-acid uptake across cellular membranes and blood-brain barrier. This study demonstrated that ATE, BATE and DPTE are potent AR antagonists and the alterations in LAT gene transcription suggest that these compounds can affect neuronal functions and should be considered as potential neurotoxic and endocrine disrupting compounds.

  • 6.
    Bereketoglu, Ceyhun
    et al.
    Örebro University, School of Science and Technology. Biology, The Life Science Center.
    Pradhan, Ajay
    Örebro University, School of Science and Technology. Biology, The Life Science Center.
    Comparative transcriptional analysis of methylparaben and propylparaben in zebrafish2019In: Science of the Total Environment, ISSN 0048-9697, E-ISSN 1879-1026, Vol. 671, p. 129-139Article in journal (Refereed)
    Abstract [en]

    Parabens are widely used as preservatives in different commercial items including food, cosmetics and pharmaceuticals, and their wide use has resulted in accumulation in the environment. Parabens have been shown to have negative effects on animals as well as human health. In this study, we carried out a comprehensive study to determine the adverse effects associated with propylparaben (PP) and methylparaben (MP) on early developmental stages of zebrafish. Mortality, hatching, developmental abnormalities and gene expression profiles were investigated in embryos exposed to both compounds. The semi-static exposure conditions showed that both MP (>= 100 mu M) and PP (>= 10 mu M) are toxic to the embryos in a concentration-dependent manner and lead to developmental abnormality. Malformations such as spinal defects, pericardial edema, and pigmentation defects were observed following both MP and PP treatments. Hatching delay, mortality and developmental abnormality data indicate that PP is more toxic than MP. For gene expression analysis, 1 and 10 mu M doses of MP and PP were analyzed. Genes from physiological pathways including stress response, cell cycle and DNA damage, inflammation, fatty acid metabolism and endocrine functions were affected by MP and PP. The gene expression profiles show that parabens cause toxicity by inducing oxidative stress, DNA double-strand breaks, apoptosis as well as by altering fatty acid metabolism. Altered expression of androgen receptor (ar) and estrogen receptor 2 alpha (esr2a) indicates an antiandrogenic and estrogenic activity of parabens in zebrafish. Overall, the present study provides considerable information on the negative effects of MP and PP using physiological endpoints and motivates further studies to explore the molecular mechanism of the toxicity associated with parabens.

  • 7.
    Elmarghani, Ahmed
    et al.
    Örebro University, School of Science and Technology.
    Pradhan, Ajay
    Örebro University, School of Science and Technology.
    Seyoum, Asmerom
    Örebro University, School of Science and Technology.
    Khalaf, Hazem
    Örebro University, School of Science and Technology.
    Ros, Torbjön
    Pelagia Miljokonsult AB, Umeå, Sweden.
    Forsberg, Lars-Håkan
    Mälarenergi AB, Vasterås, Sweden.
    Nermark, Tomas
    Karlskoga Energi MO AB, Karlskoga, Sweden.
    Osterman, Lisa
    Skebäcks Reningsverk, Örebro, Sweden.
    Wiklund, Ulf
    Tyrens AB, Umeå, Sweden.
    Ivarsson, Per
    ALS Scandinavia AB, Täby, Sweden.
    Jass, Jana
    Örebro University, School of Science and Technology.
    Olsson, Per-Erik
    Örebro University, School of Science and Technology.
    Contribution of pharmaceuticals, fecal bacteria and endotoxin to the inflammatory responses to inland waters2014In: Science of the Total Environment, ISSN 0048-9697, E-ISSN 1879-1026, Vol. 488-489, p. 228-235Article in journal (Refereed)
    Abstract [en]

    The increasing contamination of freshwater with pharmaceuticals, surfactants, pesticides and other organic compounds are of major concern. As these contaminants are detected at trace levels in the environment it is important to determine if they elicit biological responses at the observed levels. In addition to chemical pollutants, there is also a concern for increasing levels of bacteria and other microorganisms in freshwater systems. In an earlier study, we observed the activation of inflammatory systems downstream of a wastewater treatment plant (WWTP) in southern Sweden. We also observed that the water contained unidentified components that were pro-inflammatory and potentiated the immune response in human urinary bladder epithelial cells. In order to determine if these effects were unique for the studied site or represent a common response in Swedish water, we have now performed a study on three WWTPs and their recipient waters in central Sweden. Analysis of immune responses in urinary bladder epithelial cells, monocyte-like cells and blood mononuclear cells confirm that these waters activate the immune system as well as induce pro-inflammatory responses. The results indicate that the cytokine profiles correlate to the endotoxin load of the waters rather than to the levels of pharmaceuticals or culturable bacteria load, suggesting that measurements of endotoxin levels and immune responses would be a valuable addition to the analysis of inland waters.

  • 8.
    Kharlyngdoh, Joubert Banjop
    et al.
    Örebro University, School of Science and Technology.
    Asnake, Solomon
    Örebro University, School of Science and Technology.
    Pradhan, Ajay
    Örebro University, School of Science and Technology.
    Olsson, Per-Erik
    Örebro University, School of Science and Technology.
    TBECH, 1,2-dibromo-4-(1,2 dibromoethyl) cyclohexane, alters androgen receptor regulation in response to mutations associated with prostate cancer2016In: Toxicology and Applied Pharmacology, ISSN 0041-008X, E-ISSN 1096-0333, Vol. 307, p. 91-101Article in journal (Refereed)
    Abstract [en]

    Point mutations in the AR ligand-binding domain (LBD) can result in altered AR structures leading to changes of ligand specificity and functions. AR mutations associated to prostate cancer (PCa) have been shown to result in receptor activation by non-androgenic substances and anti-androgenic drugs. Two AR mutations known to alter the function of anti-androgens are the ART877A mutation, which is frequently detected mutation in PCa tumors and the ARW741C that is rare and has been derived in vitro following exposure of cells to the anti-androgen bicalutamide. AR activation by non-androgenic environmental substances has been suggested to affect PCa progression. In the present study we investigated the effect of AR mutations (ARW741C and ART877A) on the transcriptional activation following exposure of cells to an androgenic brominated flame retardant, 1,2-dibromo-4-(1,2 dibromoethyl) cyclohexane (TBECH, also named DBE-DBCH). The AR mutations resulted in higher interaction energies and increased transcriptional activation in response to TBECH diastereomer exposures. The ART877A mutation rendered AR highly responsive to low levels of DHT and TBECH and led to increased AR nuclear translocation. Gene expression analysis showed a stronger induction of AR target genes in LNCaP cells (ART877A) compared to T-47D cells (ARWT) following TBECH exposure. Furthermore, AR knockdown experiments confirmed the AR dependency of these responses. The higher sensitivity of ART877A and ARW741C to low levels of TBECH suggests that cells with these AR mutations are more susceptible to androgenic endocrine disrupters.

  • 9.
    Kharlyngdoh, Joubert Banjop
    et al.
    Glomerular Disease Therapeutics Laboratory, Department of Internal Medicine, Rush University Medical Centre, Chicago, USA.
    Pradhan, Ajay
    Örebro University, School of Science and Technology.
    Olsson, Per-Erik
    Örebro University, School of Science and Technology.
    Androgen receptor modulation following combination exposure to brominated flame-retardants2018In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 8, no 1, article id 4843Article in journal (Refereed)
    Abstract [en]

    Endocrine disrupting compounds can interfere with androgen receptor (AR) signaling and disrupt steroidogenesis leading to reproductive failure. The brominated flame-retardant (BFR) 1, 2-dibromo-4-(1, 2-dibromoethyl) cyclohexane (TBECH), is an agonist to human, chicken and zebrafish AR. Recently another group of alternative BFRs, allyl 2, 4, 6-tribromophenyl ether (ATE), and 2, 3-dibromopropyl 2, 4, 6-tribromophenyl ether (DPTE) along with its metabolite 2-bromoallyl 2, 4, 6-tribromophenyl ether (BATE) were identified as potent human AR antagonists. These alternative BFRs are present in the environment. The aim of the present study was to determine the effect of mixed exposures to the AR agonist and the AR antagonists at environmentally relevant concentrations. In vitro reporter luciferase assay showed that the AR antagonists, when present at concentration higher than TBECH, were able to inhibit TBECH-mediated AR activity. These AR antagonists also promoted AR nuclear translocation. In vitro gene expression analysis in the non-tumorigenic human prostate epithelial cell RWPE1 showed that TBECH induced AR target genes whereas DPTE repressed these genes. Further analysis of steroidogenic genes showed that TBECH up-regulated most of the genes while DPTE down-regulated the same genes. The results indicate that when TBECH and DPTE are present together they will antagonize each other, thereby reducing their individual effects.

  • 10.
    Kumar, Ranjeet
    et al.
    School of Science and Technology, The Life Science Center-Biology, Örebro University, Örebro, Sweden.
    Pradhan, Ajay
    School of Science and Technology, The Life Science Center-Biology, Örebro University, Örebro, Sweden.
    Khan, Faisal Ahmad
    Örebro University, School of Science and Technology.
    Lindström, Pia
    Boliden Mineral AB, Boliden, Sweden.
    Ragnvaldsson, Daniel
    Envix Nord AB, Umeå , Sweden.
    Ivarsson, Per
    ALS Scandinavia AB, Täby, Sweden.
    Olsson, Per-Erik
    Örebro University, School of Science and Technology.
    Jass, Jana
    Örebro University, School of Science and Technology.
    Comparative analysis of stress induced gene expression in caenorhabditis elegans following exposure to environmental and lab reconstituted complex metal mixture2015In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 10, no 7, article id e0132896Article in journal (Refereed)
    Abstract [en]

    Metals are essential for many physiological processes and are ubiquitously present in the environment. However, high metal concentrations can be harmful to organisms and lead to physiological stress and diseases. The accumulation of transition metals in the environment due to either natural processes or anthropogenic activities such as mining results in the contamination of water and soil environments. The present study used Caenorhabditis elegans to evaluate gene expression as an indicator of physiological response, following exposure to water collected from three different locations downstream of a Swedish mining site and a lab reconstituted metal mixture. Our results indicated that the reconstituted metal mixture exerted a direct stress response in C. elegans whereas the environmental waters elicited either a diminished or abrogated response. This suggests that it is not sufficient to use the biological effects observed from laboratory mixtures to extrapolate the effects observed in complex aquatic environments and apply this to risk assessment and intervention.

  • 11.
    Pradhan, Ajay
    Örebro University, School of Science and Technology.
    Molecular mechanisms of zebrafish sex differentiation and sexual behavior2015Doctoral thesis, comprehensive summary (Other academic)
    List of papers
    1. Activation of NF-kappa B Protein Prevents the Transition from Juvenile Ovary to Testis and Promotes Ovarian Development in Zebrafish
    Open this publication in new window or tab >>Activation of NF-kappa B Protein Prevents the Transition from Juvenile Ovary to Testis and Promotes Ovarian Development in Zebrafish
    Show others...
    2012 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 287, no 45, p. 37926-37938Article in journal (Refereed) Published
    Abstract [en]

    Testis differentiation in zebrafish involves juvenile ovary to testis transformation initiated by an apoptotic wave. The molecular regulation of this transformation process is not fully understood. NF-kappa B is activated at an early stage of development and has been shown to interact with steroidogenic factor-1 in mammals, leading to the suppression of anti-Mullerian hormone (Amh) gene expression. Because steroidogenic factor-1 and Amh are important for proper testis development, NF-kappa B-mediated induction of anti-apoptotic genes could, therefore, also play a role in zebrafish gonad differentiation. The aim of this study was to examine the potential role of NF-kappa B in zebrafish gonad differentiation. Exposure of juvenile zebrafish to heat-killed Escherichia coli activated the NF-kappa B pathways and resulted in an increased ratio of females from 30 to 85%. Microarray and quantitative real-time-PCR analysis of gonads showed elevated expression of NF-kappa B-regulated genes. To confirm the involvement of NF-kappa B-induced anti-apoptotic effects, zebrafish were treated with sodium deoxycholate, a known inducer of NF-kappa B or NF-kappa B activation inhibitor (NAI). Sodium deoxycholate treatment mimicked the effect of heat-killed bacteria and resulted in an increased proportion of females from 25 to 45%, whereas the inhibition of NF-kappa B using NAI resulted in a decrease in females from 45 to 20%. This study provides proof for an essential role of NF-kappa B in gonadal differentiation of zebrafish and represents an important step toward the complete understanding of the complicated process of sex differentiation in this species and possibly other cyprinid teleosts as well.

    Place, publisher, year, edition, pages
    The American Society for Biochemistry and Molecular Biology, 2012
    National Category
    Developmental Biology
    Research subject
    Biology
    Identifiers
    urn:nbn:se:oru:diva-32903 (URN)10.1074/jbc.M112.386284 (DOI)000310642200028 ()2-s2.0-84868315208 (Scopus ID)
    Funder
    Swedish Research CouncilKnowledge Foundation
    Note

    Funding Agency: Orebro University; Agri-Food and Veterinary Authority; Temasek Life Sciences Laboratory, Singapore (se även Forskningsfinansiärer)

    Available from: 2014-01-02 Created: 2014-01-02 Last updated: 2018-11-29Bibliographically approved
    2. Juvenile Ovary to Testis Transition in Zebrafish Involves Inhibition of Ptges
    Open this publication in new window or tab >>Juvenile Ovary to Testis Transition in Zebrafish Involves Inhibition of Ptges
    2014 (English)In: Biology of Reproduction, ISSN 0006-3363, E-ISSN 1529-7268, Vol. 91, no 2, p. 1-15Article in journal (Refereed) Published
    Abstract [en]

    The sex differentiation mechanisms in zebrafish (Danio rerio) remains elusive, partly due to the absence of sex chromosomes but also the process appears to depend on the synchrony of multiple genes and possibly environmental factors. Zebrafish gonadal development is initiated through the development of immature oocytes. Depending on multiple signaling cues, in about half of the individuals, the juvenile ovaries degenerate or undergo apoptosis to initiate testes development while the other half maintains the oogenic pathway. We have previously shown that activation of NFkappaB and prostaglandin synthase 2 (ptgs2) results in female biased sex ratios. Prostaglandin synthase and prostaglandins are involved in multiple physiological functions including cell survival and apoptosis. In the present study we show that inhibition of ptgs2 by meloxicam result in male biased sex ratios. On further evaluation, we observed that exposure with the prostaglandin D2 (PGD2) analogue BW-245C induced SRY-box containing gene 9a (sox9a) and resulted in male biased sex ratios. On the other hand, prostaglandin E2 (PGE2) treatment resulted in female biased sex ratios and involved activation of NFkappaB and the beta-catenin pathway as well as inhibition of sox9. Exposure to the beta-catenin inhibitor, PNU-74654, resulted in up-regulation of ptgds and male biased sex ratios which further confirmed the involvement of beta-catenin in the female differentiation pathway. In this study we show that PGD2 and PGE2 can program the gonads to either the testis or ovary differentiation pathways, indicating that prostaglandins are involved in the regulation of zebrafish gonadal differentiation.

    Keywords
    fish reproduction; gene expression; prostaglandins; sex determination; sex differentiation
    National Category
    Developmental Biology
    Research subject
    Molecular Biology
    Identifiers
    urn:nbn:se:oru:diva-35473 (URN)10.1095/biolreprod.114.119016 (DOI)000341300400009 ()24920039 (PubMedID)
    Funder
    Knowledge Foundation
    Note

    Funding Agency:

    Örebro University

    Available from: 2014-06-24 Created: 2014-06-24 Last updated: 2017-12-05Bibliographically approved
    3. Inhibition of Retinoic Acid Synthesis Disrupts Spermatogenesis and Fecundity in Zebrafish
    Open this publication in new window or tab >>Inhibition of Retinoic Acid Synthesis Disrupts Spermatogenesis and Fecundity in Zebrafish
    (English)Manuscript (preprint) (Other academic)
    National Category
    Developmental Biology
    Research subject
    Biology
    Identifiers
    urn:nbn:se:oru:diva-43321 (URN)
    Available from: 2015-03-05 Created: 2015-03-05 Last updated: 2017-10-17Bibliographically approved
    4. Zebrafish Sexual Behavior: role of sex steroid hormones and prostaglandins
    Open this publication in new window or tab >>Zebrafish Sexual Behavior: role of sex steroid hormones and prostaglandins
    (English)Manuscript (preprint) (Other academic)
    National Category
    Developmental Biology
    Research subject
    Biology
    Identifiers
    urn:nbn:se:oru:diva-43322 (URN)
    Available from: 2015-03-05 Created: 2015-03-05 Last updated: 2017-10-17Bibliographically approved
  • 12.
    Pradhan, Ajay
    et al.
    Örebro University, School of Science and Technology.
    Asnake, Solomon
    Örebro University, School of Science and Technology.
    Kharlyngdoh, Joubert Banjop
    Örebro University, School of Science and Technology.
    Modig, Carina
    Örebro University, School of Science and Technology.
    Olsson, Per-Erik
    Örebro University, School of Science and Technology.
    In silico and biological analysis of anti-androgen activity of the brominated flame retardants ATE, BATE and DPTE in zebrafishManuscript (preprint) (Other academic)
  • 13.
    Pradhan, Ajay
    et al.
    Örebro University, School of Science and Technology.
    Asnake, Solomon
    Örebro University, School of Science and Technology.
    Kharlyngdoh, Joubert Banjop
    Örebro University, School of Science and Technology.
    Modig, Carina
    Örebro University, School of Science and Technology.
    Olsson, Per-Erik
    Örebro University, School of Science and Technology.
    In silico and biological analysis of anti-androgen activity of the brominated flame retardants ATE, BATE and DPTE in zebrafish2015In: Chemico-Biological Interactions, ISSN 0009-2797, E-ISSN 1872-7786, Vol. 233, p. 35-45Article in journal (Refereed)
    Abstract [en]

    The brominated flame retardants (BFRs) 1,2-dibromo-4-(1,2-dibromoethyl)cyclohexane (TBECH or DBE-DCBH) and allyl 2,4,6-tribromophenyl ether (ATE or TBP-AE) are alternative BFRs that have been introduced to replace banned BFRs. TBECH is a potential endocrine disrupter in human, chicken and zebrafish and in a recent study we showed that ATE, along with the structurally similar BFR 2,3-dibromopropyl 2,4,6-tribromophenyl ether (DPTE or TBP-DBPE) and its metabolite 2-bromoallyl 2,4,6-tribromophenyl ether (BATE or TBP-BAE) are potential endocrine and neuronal disrupters in human. In this study we analyzed ATE, BATE and DPTE for zebrafish androgen receptor (zAR) modulating properties. In silico analysis with two softwares, Molecular Operating Environment (MOE) and Internal Coordinate Mechanics (ICM), showed that ATE, BATE and DPTE bind to zAR. In vitro AR activation assay revealed that these three BFRs down-regulate 11-ketotestosterone (KT) mediated zAR activation. Exposure to 10 mu M DPTE resulted in reduced hatching success and like TBECH, BATE and DPTE at 10 mu M also had teratogenic properties with 20% and 50% back-bone curvature respectively. Gene transcription analysis in zebrafish embryos as well as in juveniles showed down-regulation of the androgen receptor and androgen response genes, which further support that these BFRs are androgen antagonists and potential endocrine disrupting compounds. Genes involved in steroidogenesis were also down-regulated by these BFRs. In view of this, the impact of these BFRs on humans and wildlife needs further analysis.

  • 14.
    Pradhan, Ajay
    et al.
    Örebro University, School of Science and Technology.
    Ivarsson, Per
    Örebro University, School of Science and Technology. BioImpakt AB, Örebro, Sweden.
    Ragnvaldsson, Daniel
    Envix Nord AB, Umeå, Sweden.
    Berg, Håkan
    Örebro University, School of Science and Technology.
    Jass, Jana
    Örebro University, School of Science and Technology.
    Olsson, Per-Erik
    Örebro University, School of Science and Technology.
    Transcriptional responses of zebrafish to complex metal mixtures in laboratory studies overestimates the responses observed with environmental water2017In: Science of the Total Environment, ISSN 0048-9697, E-ISSN 1879-1026, Vol. 584-585, p. 1138-1146Article in journal (Refereed)
    Abstract [en]

    Metals released into the environment continue to be of concern for human health. However, risk assessment of metal exposure is often based on total metal levels and usually does not take bioavailability data, metal speciation or matrix effects into consideration. The continued development of biological endpoint analyses are therefore of high importance for improved eco-toxicological risk analyses. While there is an on-going debate concerning synergistic or additive effects of low-level mixed exposures there is little environmental data confirming the observations obtained from laboratory experiments. In the present study we utilized qRT-PCR analysis to identify key metal response genes to develop a method for biomonitoring and risk-assessment of metal pollution. The gene expression patterns were determined for juvenile zebrafish exposed to waters from sites down-stream of a closed mining operation. Genes representing different physiological processes including stress response, inflammation, apoptosis, drug metabolism, ion channels and receptors, and genotoxicity were analyzed. The gene expression patterns of zebrafish exposed to laboratory prepared metal mixes were compared to the patterns obtained with fish exposed to the environmental samples with the same metal composition and concentrations. Exposure to environmental samples resulted in fewer alterations in gene expression compared to laboratory mixes. A biotic ligand model (BLM) was used to approximate the bioavailability of the metals in the environmental setting. However, the BLM results were not in agreement with the experimental data, suggesting that the BLM may be overestimating the risk in the environment. The present study therefore supports the inclusion of site-specific biological analyses to complement the present chemical based assays used for environmental risk-assessment.

  • 15.
    Pradhan, Ajay
    et al.
    Örebro University, School of Science and Technology.
    Khalaf, Hazem
    Örebro University, School of Health and Medical Sciences, Örebro University, Sweden.
    Ochsner, Scott A.
    Baylor College of Medicine, Houston, USA.
    Sreenivasan, Rajini
    Temasek Life Sciences Laboratory, Singapore, Singapore.
    Koskinen, Jarno
    Örebro University, School of Science and Technology.
    Karlsson, Marie
    Örebro University, School of Science and Technology.
    Karlsson, Jesper
    Department of Biology, School of Science and Technology, Örebro University, Örebro, Sweden.
    McKenna, Neil J.
    Baylor College of Medicine, Houston, USA.
    Orban, Laszlo
    Temasek Life Sciences Laboratory, Singapore, Singapore; National University of Singapore, Singapore, Singapore; University of Pannonia, Keszthely, Hungary.
    Olsson, Per-Erik
    Örebro University, School of Science and Technology.
    Activation of NF-kappa B Protein Prevents the Transition from Juvenile Ovary to Testis and Promotes Ovarian Development in Zebrafish2012In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 287, no 45, p. 37926-37938Article in journal (Refereed)
    Abstract [en]

    Testis differentiation in zebrafish involves juvenile ovary to testis transformation initiated by an apoptotic wave. The molecular regulation of this transformation process is not fully understood. NF-kappa B is activated at an early stage of development and has been shown to interact with steroidogenic factor-1 in mammals, leading to the suppression of anti-Mullerian hormone (Amh) gene expression. Because steroidogenic factor-1 and Amh are important for proper testis development, NF-kappa B-mediated induction of anti-apoptotic genes could, therefore, also play a role in zebrafish gonad differentiation. The aim of this study was to examine the potential role of NF-kappa B in zebrafish gonad differentiation. Exposure of juvenile zebrafish to heat-killed Escherichia coli activated the NF-kappa B pathways and resulted in an increased ratio of females from 30 to 85%. Microarray and quantitative real-time-PCR analysis of gonads showed elevated expression of NF-kappa B-regulated genes. To confirm the involvement of NF-kappa B-induced anti-apoptotic effects, zebrafish were treated with sodium deoxycholate, a known inducer of NF-kappa B or NF-kappa B activation inhibitor (NAI). Sodium deoxycholate treatment mimicked the effect of heat-killed bacteria and resulted in an increased proportion of females from 25 to 45%, whereas the inhibition of NF-kappa B using NAI resulted in a decrease in females from 45 to 20%. This study provides proof for an essential role of NF-kappa B in gonadal differentiation of zebrafish and represents an important step toward the complete understanding of the complicated process of sex differentiation in this species and possibly other cyprinid teleosts as well.

  • 16.
    Pradhan, Ajay
    et al.
    Örebro University, School of Science and Technology.
    Kharlyngdoh, Joubert Banjop
    Örebro University, School of Science and Technology.
    Asnake, Solomon
    Örebro University, School of Science and Technology.
    Olsson, Per-Erik
    Örebro University, School of Science and Technology.
    The brominated flame retardant TBECH activates the zebrafish (Danio rerio) androgen receptor, alters gene transcription and causes developmental disturbances2013In: Aquatic Toxicology, ISSN 0166-445X, E-ISSN 1879-1514, Vol. 142, p. 63-72Article in journal (Refereed)
    Abstract [en]

    Tetrabromoethylcyclohexane (TBECH) is a brominated flame retardant that has been shown to be a potent agonist to the human androgen receptor (AR). However, while it is present in the environment, it is not known if it interacts with AR from aquatic species. The present study was therefore aimed at improving our understanding of how TBECH affects aquatic animals using zebrafish as a model organism. In silica modeling demonstrated that TBECH diastereomers bind to the zebrafish androgen receptor (zAR) and in vitro and in vivo data showed that TBECH has androgenic properties. Deleterious effects of TBECH were studied on embryonic and juvenile zebrafish and qRT-PCR analysis in vitro and in vivo was performed to determine TBECH effects on gene regulation. TBECH was found to delay hatching at 1 mu M and 10 mu M doses while morphological abnormalities and juvenile mortality was observed at 10 mu M. The qRT-PCR analysis showed alterations of multiple genes involved in chondrogenesis (cartilage development), metabolism and stress response. Thus, TBECH induces androgenic activity and has negative effects on zebrafish physiology and therefore its impact on the environment should be carefully monitored. (C) 2013 Elsevier B.V. All rights reserved.

  • 17.
    Pradhan, Ajay
    et al.
    Örebro University, School of Science and Technology.
    Olsson, Per-Erik
    Örebro University, School of Science and Technology.
    Germ cell depletion in zebrafish leads to incomplete masculinization of the brain2018In: General and Comparative Endocrinology, ISSN 0016-6480, E-ISSN 1095-6840, Vol. 265, no SI, p. 15-21Article in journal (Refereed)
    Abstract [en]

    Zebrafish sex differentiation is under the control of multiple genes, but also relies on germ cell number for gonadal development. Morpholino and chemical mediated germ cell depletion leads to sterile male development in zebrafish. In this study we produced sterile males, using a dead end gene morpholino, to determine gonadal-brain interactions. Germ cell depletion following dnd inhibition downregulated the germ cell markers, vasa and ziwi, and later the larvae developed as sterile males. Despite lacking proper testis, the gonadal 11-ketotestosterone (11-KT) and estradiol (E2) levels of sterile males were similar to wild type males. Qualitative analysis of sexual behavior of sterile males demonstrated that they behaved like wild type males. Furthermore, we observed that brain 11-KT and E2 levels in sterile males remained the same as in the wild type males. In female brain, 11-KT was lower in comparison to wild type males and sterile males, while E2 was higher when compared to wild type males. qRT-PCR analysis revealed that the liver transcript profile of sterile adult males was similar to wild type males while the brain transcript profile was similar to wild type females. The results demonstrate that proper testis development may not be a prerequisite for male brain development in zebrafish but that it may be needed to fully masculinize the brain.

  • 18.
    Pradhan, Ajay
    et al.
    Örebro University, School of Science and Technology.
    Olsson, Per-Erik
    Örebro University, School of Science and Technology.
    Inhibition of Retinoic Acid Synthesis Disrupts Spermatogenesis and Fecundity in ZebrafishManuscript (preprint) (Other academic)
  • 19.
    Pradhan, Ajay
    et al.
    Biology, The Life Science Center, School of Science and Technology, Örebro University, Örebro, Sweden.
    Olsson, Per-Erik
    Örebro University, School of Science and Technology.
    Inhibition of retinoic acid synthesis disrupts spermatogenesis and fecundity in zebrafish2015In: General and Comparative Endocrinology, ISSN 0016-6480, E-ISSN 1095-6840, Vol. 217, p. 81-91Article in journal (Refereed)
    Abstract [en]

    Timing of germ cell entry into meiosis is sexually dimorphic in mammals. However it was recently shown that germ cells initiate meiosis at the same time in male and female zebrafish. Retinoic acid (RA) has been shown to be critical for mammalian spermatogenesis. Inhibition of RA synthesis by WIN 18,446 has been reported to inhibit spermatogenesis in a wide variety of animals including humans and was once used as a contraceptive in humans. In this study we explored the role of RA in zebrafish spermatogenesis. In silico analysis with Internal coordinate mechanics docking software showed that WIN 18,446 can bind to the rat, human and zebrafish Aldh1a2 catalytic domain with equivalent potency. RA exposure resulted in upregulation of the RA metabolizing enzyme genes cyp26a1, cyp26b1 and cyp26c1 in vitro and in vivo. Exposure to WIN 18,446 resulted in down-regulation of Aldh1a2, cyp26a1 and cyp26b1 in vivo. WIN 18,446 was effective in disrupting spermatogenesis and fecundity in zebrafish but the reduction in sperm count and fecundity was only observed when zebrafish were maintained on a strict Artemia nauplii diet which is known to contain low levels of vitamin A. This study shows that RA is involved in spermatogenesis as well as oocyte development in zebrafish. As the zebrafish Aldh1a2 structure and function is similar to the mammalian counterpart, Aldh1a2 inhibitor screening using zebrafish as a model system may be beneficial in the discovery and development of new and safe contraceptives for humans.

  • 20.
    Pradhan, Ajay
    et al.
    Örebro University, School of Science and Technology.
    Olsson, Per-Erik
    Örebro University, School of Science and Technology.
    Juvenile Ovary to Testis Transition in Zebrafish Involves Inhibition of Ptges2014In: Biology of Reproduction, ISSN 0006-3363, E-ISSN 1529-7268, Vol. 91, no 2, p. 1-15Article in journal (Refereed)
    Abstract [en]

    The sex differentiation mechanisms in zebrafish (Danio rerio) remains elusive, partly due to the absence of sex chromosomes but also the process appears to depend on the synchrony of multiple genes and possibly environmental factors. Zebrafish gonadal development is initiated through the development of immature oocytes. Depending on multiple signaling cues, in about half of the individuals, the juvenile ovaries degenerate or undergo apoptosis to initiate testes development while the other half maintains the oogenic pathway. We have previously shown that activation of NFkappaB and prostaglandin synthase 2 (ptgs2) results in female biased sex ratios. Prostaglandin synthase and prostaglandins are involved in multiple physiological functions including cell survival and apoptosis. In the present study we show that inhibition of ptgs2 by meloxicam result in male biased sex ratios. On further evaluation, we observed that exposure with the prostaglandin D2 (PGD2) analogue BW-245C induced SRY-box containing gene 9a (sox9a) and resulted in male biased sex ratios. On the other hand, prostaglandin E2 (PGE2) treatment resulted in female biased sex ratios and involved activation of NFkappaB and the beta-catenin pathway as well as inhibition of sox9. Exposure to the beta-catenin inhibitor, PNU-74654, resulted in up-regulation of ptgds and male biased sex ratios which further confirmed the involvement of beta-catenin in the female differentiation pathway. In this study we show that PGD2 and PGE2 can program the gonads to either the testis or ovary differentiation pathways, indicating that prostaglandins are involved in the regulation of zebrafish gonadal differentiation.

  • 21.
    Pradhan, Ajay
    et al.
    Örebro University, School of Science and Technology.
    Olsson, Per-Erik
    Örebro University, School of Science and Technology.
    Regulation of zebrafish gonadal sex differentiation2016In: AIMS Molecular Science, ISSN 2372-028X, Vol. 3, no 4, p. 567-584Article, review/survey (Refereed)
    Abstract [en]

    While the master regulator gene Sry on the mammalian Y chromosome controls the switch for initiating male sex differentiation, many other species rely on environmental factors for gonadal sex differentiation. Yet other species, like zebrafish, appears to rely on a multitude of genetic cues for gonadal sex differentiation. Zebrafish gonadal differentiation initiates with the onset of a juvenile ovary stage and depending on the influence of unknown genetic factors either maintains oocyte development or initiate apoptotic processes to override the female differentiation pathway. In this review, we explore the role of different factors and genes that have been reported to influence zebrafish gonadal sex differentiation. We also give a brief insight of primordial germ cell (PGC) involvement in shaping male and female signaling pathway in gonadal development.

  • 22.
    Pradhan, Ajay
    et al.
    Örebro University, School of Science and Technology.
    Olsson, Per-Erik
    Örebro University, School of Science and Technology.
    Zebrafish Sexual Behavior: role of sex steroid hormones and prostaglandinsManuscript (preprint) (Other academic)
  • 23.
    Pradhan, Ajay
    et al.
    Örebro University, School of Science and Technology.
    Olsson, Per-Erik
    Örebro University, School of Science and Technology.
    Zebrafish sexual behavior: role of sex steroid hormones and prostaglandins2015In: Behavioral and Brain Functions, ISSN 1744-9081, E-ISSN 1744-9081, Vol. 11, article id 23Article in journal (Refereed)
    Abstract [en]

    Background: Mating behavior differ between sexes and involves gonadal hormones and possibly sexually dimorphic gene expression in the brain. Sex steroids and prostaglandin E-2 (PGE(2)) have been shown to regulate mammalian sexual behavior. The present study was aimed at determining whether exposure to sex steroids and prostaglandins could alter zebrafish sexual mating behavior.

    Methods: Mating behavior and successful spawning was recorded following exposure to 17 beta-estradiol (E2), 11-ketotestosterone (11-KT), prostaglandin D-2 (PGD(2)) and PGE(2) via the water. qRT-PCR was used to analyze transcript levels in the forebrain, midbrain, and hindbrain of male and female zebrafish and compared to animals exposed to E2 via the water.

    Results: Exposure of zebrafish to sex hormones resulted in alterations in behavior and spawning when male fish were exposed to E2 and female fish were exposed to 11-KT. Exposure to PGD(2), and PGE(2) did not alter mating behavior or spawning success. Determination of gene expression patterns of selected genes from three brain regions using qRT-PCR analysis demonstrated that the three brain regions differed in gene expression pattern and that there were differences between the sexes. In addition, E2 exposure also resulted in altered gene transcription profiles of several genes.

    Conclusions: Exposure to sex hormones, but not prostaglandins altered mating behavior in zebrafish. The expression patterns of the studied genes indicate that there are large regional and gender-based differences in gene expression and that E2 treatment alter the gene expression pattern in all regions of the brain.

  • 24.
    Pradhan, Ajay
    et al.
    Örebro University, School of Science and Technology. Biology, the Life Science Center.
    Olsson, Per-Erik
    Örebro University, School of Science and Technology. Biology, the Life Science Center.
    Jass, Jana
    Örebro University, School of Science and Technology. Biology, the Life Science Center.
    Di(2-ethylhexyl) phthalate and diethyl phthalate disrupt lipid metabolism, reduce fecundity and shortens lifespan of Caenorhabditis elegans2018In: Chemosphere, ISSN 0045-6535, E-ISSN 1879-1298, Vol. 190, p. 375-382Article in journal (Refereed)
    Abstract [en]

    The widespread use of phthalates is of major concern as they have adverse effects on many different physiological functions, including reproduction, metabolism and cell differentiation. The aim of this study was to compare the toxicity of the widely-used di (2-ethydlhexyl) phthalate (DEHP) with its substitute, diethyl phthalate (DEP). We analyzed the toxicity of these two phthalates using Caenorhabditis elegans as a model system. Gene expression analysis following exposure during the L1 to young adult stage showed that DEHP and DEP alter the expression of genes involved in lipid metabolism and stress response. Genes associated with lipid metabolism, including fasn-1, pod-2, fat-5, acs-6 and sbp-1, and vitellogenin were upregulated. Among the stress response genes, ced-1 wah-1, daf-21 and gst-4 were upregulated, while cd-1, cdf-2 and the heat shock proteins (hsp-16.1, hsp-16.48 and sip-1) were down regulated. Lipid staining revealed that DEHP significantly increased lipid content following 1 mu M exposure, however, DEP required 10 mu M exposure to elicit an effect. Both DEHP and DEP reduced the fecundity at 1 mu M concentration. Lifespan analysis indicated that DEHP and DEP reduced the average lifespan from 14 days in unexposed worms to 13 and 12 days, respectively. Expression of lifespan associated genes showed a correlation to shortened lifespan in the exposed groups. As reported previously, our data also indicates that the banned DEHP is toxic to C. elegans, however its substitute DEP has not been previously tested in this model organism and our data revealed that DEP is equally potent as DEHP in regulating C. elegans physiological functions.

  • 25.
    Saju, Jolly M.
    et al.
    Reproductive Genomics Group, Temasek Life Sciences Laboratory, Singapore, Singapore.
    Hossain, Mohammad Sorowar
    Reproductive Genomics Group, Temasek Life Sciences Laboratory, Singapore, Singapore; Department of Biological Sciences, National University of Singapore, Singapore, Singapore.
    Liew, Woei Chang
    Reproductive Genomics Group, Temasek Life Sciences Laboratory, Singapore, Singapore.
    Pradhan, Ajay
    Örebro University, School of Science and Technology. Biology, Örebro Life Science Center.
    Thevasagayam, Natascha May
    Reproductive Genomics Group, Temasek Life Sciences Laboratory, Singapore, Singapore.
    Tan, Lydia Shun En
    Reproductive Genomics Group, Temasek Life Sciences Laboratory, Singapore, Singapore.
    Anand, Amit
    Bioimaging and Biocomputing, Temasek Life Sciences Laboratory, Singapore, Singapore.
    Olsson, Per-Erik
    Örebro University, School of Science and Technology. Biology, Örebro Life Science Center.
    Orban, Laszlo
    Reproductive Genomics Group, Temasek Life Sciences Laboratory, Singapore, Singapore; Frontline Fish Genomics Research Group, Department of Animal Sciences, Georgikon Faculty, University of Pannonia, Keszthely, Hungary; Centre for Comparative Genomics, Murdoch University, Murdoch, Australia.
    Heat Shock Factor 5 Is Essential for Spermatogenesis in Zebrafish2018In: Cell reports, ISSN 2211-1247, E-ISSN 2211-1247, Vol. 25, no 12, p. 3252-3261Article in journal (Refereed)
    Abstract [en]

    Heat shock factors (Hsfs) are transcription factors that regulate responses to heat shock and other environmental stimuli. Four heat shock factors (Hsf1-4) have been characterized from vertebrates to date. In addition to stress response, they also play important roles in development and gametogenesis. Here, we study the fifth member of heat shock factor family, Hsf5, using zebrafish as a model organism. Mutant hsf5(-/-) males, generated by CRISPR/Cas9 technique, were infertile with drastically reduced sperm count, increased sperm head size, and abnormal tail architecture, whereas females remained fertile. We show that Hsf5 is required for progression through meiotic prophase 1 during spermatogenesis as suggested by the accumulation of cells in the leptotene and zygotene-pachytene stages and increased apoptosis in post-meiotic cells. hsf5(-/-) mutants show gonadal misregulation of a substantial number of genes with roles in cell cycle, apoptosis, protein modifications, and signal transduction, indicating an important role of Hsf5 in early stages of spermatogenesis.

  • 26.
    Seyoum, Asmerom
    et al.
    Örebro University, School of Science and Technology.
    Pradhan, Ajay
    Örebro University, School of Science and Technology.
    Effect of phthalates on development, reproduction, fat metabolism and lifespan in Daphnia magna2019In: Science of the Total Environment, ISSN 0048-9697, E-ISSN 1879-1026, Vol. 654, p. 969-977Article, review/survey (Refereed)
    Abstract [en]

    Phthalates are used as plasticizers to increase durability, resistivity and flexibility of plastic materials. The commonly used phthalate, diethylhexyl phthalate (DEHP) is used in different plastic materials like food packaging, toys and medical devices. DEHP has been linked to different toxicities in humans as well as in animals, and as a consequence other phthalates, including dibutyl phthalate (DBP) and diethyl phthalate (DEP) are being introduced. The increased use of phthalates has resulted in contamination of aquatic ecosystem and it directly threatens the aquatic life. In this study, we analyzed the effects of three phthalates DEHP, DEP and DBP using freshwater organism Daphnia magna. Although, exposure of the three phthalates at 1 and 10 μM did not result any lethality and hatching delay, the chronic exposure for 14 days resulted in reduction of body length. There was enhanced fat accumulation on exposure to all the phthalates, as indicated by oil red O staining. qRT-PCR analysis of genes involved in fat metabolism suggests that the increase in fat content could be due to inhibition of absorption and catabolism of fatty acids. Reproduction analysis showed that DBP and DEP did not alter fecundity but surprisingly, DEHP at 1 μM increased reproduction by 1.5 fold compared to control group. Phthalates also showed negative effect on lifespan as DEP at 10 μM and DBP at both 1 and 10 μM significantly reduced the lifespan. Our data indicates that along with the banned phthalate DEHP, the other substitute phthalates DEP and DBP could also have detrimental effect on aquatic organisms.

1 - 26 of 26
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