Ajay Pradhan

Activation of NF-κB Protein Prevents the Transition from Juvenile Ovary to Testis and Promotes Ovarian Development in Zebrafish

Background: NF-κB is a key regulator of anti-apoptotic processes and plays a role in gonad formation in mammals. Results: NF-κB activation leads to female-biased sex differentiation in zebrafish. Conclusion: Anti-apoptotic signaling during the juvenile ovary stage is needed for the maintenance of oocytes in zebrafish. Significance: Unraveling the regulation of apoptotic processes during gonadal transformation will facilitate understanding the molecular mechanism of zebrafish sex differentiation. 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-κB 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-Müllerian hormone (Amh) gene expression. Because steroidogenic factor-1 and Amh are important for proper testis development, NF-κB-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-κB in zebrafish gonad differentiation. Exposure of juvenile zebrafish to heat-killed Escherichia coli activated the NF-κB 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-κB-regulated genes. To confirm the involvement of NF-κB-induced anti-apoptotic effects, zebrafish were treated with sodium deoxycholate, a known inducer of NF-κB or NF-κB 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-κB using NAI resulted in a decrease in females from 45 to 20%. This study provides proof for an essential role of NF-κB 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.

The brominated flame retardant TBECH activates the zebrafish (Danio rerio) androgen receptor, alters gene transcription and causes developmental disturbances.

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 silico 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 μM and 10 μM doses while morphological abnormalities and juvenile mortality was observed at 10 μM. 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.

Juvenile Ovary to Testis Transition in Zebrafish Involves Inhibition of Ptges

ABSTRACT The sex differentiation mechanisms in zebrafish (Danio rerio) remains elusive, partly because of the absence of sex chromosomes but also because 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 NFκB 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 results 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 NFκB and the β-catenin pathway as well as inhibition of sox9. Exposure to the β-catenin inhibitor PNU-74654 resulted in up-regulation of ptgds and male-biased sex ratios, further confirming the involvement of β-catenin in the female differentiation pathway. In this study, we show that PGD2 and PGE2 can program the gonads to either the testis or the ovary differentiation pathways, indicating that prostaglandins are involved in the regulation of zebrafish gonadal differentiation.

1,2-Dibromo-4-(1,2 dibromoethyl) cyclohexane (TBECH)-mediated steroid hormone receptor activation and gene regulation in chicken LMH cells.

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.

Identification of a group of brominated flame retardants as novel androgen receptor antagonists and potential neuronal and endocrine disrupters

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), 5α-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 5α-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.

Zebrafish sexual behavior: role of sex steroid hormones and prostaglandins

BackgroundMating behavior differ between sexes and involves gonadal hormones and possibly sexually dimorphic gene expression in the brain. Sex steroids and prostaglandin E2 (PGE2) 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.MethodsMating behavior and successful spawning was recorded following exposure to 17β-estradiol (E2), 11-ketotestosterone (11-KT), prostaglandin D2 (PGD2) and PGE2 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.ResultsExposure 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 PGD2, and PGE2 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.ConclusionsExposure 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.

Inhibition of retinoic acid synthesis disrupts spermatogenesis and fecundity in zebrafish

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 up-regulation 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.

Comparative Analysis of Stress Induced Gene Expression in Caenorhabditis elegans following Exposure to Environmental and Lab Reconstituted Complex Metal Mixture

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.

Contribution of pharmaceuticals, fecal bacteria and endotoxin to the inflammatory responses to inland waters.

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.

TBECH, 1,2-dibromo-4-(1,2 dibromoethyl) cyclohexane, alters androgen receptor regulation in response to mutations associated with prostate cancer

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.