Bruna Félix Nornberg

Induction of phase II enzymes and hsp70 genes by copper sulfate through the electrophile-responsive element (EpRE): insights obtained from a transgenic zebrafish model carrying an orthologous EpRE sequence of mammalian origin

We have evaluated the homology of the electrophile-responsive element (EpRE) core sequence, a binding site for the Nrf2 transcription factor, in the proximal promoters of the mouse and zebrafish glutathione-S-transferase (gst), glutamate cysteine ligase catalytic subunit (gclc) and heat shock protein 70 (hsp70) genes. The EpRE sites identified for both species in the three analyzed genes showed a high similarity with the putative EpRE core sequence. We also produced a transgenic zebrafish model carrying a transgene comprised of the luciferase (luc) reporter gene under transcriptional control of a mouse EpRE sequence. This transgenic model was exposed to copper sulfate, and the reporter gene was significantly activated. The endogenous gst, gclc and hsp70 zebrafish genes were analyzed in the EpRE-Luc transgenic zebrafish and showed an expression pattern similar to that of the reporter transgene used. Our results demonstrate that EpRE is conserved between mouse and zebrafish for detoxification-related genes and that the development of genetically modified models using this responsive element to drive the expression of reporter genes can be an important tool in understanding the action mechanism of aquatic pollutants.

Natural occurrence of White spot syndrome virus and Infectious hypodermal and hematopoietic necrosis virus in Neohelice granulata crab

White spot syndrome virus (WSSV) and Infectious hypodermal and hematopoietic necrosis virus (IHHNV) are two infectious agents associated to economic losses in shrimp aquaculture. As virus spread occurs through vectors and hosts, this study sought to verify the presence of WSSV and IHHNV in Neohelice granulata crab from Lagoa dos Patos estuary in Brazil and nearby shrimp farms. DNA extractions were performed with phenol/chloroform protocol. Molecular diagnosis was carried out by nested PCR for WSSV and one-step PCR for IHHNV. Results showed the presence of WSSV on crabs of both Lagoa dos Patos and farms, while IHHNV was found only on crabs collected in estuary. This is the first study to report IHHNV presence in N. granulata. Moreover, as analyzed crabs had no clinical symptoms or showed in situ mortality, we suggest its use as a bioindicator for virus occurrence in aquatic environments.

ABCB1 and ABCC4 efflux transporters are involved in methyl parathion detoxification in ZFL cells

The multi-xenobiotics resistance (MXR) mechanisms are the first line of defense against toxic substances in aquatic organisms and present great importance in the adaptation related to contaminated environments. Methyl parathion (MP) is a widely used organophosphate pesticide, which has been associated to various toxic effects in organisms. In the present work, we studied the main genes related to efflux transporters in zebrafish liver (ZFL) cells exposed to MP with and without an inhibitor of ABC transporters (verapamil). The results concerning transporters activity showed that the MXR mechanism is activated to detoxify from methyl parathion. The toxic effects of MP on ZFL cells were increased in the presence of the efflux transporter inhibitor, once cell viability was significantly decreased in co-exposure experiments. The combined exposure to MP and the inhibitor caused an increase in gene expression of P-gp1 (Abcb1) and MRP4 (Abcc4), suggesting that these transporters isoforms are associated with MP efflux. In general, the expression of genes related to the antioxidant defense system (ADS) was significantly increased in ZFL cells co-exposed to MP and verapamil. These data provide useful insights for better understanding of MP detoxification mechanism in fish hepatocytes.

Expression profile of IGF paralog genes in liver and muscle of a GH-transgenic zebrafish

The objective of this study was to investigate the relationship between IGFs produced in the liver and skeletal muscle with muscle hypertrophy previously observed in a line of GH-transgenic zebrafish. In this sense, we evaluated the expression of genes related to the IGF system in liver and muscle of transgenics, as well as the main intracellular signaling pathways used by GH/IGF axis. Our results showed an increase in expression of igf1a, igf2a, and igf2b genes in the liver. Moreover, there was a decrease in the expression of igf1ra and an increase in muscle igf2r of transgenics, indicating a negative response of muscle tissue with respect to excess circulating IGFs. Muscle IGFs expression analyses revealed a significant increase only for igf2b, accompanied by a parallel induction of igfbp5a gene. The presence of IGFBP5a may potentiate the IGF2 action in muscle cells differentiation. Regarding JAK/STAT-related genes, we observed an alteration in the expression profile of both stat3 and stat5a in transgenic fish liver. No changes were observed in the muscle, suggesting that both tissues respond differently to GH-transgenesis. Western blotting analyses indicated an imbalance between the phosphorylation levels of the proliferative (MEK/ERK) and hypertrophic (PI3K/Akt) pathways, in favor of the latter. In summary, the results of this study suggest that the hypertrophy caused by GH-transgenesis in zebrafish may be due to circulating IGFs produced by the liver, with an important participation of muscle IGF2b. This group of IGFs appears to be favoring the hypertrophic intracellular pathway in muscle tissue of transgenic zebrafish.

Effects of Double Transgenesis of Somatotrophic Axis (GH/GHR) on Skeletal Muscle Growth of Zebrafish (Danio rerio)

Transgenic fish for growth hormone (GH) has been considered as a potential technological improvement in aquaculture. In this study, a double-transgenic zebrafish was used to evaluate the effect of GH and its receptor (GHR) on muscle growth. Double transgenics reached the same length of GH transgenic, but with significantly less weight, featuring an unbalanced growth. The condition factor of GH/GHR-transgenic fish was lower than the other genotypes. Histological analysis showed a decrease in the percentage of thick muscle fibers in GH/GHR genotype of ∼ 80% in comparison to GH-transgenic line. The analysis of gene expression showed a significant decrease in genes related to muscle growth in GH/GHR genotype. It seems that concomitant overexpression of GH and GHR resulted in a strong decrease of the somatotrophic axis intracellular signaling by diminishing its principal transcription factor signal transducer and activator of transcription 5.1 (STAT5.1).

Modeling Drug-Drug Interactions of AZD1208 with Vincristine and Daunorubicin on Ligand-Extrusion Binding TMD-Domains of multidrug resistance P-glycoprotein (ABCB1)

In the present study, the molecular docking mechanism based on pharmacodynamic interactions between the ligands AZD1208 and recognized chemotherapy agents (Vincristine and Daunorubicin) with human ATP-binding cassette (ABC) transporters (ABCB1) was investigated. For the first time, were combined an in silico approaches like molecular docking and ab initio computational simulation based on Density Functional Theory (DFT) to explain the drug-drug interaction mechanism of aforementioned chemotherapy ligands with the transmembrane ligand extrusion binding domains (TMDs) of ABCB1. In this regard, the theoretical pharmacodynamic interactions were characterized by using the Gibbs free energy (FEB, kcal/mol) from the best ABCB1-ligand docking complexes. The molecular docking results pointing that for the three chemotherapy ABCB1-ligand complexes are mainly based in non-covalent hydrophobic and hydrogen-bond interactions showing a similar toxicodynamic behavior in terms of strength of interaction (FEB, kcal/mol) and very close free binding energies when compared with the FEB-values of the ABCB1 specific-inhibitor (Rhodamine B) = -6.0 kcal/mol used as theoretical docking control to compare with FEB (AZD1208-ABCB1) ∼ FEB (Vincristine-ABCB1) ∼ FEB (Daunorubicin-ABCB1) -6.2 kcal/mol as average. Ramachandran plot suggests that the 3D-crystallographic structure from ABCB1 transporter can be efficiently-modeled with conformationally-favored Psi versus Phi dihedral angles for all key TMDs-residues. Though, the results of DFT-simulation corroborate the existence of drug-drug interaction between (AZD1208/Vincristine) > (AZD1208/Daunorubicin). These theoretical pieces of evidence have preclinical relevance potential in the design of the new drugs to understand the polypharmacology influence in the molecular mechanism of multiple-drugs resistance, contributing with a higher success in chemotherapy and prognosis of cancer patients.

Reproductive parameters of double transgenic zebrafish ( Danio rerio ) males overexpressing both the growth hormone (GH) and its receptor (GHR)

Growth hormone (GH) transgenesis presents a high potential application in aquaculture. However, excess GH may have serious consequences due to pleiotropic actions. In order to study these effects in zebrafish (Danio rerio), two transgenic lines were developed. The first expresses GH ubiquitously and constitutively (F0104 line), while the second expresses the GH receptor in a muscle-specific manner (Myo-GHR line). Results from the F0104 line showed accelerated growth but increased reproductive difficulties, while Myo-GHR did not show the expected increase in muscle mass. Since the two lines appeared to display complementary characteristics, a double transgenic (GH/GHR) was created via crossing between them. This double transgenic displayed accelerated growth, however reproductive parameters remained uncertain. The objective of the present study was to determine the reproductive capacity of males of this new line, by evaluating sperm parameters, expression of spermatogenesis-related genes, and reproductive tests. Double transgenics showed a strong recovery in almost all sperm parameters analyzed when compared to the F0104 line. Gene expression analyses revealed that Anti-Müllerian Hormone gene (amh) appeared to be primarily responsible for this recovery. Reproductive tests showed that double transgenic males did not differ from non-transgenics. It is possible that GHR excess in the muscle tissues of double transgenics may have contributed to lower circulating GH levels and thus reduced the negative effects of this hormone with respect to reproduction. Therefore, it is clear that GH-transgenesis technology should take into account the need to obtain adequate levels of circulating hormone in order to achieve maximum growth with minimal negative side effects.

GH indirectly enhances the regeneration of transgenic zebrafish fins through IGF2a and IGF2b

The somatotropic axis, composed essentially of the growth hormone (GH) and insulin-like growth factors (IGFs), is the main regulator of somatic growth in vertebrates. However, these protein hormones are also involved in various other major physiological processes. Although the importance of IGFs in mechanisms involving tissue regeneration has already been established, little is known regarding the direct effects of GH in these processes. In this study, we used a transgenic zebrafish (Danio rerio) model, which overexpresses GH from the beta-actin constitutive promoter. The regenerative ability of the caudal fin was assessed after repeated amputations, as well as the expression of genes related to the GH/IGF axis. The results revealed that GH overexpression increased the regenerated area of the caudal fin in transgenic fish after the second amputation. Transgenic fish also presented a decrease in gene expression of the GH receptor (ghrb), in opposition to the increased expression of the IGF1 receptors (igf1ra and igf1rb). These results suggest that transgenic fish have a higher sensitivity to IGFs than to GH during fin regeneration. With respect to the different IGFs produced locally, a decrease in igf1a expression and a significant increase in both igf2a and igf2b expression was observed, suggesting that igf1a is not directly involved in fin regeneration. Overall, the results revealed that excess GH enhances fin regeneration in zebrafish through igf2a and igf2b expression, acting indirectly on this major physiological process.