Modelos in vivo e in vitro geneticamente modificados como biomarcadores de poluição aquática

Dissertação (mestrado)-Universidade Federal do Rio Grande, Programa de Pós-Graduação em Ciências Fisiológicas – Fisiologia Animal Comparada, Instituto de Ciências Biológicas, 2007.A indução de vários genes que codificam para enzimas envolvidas no processo de detoxificação celular e resposta antioxidante é mediada através de um mecanismo comum, o qual depende da presença dos elementos de resposta eletrofílicos (EpRE) na região promotora destes genes. No presente estudo foi analisada a homologia da seqüência central do EpRE, a qual representa um sítio de ligação para o fator de transcrição Nrf2, de regiões conservadas de promotores das enzimas glutationa S-transferase (Gsta1), subunidade catalítica da glutamato cisteína ligase (Gclc) e proteína de choque térmico (Hsp70) de zebrafish (Danio rerio) e camundongo (Mus musculus). A seqüência EpRE foi identificada para ambas as espécies em todos os genes comparados, mostrando uma alta similaridade com a seqüência central do EpRE. Adicionalmente, foi produzido um modelo de zebrafish transgênico carregando um transgene que contém um EpRE obtido do promotor da Gsta1 de camundongo fusionado ao promotor mínimo da metalotioneína (mt1) do camundongo direcionando a expressão do gene repórter da luciferase (luc). Este modelo in vivo foi exposto ao sulfato de cobre e o gene repórter foi significativamente ativado. Os genes gclc e hsp70 do zebrafish foram analisados nos peixes transgênicos carregando a construção EpRE-Luc e mostraram um padrão similar de expressão com o gene da luciferase. Também foi produzido um modelo alternativo carregando o transgene EpRE-GFP. Nesta construção genética o gene repórter da luc foi substituído pelo gene da proteína verde fluorescente (gfp). Os peixes carregando o transgene EpRE-GFP foram expostos ao cobre e a expressão da Gfp foi diretamente quantificada nas larvas vivas em fluorímetro. Neste caso, um aumento na expressão da Gfp só foi detectado na maior concentração de cobre, provavelmente, devido ao efeito do mosaismo ou da fluorescência basal das larvas. Na última etapa deste trabalho, foi produzida uma linhagem estável de hepatoma (HTC) carregando o transgene EpRE-GFP. A linhagem EpRE-GFP HTC foi tratada com sulfato de cobre e metil paration por 48 h. Para avaliar as respostas genéticas produzidas por estes poluentes, a expressão da Gfp foi diretamente quantificada e os resultados mostraram que a expressão da Gfp é significativamente aumentada somente nas maiores concentrações de ambos os poluentes. Desta forma, os resultados obtidos neste estudo demonstram que o uso de EpRE associado a genes repórteres pode ser um modelo interessante para estudo de respostas genéticas das células frente a poluentes aquáticos, bem como uma ferramenta adequada para estudos toxicológicos in vivo e in vitro

mRNA expression and activity of ion-transporting proteins in gills of the blue crab callinectes sapidus: effects of waterborne copper

Waterborne Cu effects on the transcription of genes encoding ion-transporting proteins and the activities of these proteins were evaluated in gills of the blue crab Callinectes sapidus acclimated to diluted (2%) and full (30%) seawater. Crabs were exposed (96 h) to an environmentally relevant concentration of dissolved Cu (0.78 mM) and had their posterior (osmoregulating) gills dissected for enzymatic and molecular analysis. Endpoints analyzed were the activity of key enzymes involved in crab osmoregulation (sodiumpotassium adenosine triphosphatase [Naþ/Kþ-ATPase], hydrogen adenosine triphosphatase [Hþ-ATPase], and carbonic anhydrase [CA]) and the mRNA expression of genes encoding these enzymes and the sodium-potassium-chloride (Naþ/Kþ/2Cl_) cotransporter. Copper effects were observed only in crabs acclimated to diluted seawater (hyperosmoregulating crabs) and were associated with an inhibition of the expression of mRNA of genes encoding the Naþ/Kþ-ATPase and the Naþ/Kþ/2Cl_ cotransporter. However, Cu did not affect Naþ/Kþ-ATPase activity, indicating that the gene transcription is downregulated before a significant inhibition of the enzyme activity can be observed. This also suggests the existence of a compensatory response of this enzyme to prevent osmoregulatory disturbances after short-term exposure to environmentally relevant Cu concentrations. These findings suggest that Cu is a potential ionoregulatory toxicant in blue crabs C. sapidus acclimated to low salinity. The lack of Cu effect on blue crabs acclimated to full seawater would be due to the reduced ion uptake needed for the regulation of the hemolymph osmotic concentration in full seawater (30%). Also, this could be explained considering the lower bioavailability of toxic Cu (free ion) associated with the higher ionic content and dissolved organic matter concentration in high salinity (30%) than in diluted seawater (2%)

Initial results in the development of a reporter cell line for toxicology studies at gene expression level: Activation of the electrophile-responsive element by copper and methyl parathion

Induction of many genes encoding detoxifying enzymes and antioxidant proteins is mediated through a common mechanism, which is controlled by electrophile-responsive elements (EpRE)within the regulatory region of those genes. Copper and methyl parathion are environmental pollutants known to induce the expression of EpRE-mediated genes. In order to evaluate the molecular response triggered by these pollutants, a stable cell line was produced, which carries a transgene comprised of the green fluorescent protein (GFP) reporter gene under transcriptional control of the mouse glutathione-S-transferase (gst1)electrophile-responsive element fused to the mouse metallothionein (mt1) minimal promoter. The rat HTC hepatoma cells were transfected with the EpREmt–GFP construct and successfully selected with G418 antibiotic. EpREmt–GFP HTC cells were treated with 0.002 mg L 1, 0.02 mg L 1, 0.2 mg L 1 and 2mg L 1 copper sulfate and 0.001 mg L 1,0.01 mg L 1, 0.1 mg L 1 and 1 mg L 1 methyl parathion for 48 h. GFP expression was directly quantified in living cells using a microplate fluorimeter. GFP expression was significantly increased in higher concentrations of both pollutants, showing a 1.80- and 2.58-fold induction of GFP at 2 mg copper L 1 and 1 mg methyl parathion L 1, respectively (p < 0.01). The results obtained in the present study demonstrate that the EpREmt–GFP HTC cell line can be an interesting model for further development for the study of the cellular response to aquatic pollutants as well as a new tool for environmental monitoring at the molecular level

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 EpRELuc 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 detoxificationrelated 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

Genotype-dependent gene expression profile of the antioxidant defense system (ADS) in the liver of a GH-transgenic zebrafish model

The aim of this study was to evaluate the effects of growth hormone (GH)overexpression on the gene expression profile of multiple components of the antioxidant defense system(ADS)of different genotypes of a GH transgenic zebrafish (Danio rerio)model. Several ADS-related genes were analyzed by semiquantitative reverse transcription–PCR in the liver of hemizygous (HE) and homozygous (HO)transgenic zebrafish. The results showed a significant reduction in the glutamate cysteine ligase catalytic subunit (GCLC) and the gene expression of two glutathione S-transferase (GST) isoforms and an increase in the glutathione reductase gene in the HO group compared to non-transgenic controls. The expression of the Cu, Zn-superoxide dismutase (SOD1) and catalase (CAT) genes was reduced in HO and HE groups, respectively. Among the ten genes analyzed, two were altered in HE transgenic zebrafish and five were altered in HO transgenic zebrafish. These findings indicate a genotypedependent gene expression profile of the ADS-related genes in the liver of our GH-transgenic zebrafish model and are in agreement with the general effects of GH hypersecretion in the fish and mouse,which involves a reduction in the capability of the tissues to deal with oxidative stress situations. The GH-transgenic zebrafish model used here seems to be an interesting tool for analyzing the effect of different GH expression levels on physiological processes

SOCS1 and SOCS3 are the main negative modulators of the somatotrophic axis in liver of homozygous gh-transgenic zebrafish (danio rerio)

Homozygote individuals (HO) of the GH-transgenic zebrafish lineage (F0104), despite expressing double the amount of growth hormone (GH) in relation to the hemizygote (HE) individuals, presented smaller growth in relation to the last, and similar to the non-transgenic (NT) group. Through the analysis of the expression of genes of the somatotrophic axis in the livers of HO and NT individuals, it was verified that GHR, JAK2 and STAT5.1 did not present significant differences among the analyzed genotypes (NTand HO). However, in the IGF-I gene expression, an accentuated decrease was observed in group HO(p < 0.01), suggesting a resistance effect to excess GH. This resistance could be related to the insufficient amount of energy for supporting the accelerated metabolic demand caused by excess circulating GH. Analysis of the genes involved in the regulation of GH signalization by dephosphorylation (PTP-H1 and PTP-1B) did not show any significant alteration when comparing groups HO and NT. However, the analysis of the SOCS1 and SOCS3 genes showed an induction in homozygotes of 2.5 times (p < 0.01) and 4.3 times (p < 0.05), respectively, in relation to non-transgenics. The results of the present work demonstrate that, in homozygotes, GH signaling is reduced by the action of the SOCS1 and SOCS3 proteins

Growth hormone transgenesis affects osmoregulation and energy metabolism in zebrafish (Danio rerio)

Growth hormone (GH) transgenic fish are at a critical step for possible approval for commercialization. Since this hormone is related to salinity tolerance in fish, our main goal was to verify whether the osmoregulatory capacity of the stenohaline zebrafish (Danio rerio) would be modified by GH-transgenesis. For this, we transferred GH-transgenic zebrafish (T) from freshwater to 11 ppt salinity and analyzed survival as well as relative changes in gene expression. Results show an increased mortality in T versus non-transgenic (NT) fish, suggesting an impaired mechanism of osmotic acclimation in T. The salinity effect on expression of genes related to osmoregulation, the somatotropic axis and energy metabolism was evaluated in gills and liver of T and NT. Genes coding for Na(+), K(+)-ATPase, H(+)-ATPase, plasma carbonic anhydrase and cytosolic carbonic anhydrase were up-regulated in gills of transgenics in freshwater. The growth hormone receptor gene was down-regulated in gills and liver of both NT and T exposed to 11 ppt salinity, while insulin-like growth factor-1 was down-regulated in liver of NT and in gills of T exposed to 11 ppt salinity. In transgenics, all osmoregulation-related genes and the citrate synthase gene were down-regulated in gills of fish exposed to 11 ppt salinity, while lactate dehydrogenase expression was up-regulated in liver. Na(+), K(+)-ATPase activity was higher in gills of T exposed to 11 ppt salinity as well as the whole body content of Na(+). Increased ATP content was observed in gills of both NT and T exposed to 11 ppt salinity, being statistically higher in T than NT. Taking altogether, these findings support the hypothesis that GH-transgenesis increases Na(+) import capacity and energetic demand, promoting an unfavorable osmotic and energetic physiological status and making this transgenic fish intolerant of hyperosmotic environments

Metabolic rate and reactive oxygen species production in different genotypes of GH-transgenic zebrafish

Growth hormone overexpression increases growth and consequently increases the metabolic rate in fishes. Therefore, the objective of this study was to evaluate the effects of growth hormone overexpression in zebrafish Danio rerio in terms of growth, oxygen consumption, reactive oxygen species production, lipid hydroperoxide content, antioxidant enzyme activity and glutamate-cysteine ligase catalytic subunit gene expression. The employed models were wild type and transgenic (hemizygous and homozygous) zebrafish expressing the Odonthestes argentinensis growth hormone gene directed by the Cyprinus carpio beta-actin promoter. Higher growth parameters were observed in the hemizygous group. The homozygous group possessed higher oxygen consumption and reactive oxygen species production. Growth hormone transgenesis causes a decrease in glutamate-cysteine ligase catalytic subunit expression, an enzyme responsible for glutathione synthesis. Although the lipid hydroperoxide content was similar between groups, we demonstrate that growth hormone overexpression has the potential to generate oxidative stress in fishes

In vitro culture of Neoechinorhynchus buttnerae (Acanthocephala: Neoechinorhynchidae): Influence of temperature and culture media

Abstract Infection by the acantocephalan Neoechinorhynchus buttnerae is considered one of most important concerns for tambaqui fish (Colossoma macropomum ) production. Treatment strategies have been the focus of several in vivo studies; however, few studies have been undertaken on in vitro protocols for parasite maintenance. The aim of the present study was to develop the best in vitro culture condition for N. buttnerae to ensure its survival and adaptation out of the host to allow for the testing of substances to be used to control the parasite. To achieve this, parasites were collected from naturally infected fish and distributed in 6-well culture plates under the following treatments in triplicate: 0.9% NaCl, sterile tank water, L-15 Leibovitz culture medium, L-15 Leibovitz + agar 2% culture medium, RPMI 1640 culture medium, and RPMI 1640 + agar 2% culture medium. The plates containing the parasites were maintained at 24 °C, 28 °C, and 32 °C. The RPMI 1640 + agar 2% culture medium showed the best survival of 24 days at 24 °C. No body alterations such as swollen parasites, body deformation, dehydration and hardening were observed in the RPMI 1640 + 2% culture medium