RESUMO
A novel cell line, Danio rerio gill (DrG), derived from the gill tissue of zebrafish, was established and characterized. The cells were able to grow at a wide range of temperatures from 25°C to 32°C in Leibovitz's L-15 medium. The DrG cell line consists of epithelial-like cells with a diameter of 18-22µm. The cell line was characterized by mitochondrial 12S rRNA gene. Acute toxicity tests were conducted on D. rerio by exposing them to nicotine for 96h under static conditions. In vitro cytotoxicity of nicotine was assessed in DrG cell line using multiple endpoints such as 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), Neutral Red assay, Alamar Blue assay and Coomassie Blue protein assay. Linear correlations between each in vitro cytotoxicity assay and the in vivo mortality data were highly significant. Nicotine induced intracellular reactive oxygen species generation in DrG cell line in a concentration dependent manner. DrG cell line and zebrafish exposed to nicotine significantly increased the elevation of lipid peroxidation (LPO) while depletion of reduced glutathione (GSH), manganese superoxide dismutase (MnSOD), catalase (CAT), glutathione S-transferase (GST) and glutathione peroxidise(GPx1a) was observed. In nicotine treated fish and cells a negative correlation between reduced glutathione and LPO was observed. In addition, the production of ROS and the resulting oxidative stress resulted in increased expression of apoptosis related genes p53 and cas3.Collectively, our result suggests that nicotine has the potential to induce reactive oxygen species (ROS) production, oxidative stress and apoptosis in DrG cell line and zebrafish.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Brânquias/metabolismo , Nicotina/farmacologia , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Catalase/genética , Catalase/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Relação Dose-Resposta a Droga , Estimulantes Ganglionares/farmacologia , Estimulantes Ganglionares/toxicidade , Brânquias/citologia , Glutationa/metabolismo , Glutationa Peroxidase/genética , Glutationa Peroxidase/metabolismo , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Nicotina/toxicidade , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Temperatura , Testes de Toxicidade Aguda/métodos , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Glutationa Peroxidase GPX1RESUMO
An attempt was made to determine the replication efficiency of white spot syndrome virus (WSSV) of shrimp in different organs of freshwater rice-field crab, Paratelphusa hydrodomous (Herbst), using bioassay, PCR, RT-PCR, ELISA, Western blot and real-time PCR analyses, and also to use this crab instead of penaeid shrimp for the large-scale production of WSSV. This crab was found to be highly susceptible to WSSV by intramuscular injection. PCR and Western blot analyses confirmed the systemic WSSV infection in freshwater crab. The RT-PCR analysis revealed the expression of VP28 gene in different organs of infected crab. The indirect ELISA was used to quantify the VP28 protein in different organs of crab. It was found that there was a high concentration of VP28 protein in gill tissue, muscle, haemolymph and heart tissue. The copy number of WSSV in different organs of infected crab was quantified by real-time PCR, and the results revealed a steady increase in copy number in different organs of infected crab during the course of infection. The viral inoculum prepared from different organs of infected crab caused significant mortality in tiger prawn, Penaeus monodon (Fabricius). The results revealed that this crab can be used as an alternate host for WSSV replication and production.
Assuntos
Braquiúros/virologia , Replicação Viral , Vírus da Síndrome da Mancha Branca 1/fisiologia , Animais , Água Doce , Perfilação da Expressão Gênica , Regulação Viral da Expressão Gênica , Fatores de Tempo , Proteínas do Envelope Viral/genética , Vírus da Síndrome da Mancha Branca 1/genéticaRESUMO
Explants from different organs of freshwater crab, Paratelphusa hydrodomous were prepared to establish an in vitro system for replication of white spot syndrome virus (WSSV) of shrimp. Heart explants were maintained for 53 days without any morphological changes in EX-CELL™ 405 medium with and without serum whereas the explants of eye muscle, gill, shell membrane and appendage muscle died within 15 days of culture period. The heart explants on different days of culture were exposed to WSSV for 10 days to study the viral replication. The infection of WSSV in explants of the heart was confirmed by PCR, RT-PCR, Western blot, histology, immunohistochemistry, bioassay and transmission electron microscopy. The WSSV was quantified by real-time PCR and indirect ELISA. The WSSV inoculum prepared from the heart explants of crab caused significant mortality in Penaeus monodon in challenge experiments and the results indicate that the WSSV which replicated in the heart explants of freshwater crab maintains its infectivity as in marine shrimp. The results indicate that the heart explants of P. hydrodomous would be a good alternative to whole animals for production of WSSV.