RESUMO
This study investigated the potentially detrimental effects of copper and elevated aquatic CO(2) (hypercarbia), alone or in combination, on pacu, Piaractus mesopotamicus. Fish were exposed for 48 h to control (no copper addition in normocarbia), to 400 µg Cu(2+)L(-1), to hypercarbic (1% CO(2); PCO(2) = 6.9 mm Hg) water and to 400 µg Cu(2+)L(-1) + hypercarbia. In liver the single factors caused an increase in lipid hydroperoxide concentration that was not observed when the factors were combined. Copper exposure elicited increased hepatic superoxide dismutase activity, irrespective of aquatic CO(2) level. On the other hand, the effects of copper on hepatic glutathione peroxidase activity were dependent on water CO(2) levels. The two stressors combined did not affect hepatic catalase activity. Hypercarbic water caused a decline in plasma glucose concentration, but this was not observed when hypercarbia was combined with copper exposure. Copper caused a decrease in branchial Na(+)/K(+)-ATPase activity that was independent of water CO(2) level. Copper caused an increase in branchial metallothionein concentration that was independent of water CO(2) level. Thus, branchial metallothionein and Na(+)/K(+)-ATPase were effective biomarkers of copper exposure that were not affected by water CO(2) level.
Assuntos
Antioxidantes/metabolismo , Dióxido de Carbono/metabolismo , Characidae/metabolismo , Cobre/toxicidade , Exposição Ambiental/análise , Animais , Análise Química do Sangue/métodos , Catalase/metabolismo , Brânquias/efeitos dos fármacos , Brânquias/enzimologia , Brânquias/metabolismo , Fígado/efeitos dos fármacos , Fígado/enzimologia , Fígado/metabolismo , Metalotioneína/metabolismo , Oxirredução , ATPase Trocadora de Sódio-Potássio/metabolismo , Superóxido Dismutase/metabolismo , Testes de Toxicidade/métodos , Poluentes da Água/toxicidadeRESUMO
BACKGROUND: Thyroid hormones (THs) are known to regulate protein synthesis by acting at the transcriptional level and inducing the expression of many genes. However, little is known about their role in protein expression at the post-transcriptional level, even though studies have shown enhancement of protein synthesis associated with mTOR/p70S6K activation after triiodo-L-thyronine (T3) administration. On the other hand, the effects of TH on translation initiation and polypeptidic chain elongation factors, being essential for activating protein synthesis, have been poorly explored. Therefore, considering that preliminary studies from our laboratory have demonstrated an increase in insulin content in INS-1E cells in response to T3 treatment, the aim of the present study was to investigate if proteins of translational nature might be involved in this effect. METHODS: INS-1E cells were maintained in the presence or absence of T3 (10(-6) or 10(-8) M) for 12 hours. Thereafter, insulin concentration in the culture medium was determined by radioimmunoassay, and the cells were processed for Western blot detection of insulin, eukaryotic initiation factor 2 (eIF2), p-eIF2, eIF5A, EF1A, eIF4E binding protein (4E-BP), p-4E-BP, p70S6K, and p-p70S6K. RESULTS: It was found that, in parallel with increased insulin generation, T3 induced p70S6K phosphorylation and the expression of the translational factors eIF2, eIF5A, and eukaryotic elongation factor 1 alpha (eEF1A). In contrast, total and phosphorylated 4E-BP, as well as total p70S6K and p-eIF2 content, remained unchanged after T3 treatment. CONCLUSIONS: Considering that (i) p70S6K induces S6 phosphorylation of the 40S ribosomal subunit, an essential condition for protein synthesis; (ii) eIF2 is essential for the initiation of messenger RNA translation process; and (iii) eIF5A and eEF1A play a central role in the elongation of the polypeptidic chain during the transcripts decoding, the data presented here lead us to suppose that a part of T3-induced insulin expression in INS-1E cells depends on the protein synthesis activation at the post-transcriptional level, as these proteins of the translational machinery were shown to be regulated by T3.
Assuntos
Fator de Iniciação 2 em Eucariotos/metabolismo , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Fatores de Iniciação de Peptídeos/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Tri-Iodotironina/farmacologia , Animais , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Células Secretoras de Insulina/patologia , Insulinoma/metabolismo , Insulinoma/patologia , Peptídeos e Proteínas de Sinalização Intracelular , Modelos Animais , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Fosfoproteínas/metabolismo , Fosforilação/efeitos dos fármacos , Biossíntese de Proteínas , Ratos , Fator de Iniciação de Tradução Eucariótico 5ARESUMO
Copper sulfate is widely used in aquaculture. Exposure to this compound can be harmful to fish, resulting in oxidative metabolism alterations and gill tissue damage. Pacu, Piaractus mesopotamicus, (wt = 43.4 +/- 3.35 g) were distributed in experimental tanks (n = 10; 180 l) and exposed for 48 h to control (without copper addition), 0.4Cu (0.4 mg l(-1)), 0CupH (without copper addition, pH = 5.0) and 0.4CupH (0.4 mg l(-1), pH = 5.0). In liver and red muscle, the superoxide dismutase (SOD) was responsive to the increases in the aquatic copper. The plasmatic intermediary metabolites and hematological variables in the fish of group 0.4Cu were similar to those of the control group. Conversely, the exposure to 0.4CupH caused an increase in the plasmatic lactate, number of red blood cells (RBC) and hemoglobin (Hb). Plasmatic copper concentration [Cu(p)] increased in group 0.4Cu and 0.4CupH, which is higher in group 0.4CupH, suggests an effect of water pH on the absorbed copper. Exposure to 0.4Cu and 0.4CupH resulted in a reduction in the Na(+)/K(+)-ATPase activity and an increase in metallothionein (MT) in the gills. Exposure to 0CupH caused a decrease in glucose and pyruvate concentrations and an increase in RBC, Hb, and the branchial Na(+)/K(+)-ATPase activity. These responses suggest that the fish triggered mechanisms to revert the blood acidosis, save energy and increase the oxygen uptake. MT was an effective biomarker, responding to copper in different pHs and dissolved oxygen. Combined-factors caused more significant disturbance in the biomarkers than single-factors.