Efeitos toxicológicos de antinflamatórios não esteroidais em peixes de água doce

Abstract

Resumo: Farmacos antinflamatorios nao-esteroidais (AINEs) sao amplamente empregados na medicina humana e veterinaria e apresentam potencial de contaminar agua e sedimentos atraves de entradas de estacoes de tratamento de esgoto. No presente estudo, os efeitos de alguns AINEs foram analisados em peixes nativos (Hoplias malabaricus e Rhamdia quelen). A toxicidade do paracetamol, do diclofenaco e do ibuprofeno foi avaliada em cultura primaria da linhagem macrofagica de rim anterior de H. malabaricus. Seus efeitos na viabilidade celular, producao de oxido nitrico (NO) induzida por lipopolissacarideo (LPS) e genotoxicidade foram analisados. Na cultura celular primaria, para padronizacao, a analise por citometria CD11b+ mostrou 71,5 % de celulas progenitoras, 19,5 % de macrofagos e 9,0 % de monocitos. A producao de oxido nitrico induzida por LPS por essas celulas foi bloqueada apos tratamento com dexametasona e L-NMMA. Apos 24h de exposicao das celulas ao diclofenaco (0,2- 200 ng/mL), paracetamol (0,025-250 ng/mL) e ibuprofeno (10-1000 ng/mL), houve reducao na producao basal de NO e inibicao da producao de NO induzida por LPS em todas as concentracoes testadas. A genotoxicidade ocorreu na maior concentracao de diclofenaco, nas concentracoes intermediarias de paracetamol e tambem com ibuprofeno. A toxicidade trofica do diclofenaco em H. malabaricus foi avaliada, sendo os peixes alimentados duas vezes por semana com Astyanax sp. previamente submetido a inoculacao intraperitoneal (IP) com diclofenaco (0; 0,2; 2,0 ou 20,0 ?g/Kg), totalizando 12 doses. A metade dos peixes recebeu carragenina IP a 1 mg/Kg e depois de 4 horas, os mesmos foram anestesiados e eutanasiados para estimativa da migracao celular. Nos outros peixes (sem carragenina), os parametros hematologicos, a producao de NO basal e apos estimulacao por LPS em rim anterior, o indice hepatossomatico (HSI) e a analise hepatica das atividades de superoxido dismutase (SOD), glutationa peroxidase (GPx), glutationa S-transferase (GST), etoxiresorufina-O-deetilase (EROD) e catalase (CAT) foram determinadas. A glutationa reduzida (GSH) e a lipoperoxidacao (LPO) foram tambem avaliados. Houve aumento na contagem eritrocitaria e no hematocrito na menor dose de diclofenaco. A hemoglobina diminuiu na maior dose. A contagem de trombocitos aumentou em todos os grupos expostos ao diclofenaco e a contagem de leucocitos sanguineos totais diminuiu seguindo a reducao de neutrofilos. Os monocitos reduziram na maior dose. O numero de macrofagos peritoneais residentes nao diferiu entre os grupos, mas a migracao celular reduziu apos a administracao de carragenina, com uma significante diminuicao na migracao dos polimorfonucleares. A sintese basal de NO das culturas celulares de rim anterior dos animais tratados com diclofenaco foi significantemente menor nas celulas dos grupos de 2 e 20 ?g/Kg. A producao de NO estimulada por LPS decresceu em todos os grupos tratados. No figado, o diclofenaco causou estresse oxidativo com aumento de LPO e de atividade da GPx. Em contraste, a atividade da GST reduziu. Os efeitos do diclofenaco em componentes do sistema imune tambem foram avaliados apos exposicao hidrica de R. quelen ao diclofenaco a 0,2, 2,0 e 20,0 ?g/L durante 14 dias. Os peixes foram anestesiados, o sangue retirado e apos eutanasia o rim anterior foi coletado. As proteinas do plasma e rim anterior envolvidas na producao de NO, migracao celular e ativacao do sistema complemento foram analisadas por cromatografia liquida acoplada a espectrometria de massas do tipo tandem. No plasma foi observada a0 inibicao da expressao de receptor toll like 2 (Tlr2), fosfolipase C (Plc ), quinase quinase quinase 3 (Mekk), 1-fosfatidilinositol 3-quinase (Pi3k), proteina ativadora-1 (Ap-1), fator nuclear de polipeptideo kappa light (Nf-kb) e proteina NO sintase induzivel (iNOS). No rim anterior, a expressao de Tlr2, Plc , Mekk, Pi3k, Ap1 e Nf-kb tambem foi significantemente inibida. Varias proteinas envolvidas na migracao celular foram detectadas no plasma. Nos peixes machos, a expressao da proteina receptora de quimiocina 4 (Cxcr4), Integrina ?1 (It?1), Radixina (Rdx) e Metalopeptidase de matriz (Mmp)-17 foi inibida. Nos peixes femeas, a expressao de Cxcr4, Itga1, Rdx, Mmp17 e Mmp1 reduziu. No presente estudo, houve modificacao na expressao da proteina componente do complemento 3 (C3), do fator B do complemento (Cfb) e da serina peptidase 1 associada a manana (Masp1), bem como de C1q e do componente do complemento 7 (C7). Adicionalmente, MHC1 no plasma diminuiu significativamente. Em sintese, os NSAIDs estudados influenciaram a producao de NO e causaram danos ao DNA nas celulas monociticas oriundas de H. malabaricus. Os peixes apresentaram modificacoes hematologicas e bioquimicas quando submetidos a exposicao ao diclofenaco. A inibicao da expressao de muitas proteinas envolvidas na sintese de NO, migracao celular e ativacao do sistema complemento foi observada nos peixes estudados, o que pode comprometer os mecanismos de defesa imune inata destes animais. Palavras-chave: Farmacos. Cultura celular. Imunotoxicidade. Genotoxicidade. Macrofago. Estresse oxidativo. Migracao celular. Parametros hematologicos.

Abstract: Non-steroidal anti-inflammatory drugs (NSAIDs) are largely employed in human and veterinary medicine and have the potential to contaminate water and sediments via inputs from sewage treatment plants. Their impacts on humans and ecosystems are emerging issues in environmental health. In the present study, the effects of some NSAIDs were analyzed in native fish species (Hoplias malabaricus and Rhamdia quelen). The toxicity of acetaminophen, diclofenac and ibuprofen was evaluated on primary culture of monocytic lineage of anterior kidney from H. malabaricus. Their effects in cell viability, lipopolysaccharide (LPS)-induced nitric oxid (NO) production and genotoxicity were analyzed. In primary culture cell, cytometry analysis CD11b+ cells showed 71.5 % of stem cells, 19.5 % of macrophages and 9.0 % of monocytes. Cell viability was lower in the Ficoll compared to Percoll separation. LPS-induced NO production by these cells was blocked after treatment with dexamethasone and LNMMA. After 24 h of cell exposure to diclofenac (0.2-200 ng/mL), acetaminophen (0.025-250 ng/mL) and ibuprofen (10-1000 ng/mL), there was a reduction in basal NO production and an inhibition of LPS-induced NO production at all tested concentrations. Genotoxicity occurred at the highest concentration of diclofenac, at the intermediary concentrations of acetaminophen and also with ibuprofen. The toxicity of diclofenac was also evaluated in H. malabaricus after trophic exposure, where fish were fed twice every week with Astyanax sp. previously submitted to intraperitoneal inoculation (IP) with diclofenac (0; 0.2; 2.0 or 20.0 ?g/Kg), totaling 12 doses. In sequence, half of fish received 1 mg/Kg of carrageenan IP and after 4 hours, they were anesthetized and euthanized for cell migration estimation. In the other fish (without carrageenan), the hematological parameters, NO basal production and after LPS-stimulate in head kidney, hepatosomatic index (HSI) and liver biochemical analysis, such as activities of superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione Stransferase (GST), ethoxyresorufin-O-deethylase (EROD) and catalase (CAT) were measured. Reduced glutathione (GSH) and lipoperoxidation (LPO) were also determined. The results of trophic exposure of H. malabaricus showed increases in red blood cells count and in the hematocrit at the low dose of diclofenac. In contrast, the hemoglobin reduced at the highest dose. Thrombocyte count increased in all groups exposed to diclofenac and the total blood leukocyte counts decreased following the neutrophil's reduction. Monocytes decreased at the highest dose. The number of resident peritoneal cells did not differ among the groups, but the cell migration reduced after carrageenan administration, with a significant decrease in the migration of polymorphonuclear cells. The basal NO synthesis of anterior kidney cell cultures from diclofenac-treated animals was significantly lower in the cells from the groups 2 and 20 ?g/Kg. The LPS-stimulated NO production reduced in all of the diclofenac-treated groups. Diclofenac also reduced HSI at the 0.2 ?g/Kg. In liver, diclofenac caused oxidative stress with increased LPO and GPx activity. In contrast, GST activity decreased. The effects of diclofenac in components of the immune system were also evaluated after hydric exposure of R. quelen to diclofenac at 0.2, 2.0 and 20.0 ?g/L during 14 days. After the exposure, fish were anesthetized and blood was taken from caudal vein. After this, fish were euthanized and the anterior kidney was collected. Plasma and kidney proteins involved in NO production, cell migration and complement system activation were analyzed using liquid chromatography tandem mass spectrometry in a shotgun proteomic approach. Results obtained after hydric exposure of R. quelen to diclofenac for plasma samples showed significant inhibition in the expression of toll like receptor 2 (Tlr2), phospholipase C (Plc ), kinase kinase kinase 3 (Mekk), 1-phosphatidylinositol 3-kinase (Pi3k), activator protein-1 (Ap-1), nuclear factor of Kappa light polypeptide (Nf-kb), and the NO synthase inducible protein (iNOS). In the head kidney, the expression of Tlr2, Plc , Mekk, PI3K, Ap1 and Nf-kb was also significantly inhibited. Various proteins involved in cell migration were detected in the plasma. In male fish, the expression of Chemokine receptor 4 protein (Cxcr4), Integrin ?1 (It?1), Radixin (Rdx) and Matrix Metallopeptidase (Mmp)-17 was inhibited. In female fish, the expression of Cxcr4, Itga1, Rdx, Mmp17 and Mmp1 decreased. In the present study, the expression of complement component 3 protein (C3), complement factor B (Cfb) and mannan-binding lectin serine peptidase 1 (Masp1) changed as well as C1q and complement component 7 (C7). Additionally, MHC1 in plasma significantly decreased. In summary, the studied NSAIDs influenced NO production and caused DNA damage in monocytic cells from H. malabaricus. Fish presented hematological and biochemical changes when submitted to diclofenac exposure. The expression inhibition of many proteins involved in NO synthesis, cell migration and activation of the complement system was observed in the studied fish, which may compromise innate immune defense mechanisms of these animals. Keywords: Pharmaceuticals. Cell culture. Immunotoxicity. Genotoxicity. Macrophage. Oxidative stress. Cell migration. Hematological parameters.