Developmental, behavioral, biochemical and epigenetic effect of pharmaceuticals in larvae and embryos of Danio rerio

• Autor(a) principal: Nogueira, Ana Filipa Ferreira
• Data de Publicação: 2017
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10773/22024
• topic: Medicamentos - Toxicologia ambiental; Larvas; Marcadores bioquímicos; Epigenética

The scientific community has been concerned for several years about the presence of pharmaceuticals in the wild, since these compounds may have deleterious or unpredictable effects on living organisms. The scientific knowledge on the fate and effect of drug residues in the environment has evolved considerably in recent years, revealing that drug residues do indeed pose a threat to the aquatic ecosystem. Two examples of widely used pharmaceuticals that are present in the environment are paracetamol (acetaminophen) and ciprofloxacin, Paracetamol is a drug with analgesic properties, used for the temporary relief of mild to moderate pain associated with common colds, and in the reduction of fever. Ciprofloxacin is an antibiotic from the chemical class of the fluoroquinolones which presents a broad antibacterial spectrum. Firstly we decided the range of concentrations for each drug. In the case of paracetamol, the tested concentrations were 0.005 mg/L, 0.025 mg/L, 0.125 mg/L, 0.625 mg/L, and 3.125 mg/L, plus a control treatment with water from the facility system. And for ciprofloxacin, the tested concentrations were 0.005 μg/L, 0.013 μg/L, 0.031 μg/L, 0.078 μg/L, 0.195 μg/L, and 0.488 μg/L. These concentrations were chosen as for their environmental relevance since they are close to the real concentrations of these pharmaceuticals found in surface waters and effluents. This work aims to characterize the effects of both drugs in zebrafish embryos and larvae, not only in developmental and behaviour parameters, but also using a biomarker-based approach, namely by quantifying the activities of catalase (CAT), glutathione-S-transferase (GST’s), cholinesterases (ChE’s), glutathione peroxidase (GPx), and determining the TBARS level. Exposure to paracetamol caused an increase in the percentage of organisms with morphological deformations, but no morphological deformations were observed in organisms exposed to ciprofloxacin. Concerning larvae behavioural tests, significant differences were observed for larvae exposed to paracetamol but not for larvae exposed to ciprofloxacin. In the biomarker determination, both drugs caused a statistically significant increase in ChE activity; in CAT only ciprofloxacin caused a significant difference, a decrease in its activity. Paracetamol induced an increase in activity for total GPx and in GST’s. The TBARS levels significantly increased in the exposure to paracetamol, but they significantly decreased in the exposure to ciprofloxacin. In this work, we also prepared an immunohistochemical detection of global methylation, which allowed to observe that, in embryos, the highest concentration of paracetamol caused a slight increase in the intensity of the 5- mdC signal, which can be translated into a slight increase in the DNA methylation. On the other hand, the embryos exposed to ciprofloxacin did not appear to have any difference from control. The comparison of the here-obtained results for the two different drugs allows observing that zebrafish larvae were more sensitive to the exposure to paracetamol, being the most sensitive methodology the biomarker determination. We can also conclude that oxidative stress occurred as a consequence of the exposure to both pharmaceuticals, being more evident in the case of paracetamol. We also showed that the oxidative stress created by the two pharmaceuticals may be the cause of all the other observations.