Zebrafish as a model for toxicological studies: is there a role for Paraoxonase?
Year: 2015
Type: dissertation
Abstract: Human PON1 enzyme is a multifaceted biomolecule with antioxidant, anti-atherogenic and detoxifying properties. In that way, it is considered one of the main human body endogenous free-radical scavenging system. PON1 actions are related with the three activities assigned to it: paraoxonase (POase), arylesterase (AREase) and lactonase (LACase). Due to the easy measurement of PON1 activities and to its association to several pathologies linked to oxidative stress, PON1 represents a potential biomarker of disease, in particular for renal disease. The general goal of this exploratory and pioneer study was to explore the PON1 activities in zebrafish animal model. Zebrafish is an emergent animal model that is becoming to be largely used as a model in human disease and toxicological studies. Zebrafish presents several characteristics that turns it in an extraordinary, even a unique, model for the toxicological study. Among these characteristics, we can identify the reduced size, the transparency of the embryo, the rapid organogenesis, the low cost of maintenance and the complete sequencing of its genome, where PON1 gene is included. The knowledge on the homology between the zebrafish and human at genetic, molecular and physiological levels is increasing. A good correlation between zebrafish and man has been found for organ toxicity as well. In the present study, the homology between human (HGNC:9204) and zebrafish (Zgc:91887) enzymes was firstly stablished, supporting that PON1 is highly preserved on the zebrafish, presenting 49% of similitude with the man. All experiments involving zebrafish were previously approved by the Ethical Committee at the Instituto Gulbenkian de Ciência (IGC). With the objective of determine if human PON1 activities were present on zebrafish, in the first part of this study, the quantification of the activities POase, AREase and LACase activities were carried out in the whole animal extract, using the same spectrophotometric methods which were validated for human plasma samples. Both AREase and LACase activities were determined while POase was not quantifiable. As the homogenate of whole animal was used, the contribution of other enzymes should not be excluded. For instance, the esterases, as the cholinesterases and the carboxylesterase, due to their capacity of hydrolyze the phenyl acetate, might increase the AREase activity. Also, the other paraoxonases of PON family (PON2 and PON3) may contribute for the AREase and LACase activities, when whole animal extract is used, comparatively to plasma. Aiming to exclude the contribution of the esterases, other than PON enzymes, and as anti-PON1 for the zebrafish was not available, a pharmacological approach was followed by using the paraoxon as an inhibitor of cholinesterase and carboxylesterase enzymes. A significant diminution of the AREase activity was observed when the zebrafish larvae were exposed to the xenobiotic. In attempting to clarify the contribution of PON1 for whole animal AREase and LACase activities, zebrafish larvae were exposed to acetylsalicylic acid, which is an inducer of PON1. No changes in AREase or LACase activities changes were observed upon exposition to the PON1 inducer. There are many factors that might influence the PON1 status. However, what turns out to be more dominant is age. In that regard, the AREase, LACase and POAse activities during the zebrafish development were explored in the second part of this study. The PON1 activities were determined in zebrafish larvae from day 1 to day 7 post fertilization (dpf). The contribution of chorion – structure that evolves zebrafish embryo – for the AREase and LACase activities was also tested. At 24 hours post fertilization, the zebrafish embryos are evolved by the chorion and within 48 hours post fertilization they may or may not present that structure. The comparison of AREase and LACase activities between groups with and without chorion, at 24 and 48 hours post fertilization disclosed that only AREase activity is influenced by the presence of chorion for embryos with 24 hours post fertilization. These results suggest that proteins with AREase activity integrate the chorion. The evaluation of AREase and LACase activities until 7 dpf pointed to an increase of both activities during the zebrafish development, in particular for the AREase activity. The zebrafish is being postulated as a good model for nephrotoxicity studies due to the similarity of its pronephro to the human kidney. As PON1 is being studied as a potential biomarker of renal disease, the third objective stablish was to evaluate the relation between PON1 activities and drugs associated to nephrotoxicity. Tenofovir disoproxil fumarate (TDF) and paracetamol (PCM), as drugs with proved association to nephrotoxicity on zebrafish and man, were tested in two concentrations. For both drugs, a decrease in the AREase activity was observed. The results herein presented show, for the first time, that the zebrafish presents AREase and LACase activities, although they are not to exclusively assign to PON1 enzyme. The POAse activity might not be present in this model. The LACase activity, and in particular the AREase activity, increase during zebrafish development. Moreover, it was for the first time showed that proteins with AREase activity are presented in zebrafish chorion. The characterization of this enzyme and other important enzymes for xenobiotics and drug detoxification is an important step for the proposal of zebrafish as a model for translational research and to disclose its applicability to pre-clinical studies. In this regard, the present work, give new insights on the three activities involved in endogenous and exogenous compounds metabolism.
Author: Lara Andreia Pires Afonso
Language: English
Subfield: Clinical Biochemistry
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W2273412646