Zebrafish (Danio rerio) early-life stage exposure to triclosan

: immediate and delayed effects

Student thesis: Doc typesDoctor of Sciences

Abstract

Personal care products have been detected in the aquatic environment across the world. However, as emerging contaminants, there is still a lack of knowledge about their ecological impacts, environmental fate and effects. In this context, there is a pressing need to understand the underlying molecular mechanisms of their toxicity to be able to predict the adverse effects on the survival and the fitness. Triclosan (TCS) is a broad spectrum antibacterial agent widely used in personal care products and present in most aquatic ecosystems. The main objective of the present study was to characterize the effects of TCS exposure during early life stages (ELS), using zebrafish as model species. As a first step, we aimed to characterize the immediate effects of TCS-induced toxicity. To fulfil this goal, we first investigated the occurrence of triclosan acclimation using time-to-death approach. As a result, larvae exposed to 50 µg/L of TCS demonstrated higher sensitivity (delayed hatching and decreased survival), whereas fish exposed to the highest concentration of TCS (100 µg/L) presented a similar phenotype than controls suggesting that acclimation process has been triggered at this concentration.
Secondly, we aimed to understand the biological mechanisms underlying the responses at these two sublethal concentrations in zebrafish ELS. As a first step, without a priori proteomic analysis was performed together with the monitoring of the response of several enzymes involved in oxidative stress (GST, GPx, GR) and neurotoxicity (AChE). Our integrative approach allowed the identification of 1° a complex non-monotonic dose-related effect of TCS ; 2° proteins altered by TCS exposure, either belonging to the common cellular response such as cytoskeletal and stress response proteins, or being more specific to the chemical as for example proteins involved in vision and neuronal development ; 3° impairment in glutathione metabolism and increased AChE activity following TCS exposure, suggesting that the major toxicity mechanisms were oxidative stress and neurotoxicity. As a second step, to supplement proteomics and enzymatic data, the direct effects of TCS on DNA methylation (one of the main epigenetic mechanisms) and their consequences on gene expression were undertaken. To achieve this part we used a recent and promising high-throughput techniques: reduced representative bisulfite sequencing (RRBS) which is, up to know, the only approach allowing genome-wide DNA methylation study at a base pair resolution for multiple samples. Our results demonstrated that TCS impacts DNA methylation levels of at least 171 differentially methylated fragments (DMFs). Among them, pathways involved in TGF-beta signaling were enriched in larvae exposed to 50 µg/L, while de novo pyrimidine biosynthesis functions were overrepresented in fish exposed to 100 µg/L of TCS. In addition, we revealed a positive correlation between DNA methylation patterns and gene expression levels for introns together with significant expression alterations of genes involved in dendrite development (grip1), differentiation of serotonergic neurons (irx1a) and neurotransmitter (ACh) transport (slc18a3b), as well as a transcriptional factor (gli2b) involved in cell proliferation and expressed mainly in the neural plate and the central nervous.
Lastly, we aimed to characterize the delayed effects of TCS-induced toxicity. In this context, fertilized eggs were exposed until 5 days post-fertilization (dpf) to 50 and 100 µg/L of TCS and larvae were then subsequently transferred into clean water and raised until 30 dpf. Delayed mortality as well as growth impairment and delayed development were observed during the clean water period at both concentrations.
In conclusion, our study provides insights into the molecular mechanisms underlying TCS-induced toxicity in zebrafish early-life stages. We demonstrated for the first time that TCS exposure altered DNA methylation and has the potential to lead to severe delayed effects. Our results support the importance to consider TCS as an important candidate for prioritisation in the Water Framework Directive and proposed a new integrative approach to gain ecotoxicological data for ERA.
Date of Award2 Feb 2018
Original languageEnglish
Awarding Institution
  • University of Namur
SupervisorFrédéric Silvestre (Supervisor), Carolina Di Paolo (Jury), Ansuman Chatterjee (Jury), Krishna Das (Jury) & Patrick Kestemont (Jury)

Keywords

  • ecotoxicology
  • Danio rerio
  • Epigenetics
  • Triclosan
  • toxicity
  • RRBS
  • Proteomics

Attachment to an Research Institute in UNAMUR

  • ILEE

Cite this

Zebrafish (Danio rerio) early-life stage exposure to triclosan: immediate and delayed effects
Falisse, É. (Author). 2 Feb 2018

Student thesis: Doc typesDoctor of Sciences