Non destructive in vivo measurement of ethoxyresorufin biotransformation by zebrafish prolarva: Development and application

Abstract

Biotransformation enzymatic activities, such as the cytochrome P450 one, have been used as biomarkers for pollution assessment for a long time. Nevertheless, such biochemical tools are destructive processes, because they are performed on fish liver or total larvae homogenates. Moreover, the adaptation of this bioassay to some fish larvae, like Danio rerio ones, is ineffective because it needs a lot of organisms. We thus developed an original, nondestructive method to detect the induction of a biotransformation activity in the prolarva of the fish, Danio rerio, exposed to chemicals. This methodology is based on the assessment of EROD activity, by measurement in the culture medium of the fluorescence increase due to the excretion of resorufin by fish during an ethoxyresorufin exposure. After exposure of fish embryos to known inducers (BaP and beta-naphtoflavone), the prolarvae were exposed to the substrate (ethoxyresorufin), and the kinetic of the fluorescence increase was measured. A dose-effect relationship was observed, with a three to five fold increase of EROD basal activity. This methodology also allowed us to compare between EROD activity induction by different environmental samples. The proposed methodology thus allows to perform a simple, sensitive, and reproducible microbiotest for the detection of sublethal concentrations of AhR chemical inducers in environmental samples.

Influence of sediment composition on PAH toxicity using zebrafish (Danio rerio) and Japanese medaka (Oryzias latipes) embryo-larval assays

“…The EROD activity modulation by BaP is in general agreement with AhR agonist properties of this compound. The ability of BaP to induce EROD activity (indicator of phase I biotransformation activities) through the AhR2 pathway has been established in marine fish (Au et al 2004;Barron et al 2004) and freshwater fish (Bols et al 1999;Costa et al 2011;Incardona et al 2011;Noury et al 2006). Regarding the results of Fluo exposure, no significant induction of EROD activity was observed compared to the control treatment.…”

GFP Transgenic Medaka (Oryzias latipes) under the Inducible cyp1a Promoter Provide a Sensitive and Convenient Biological Indicator for the Presence of TCDD and Other Persistent Organic Chemicals

“…Thirdly, external parameters such as temperature, pH, etc. can also influence the outcome of EROD assay [7], [25], [28]. Transgenic fish assay can avoid these problems as it monitors TCDD more directly from the upstream transcriptional activity, which is controlled by TCDD-AhR interaction.…”

Zebrafish: A Marvel of High-Throughput Biology for 21st Century Toxicology

“…For example, transgenic zebrafish expressing green fluorescent protein (GFP) regulated by the cytochrome P450 1A (cyp1a) promoter can be used as a rapid, non-destructive in vivo screen for induction of phase I metabolism (91, 92). Other rapid in vivo non-destructive techniques that are amenable to HT screening include the ethoxyresorufin-O-deethylase (EROD) assay, which can be visualized and quantified by fluorescence microscopy (93). Using this technique, many chemicals can be screened for cytochrome P450-specific activity.…”