Sidika Sakalli: Usingthe EROD assay to determinetheAhRmediated dioxin-likeactivityofenvironmentalsamples
Dioxin-likecompounds are a classofenvironmentalcontaminantsthatpotentiallycanbefound in water and sediment atcontaminatedsites. These compounds are characterized by theirability to activatethe aryl hydrocarbon receptor (AhR)-signaltransductionpathway.
Yangtze River is the longest river in Asia and the third longest river in the world. It has important place in theculture and economyofChina. The Yangtze River basin
is an elemental source for drinking water for millions of people and plays a significant role in freshwater fishery of the country however, river is suffering from industrial
and domestic pollution. Organic pollutants, particularly aryl hydrocarbon receptor (AhR) agonists, appeared to play a crucial role in the Yangtze River.
Cytochrome P450 (CYP) is a major enzyme family for the metabolism of lipophilic xenobiotics that plays a central role in detoxification mechanisms in organisms. EROD assay is one of the most commonly used methods for determination cytochrome P450 activity and applied in many organisms. The EROD activity can be used in in vitro
to determine the AhR mediated dioxin-like activity of environmental samples like water and sediment. Activityof EROD describestherateofthe CYP1A mediateddeethylation
ofthesubstrate 7-ethoxyresorufin (7-ER) to formtheproductresorufin. Thecatalyticactivitytowardsthissubstrateisanindicationoftheamountofenzymepresent
and ismeasured as theconcentrationofresorufinproduced per mg protein per minute (mol/mg/min).
In this study, sediment extracts from Yangtze River will be tested in vitro with the EROD induction assay using fish cell line (RTL-W1, rainbowtrout liverfibroblasts) for AhR-mediated dioxin-like activity. The evaluation of sample’s toxicity will be carried out
in a comparison to the values of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), thestrongestknowninducer. To avoid cytotoxic effects in the EROD assay, the neutral red retention assay (NR) will be performed beforehand to assess the initial concentrations
of the sediment extracts set by the NR50 value. Alloftheexperimentswillbeperformed in independent triplicates. In short, during this experiment, students will gain the knowledge about basic cell culture, NR toxicity test and EROD assay as well as evaluating the data.