Detection of reactive oxygen/nitrogen species and determination of cellular effects in rainbow trout (Oncorhynchus mykiss) hepatocytes

Abstract

ABSTRACT

Cell-based assays are useful and effective for biomonitoring and toxicity screening purposes, and there is a need for fast, routine and simple screening assays. An assay to detect the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) and oxidative DNA damage was optimised for use in primary cultures of isolated rainbow trout (Oncorhynchus mykiss) hepatocytes. The fluorescent probes 2 ,7 -dichlorofluorescin diacetate (DCF), 5-(& 6)-chloro-methyl-2' 7'-dichlorodihydro-fluorescein diacetate (CM-DCF) and Dihydrorhodamine 123 (DHR123) were used to detect ROS/RNS production in cells exposed to the model substances H2O2, Fe2+, 2,2 -azobios(2-amidino)propane (AAPH) and 3-morpholinosydnonimine hydrochloride (Sin-1), metals (Cu2+, Cd2+), organic compounds (Paraquat and Benzo[a]pyrene) and various environmental samples. Acute toxicity caused by the same substances and extracts was examined using the assays for metabolic inhibition (Alamar blue) and membrane integrity (CFDA-AM) as well as determination of oxidative DNA damage by an alkaline unwinding method (Fast Micromethod). Significant increases in ROS production were seen after 30 minutes exposure to the most potent model substances and xenobiotics. No ROS production was observed after 24 hours of exposure to these chemicals. Of the tested environmental extracts, only one of the samples caused significant ROS/RNS production after 2 hour of exposure. The Fast Micromethod detected a significant increase in oxidative DNA damage after 2 hours of exposure to H2O2 and Fe2+.Cytotoxicity in terms of metabolic inhibition and loss of cellular integrity was only observed after exposure to some of the test compounds and samples tested. The ROS-detection assay and the fast micromethod provide useful methods for the prediction of toxic potency of xenobiotics and environmental samples. However, further work is needed improve the sensitivity of both methods for future use in environmental monitoring and chemical screening.