Short communication: Evaluation of mitochondrial surface area of selected metals and pesticides in RTL-W1 cells: A high-content imaging approach

Abstract

In recent years, there has been increasing interest in utilizing fish cell culture in vitro assays, as alternatives to whole fish assays, for assessing the toxicity of aquatic pollutants. The fish cell line RTL-W1, derived from rainbow trout (Oncorhynchus mykiss) liver, was shown to retain several tissue specific features and to respond to chemical insults similarly to fish in vivo. In our study, we investigated the toxicity of two metals silver and cadmium and two pesticides azoxystrobin and paraquat using a cytotoxicity assay that measures simultaneously cell metabolic activity and cell membrane integrity. Moreover, we developed a novel ‘high-content imaging’ approach to evaluate if mitochondria's surface area is a sensitive and specific indicator of mitochondria toxicity. Initially, the cytotoxicity assay was used to determine the chemicals' effective concentrations (EC₅₀). Subsequently, we assessed the mitochondria surface area at different toxicity level (i.e., EC₅₀, EC₂₅ and EC₁₀) to compare the sensitivity and specificity of this method. The EC_50s measured by cell metabolic activity, for silver, cadmium, azoxystrobin and paraquat were 0.71, 29.05, 2.34 and 1260 μM, respectively. Mitochondria surface area was reduced by all chemicals at the EC₅₀, and by silver and azoxystrobin at the EC₁₀ and EC₂₅; indicating that the latter chemicals affect mitochondria more specifically. In conclusion, our study demonstrated that mitochondrial surface area serves as a sensitive marker for chemicals inducing mitochondria toxicity in fish liver cells. Additionally, assays using RTL-W1 cells proved to be effective for detecting the hepatic cytotoxicity of environmental contaminants.