SETAC Europe 35th Annual Meeting

Comparing the Sensitivity of Three Rainbow Trout Cell Lines Through Cytotoxicity and Targeted Gene Expression For Aquatic Testing

Abstract: The International Organization for Standardization recently approved the RTgill-W1 cell line as an in vitro alternative for acute toxicity testing in fish. While RTgill-W1 reflects acute toxicity in rainbow trout (Oncorhynchus mykiss), it represents only one tissue type. Testing with various tissue types can elucidate tissue-specific mechanisms of toxicity that may be overlooked when using a single cell type. In this study, the rainbow trout cell lines RTL-W1 (liver), RTgut-GC (intestine), and RTgill-W1 (gill) were exposed to a range of 3,4-dichloroaniline (DCA) and cadmium (Cd) concentrations. Cytotoxicity levels and effective concentrations for 50% cell viability (EC50) of Cd and DCA were determined using a multi-endpoint viability assay with alamarBlue, CDFA-AM, and neutral red as markers of cell metabolic activity, cell membrane integrity, and lysosomal integrity, respectively. Results indicated Cd EC50 values of 72.5 µM, 92.6 µM, and 234.5 µM for RTgill-W1, RTgut-GC, and RTL-W1 cells, respectively. For 3,4 DCA, EC50 values were 442.8 µM, 622.3 µM, and 653.3 µM for RTgill-W1, RTgut-GC, and RTL-W1 cells, respectively. Across all cell types, RTgill-W1 was the most sensitive to both 3,4 DCA and Cd, with cadmium exhibiting higher toxicity in all cell lines compared to 3,4 DCA. RTL-W1 cells displayed greater resistance to these chemicals, suggesting that, similar to tissues in vivo, this cell line may possess more robust detoxification mechanisms. To test this hypothesis, cells were exposed to non-toxic and toxic concentrations of cadmium (18.5 µM and 185 µM, respectively) and DCA (0.445 µM and 6.67 µM, respectively) for 24 hours. Following exposure, total RNA from each cell type was extracted and reverse-transcribed for quantitative PCR (qPCR) analysis. Metallothionein and Cytochrome P450 1A messenger RNA levels will be measured by qPCR as biomarkers of cadmium and DCA exposure and toxicity. With this approach, we aim to assess variability in the sensitivity of different cell types to chemical toxicity and determine whether differences in sensitivity can be explained by molecular detoxification mechanisms across cell types.

Disclaimer/ Disclosure (optional):

Author(s):

Bennett Upton Oklahoma State University United States This Author Is the Presenter

Justin Scott Oklahoma State University United States

Matteo Minghetti Oklahoma State University United States