Characterization of rainbow trout (Oncorhynchus mykiss) and fathead minnow (Pimephales promelas) cell lines as models to study pulp and paper effluent effects in fish

Abstract

The effects of aquatic toxicants on fish growth and development have been well-documented, in contrast to the changes in gene expression that necessarily precede them. Many of these toxicants are exogenous compounds that possess the ability to mimic steroid hormones or inhibit normal endocrine functions, which are known as endocrine disrupting compounds (EDCs). The Law laboratory previously obtained mRNA expression data indicating that exposure to aquatic effluents from a combined news/kraft pulp and paper mill changed the expression of genes associated with EDC exposure in the livers of fathead minnows (Pimephales promelas). Societal desire to reduce the use of animals in toxicity testing has encouraged the development and use of in vitro systems, including vertebrate cell models. In this study, the gene expression of the rainbow trout (Oncorhynchus mykiss) liver cell line RTL-W1 and the fathead minnow liver cell line FHM-L was examined following effluent exposure. In the RTL-W1 and FHM-L cell lines, 24 h exposure to effluents was sufficient for assessing changes in gene expression, with maximum changes observed between 4 and 6 h after exposure. Both cell lines showed upregulation of the estrogenic and androgenic-related genes, but this upregulation did not follow any consistent pattern in any of the effluent treatments. However, both lines were responsive to toxic compounds in the effluents in a time-dependent fashion, as indicated by the induction of cytochrome P450 1A (CYP1A). The expression of CYP1lA was gradually up-regulated over time, peaking between 4 and 6 h after exposure and declining thereafter. Secondary treatment effectively removed toxic compounds from the effluent, as demonstrated by the decrease in fold induction of CYP1A mRNA levels after 6 h of exposure to combined mill outfall effluent compared to that of cells exposed to untreated and secondary treated kraft effluents This work, along with subsequent correlation analysis of gene expression in cell lines to that in the parent tissue, begins to validate fish cell lines as predictors of changes in aquatic vertebrate health upon exposure to pulp and paper mill effluents and represents a critical step towards the Law lab goal of developing their use as a complement to whole fish life cycle testing that is currently employed in ecotoxicology studies.