Application of the rainbow trout derived intestinal cell line (RTgutGC) for ecotoxicological studies: molecular and cellular responses following exposure to copper

Abstract

There is an acknowledged need for in vitro fish intestinal model to help understand dietary exposure to chemicals in the aquatic environment. The presence and use of such models is however largely restrictive due to technical difficulties in the culturing of enterocytes in general and the availability of appropriate established cell lines in particular. In this study, the rainbow trout (Oncorhynchus mykiss) intestinal derived cell line (RTgutGC) was used as a surrogate for the “gut sac” method. To facilitate comparison, RTgutGC cells were grown as monolayers (double-seeded) on permeable Transwell supports leading to a two-compartment intestinal model consisting of polarised epithelium. This two-compartment model divides the system into an upper apical (lumen) and a lower basolateral (portal blood) compartment. In our studies, these cells stained weakly for mucosubstances, expressed the tight junction protein ZO-1 in addition to E-cadherin and revealed the presence of polarised epithelium in addition to microvilli protrusions. The cells also revealed a comparable transepithelial electrical resistance (TEER) to the in vivo situation. Importantly, the cell line tolerated apical saline (1:1 ratio) thus mimicking the intact organ to allow assessment of uptake of compounds across the intestine. Following an exposure over 72 h, our study demonstrated that the RTgutGC cell line under sub-lethal concentrations of copper sulphate (Cu) and modified saline solutions demonstrated uptake of the metal with saturation levels comparable to short term ex situ gut sac preparations. Gene expression analysis revealed no significant influence of pH or time on mRNA expression levels of key stress related genes (i.e. CYP3A, GST, mtA, Pgp and SOD) in the Transwell model. However, significant positive correlations were found between all genes investigated suggesting a co-operative relationship amongst the genes studied. When the outlined characteristics of the cell line are combined with the division of compartments, the RTgutGC double seeded model represents a potential animal replacement model for ecotoxicological studies. Overall, this model could be used to study the effects and predict aquatic gastrointestinal permeability of metals and other environmentally relevant contaminants in a cost effective and high throughput manner.

Assessing the impact of benzo[ a ]pyrene with the in vitro fish gut model: An integrated approach for eco-genotoxicological studies

“…Later work demonstrated trans-epithelial electrical resistance (TEER) comparable to values measured in vivo; apical localisation of tight junction proteins; and the induction of Na + /K + -ATPase mRNA following exposure to saltwater buffer [34]. Recent work published by our laboratory has demonstrated the direct comparability of this model to existing ex situ gut sac preparation methods when exposed to copper, a ubiquitous environmental contaminant [35]. However, the suitability of this cell line for toxicological assessment and as an in vitro animal replacement tool needs to be further examined using more general environmental contaminants, especially organic contaminants which require metabolic activation, to establish its sensitivity and comparability before incorporation into a regulatory framework.…”

“…Prior studies, including our own, have established the comparability of this intestinal in vitro model to native tissue cultured as monolayers on Transwell inserts [34,35]. In this study, we focused on the metabolic activity of the system following exposure to a known carcinogen, B[a]P, as mentioned above.…”

“…In addition, genotoxic response was determined using the alkaline single cell electrophoresis or comet assay. As this study was carried out to ascertain the potential of this method to act as a screening tool for environmental contaminants, these experiments were not carried out on Transwell inserts as done in our earlier studies [35] and are instead representative of cells grown as monolayers on 12-well plates.…”

“…The primary objective of this study was to test whether the RTgutGC cell line can be used as robust environmental monitoring tool. As the bottleneck in in vitro toxicological studies is reported by ECVAM to be functional metabolism [42], we focussed on characterising metabolism and evaluating genotoxic damage within this system, building up from our previous study, which included thorough morphological and histological characterisation of this cell line [35]. In this work, uptake of a known genotoxicant and carcinogen, B[a]P), was verified using Gas Chromatography-Mass Spectrometry (GC-MS) quantification of the removal of the parent compound and immunofluorescence identification in the cells.…”

“…Furthermore, external pH is known to partly determine the cytoplasmic or intracellular pH and can have a subsequent effect (beneficial or detrimental) on enzymatic activity, reaction rate, protein stability, and other biological cascade processes, across numerous taxonomic groups. Following initial characterisation of the activity, therefore, B[a]P exposures were also carried out using three exposure solutions with varying pH (L-15 medium pH 7.5 and a L-15: saline exposure of pH 7.7 and 7.4) as detailed in our earlier studies [35]. Using B[a]P as model environmental contaminant, we probed the ability of this cell line for active xenobiotic metabolism, the resulting genotoxicity, and its comparability to information available in the literature.…”