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

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

The effect of a glyphosate-based herbicide on acetylcholinesterase (AChE) activity, oxidative stress, and antioxidant status in freshwater amphipod: Gammarus pulex (Crustacean)

This study had determined the effect of glyphosate-based herbicide (GBH) on acetylcholinesterase (AChE) enzyme activity, oxidative stress, and antioxidant status in Gammarus pulex. Firstly, the 96-h LC50 value of glyphosate on G. pulex was determined and calculated as 403 μg/L. Subsequently, the organisms were exposed to sub-lethal concentrations (10, 20, and 40 μg/L) of the determined GHB for 24 and 96 h. The samples were taken from control and GBH-treated groups at 24 and 96 h of study and analysed to determine the malondialdehyde (MDA) and reduced glutathione (GSH) levels, the AChE, superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) enzyme activities. In the G. pulex exposed to GBH for 24 and 96 h, the MDA level increased significantly (p < 0.05). The GSH level, the AChE, the CAT, and the GPx activities decreased compared with the control group (p < 0.05). G. pulex exposure to GBH for 24 h showed a temporary reduction in the SOD. GBH exposure led to oxidative stress in the G. pulex as well as affected the cholinergic system of the organism. These results indicated that the parameters measured may be important indicators of herbicide contamination in G. pulex