Fish are the most frequently used vertebrates in regulatory ecotoxicology. Therefore, thousands of animal tests are carried out every year. Alternatives to reduce or replace fish tests for risk assessment of chemicals and industrial effluents (e.g. OECD236, Fish embryo toxicity test) are of high societal importance. A real animal free test approach are tests with fish cell lines.
- A cell line is a culture of cells in a culture dish, derived from a tissue of origin – e.g. the gills of fish.
- We work with immortal cell lines: they can be cultured indefinitely; no further fish need to be sacrificed.
- As the basic unit of life, and as targets of chemical exposure, we use cells as surrogate for what happens in whole fish.
Prediction of acute fish toxicity:
Several studies have shown that the acute toxicity of chemicals to fish can be reliably predicted with a rainbow trout gill cell line.
We offer the fish gill cell line viability test to predict the acute toxicity of chemicals and effluents to fish.
- See Bols et al., 1994, J. Fish Dieseases 17: 601-611 for basic characterization of this cell lines.
- See Tanneberger et al., 2013, Env. Sci. Technol. 2013, 47: 1110−1119 for the prediction of acute fish toxicity using the RTgill-W1 cell line assay.
Additionally, we offer gene expression analysis as an additional end point.
Induction of CYP1A as marker of biotransformation and toxicity: EROD assay
The biotransformation of lipophilic xenobiotics occurs mainly via the cytochrome P450 enzyme family. The CYP1A subfamily metabolizes PAHs, PCBs, dioxins and furans. These compounds activate the CYP1A enzyme system over the cytosolic aryl hydrogen receptor. The binding strength of the ligand is proportional to the gene expression and correlates with the toxicity.
The induction of CYP1A is measured by the activity of the biotransformation enzyme 7-ethoxyresorufin-O-deethylase (EROD). In presence of NADPH (β-nicotinamide adenine dinucleotide phosphate), the enzyme converts the artificial substrate, 7-ethoxyresorufin, to resorufin, which can be measured via fluorescence spectroscopy. EROD induction in the sample can be compared to a standard, such as TCDD (Dioxin) or Benzo(a)pyrene (Polycyclic Aromatic Hydrocarbon).
We offer the EROD assay with the rainbow trout liver cell line, RTL-W1
- See Lee et al., 1993, Cell Biol. Toxicol. 9: 279-294 for basic characterization of this cell lines.
- See Bols et al., 1999, Ecotoxicol. Environ. Safety 44: 118-128 and Schirmer et al. 2004, Toxicol. 205: 211-221 for assessing CYP1A induction in RTL-W1 cells of individual chemicals and water samples, respectively.