Toxicological application of primary hepatocyte cell cultures of Atlantic cod (Gadus morhua)--effects of BNF, PCDD and Cd.

Fish primary hepatocyte cultures are commonly used for toxicological assessment of contaminants. So far no one has described a protocol on how to use Atlantic cod hepatocytes in bioassays. In this work we describe an experiment in which we were able to isolate intact liver cells from mature individuals. Hepatic cytochrome P450 1A (CYP1A) expression in the isolated cells was evaluated with in situ hybridization after intraperitoneal injection with the strong CYP1A inducer ss-naphthoflavone (BNF). Cod hepatocytes were further exposed to 1,2,3,7,8-polychlorinated dibenzo-p-dioxin (PCDD) and cadmium (Cd). Transcriptional responses of 11 genes were quantified (CYP1A, metallothionein (MT), aryl hydrocarbon receptor 2 (AhR2), UDP-glucuronosyltransferase (UGT), glutathione S-transferase (GST), vitellogenin B (VTGB), hypoxia-inducible factor 1 (HIF1), heme oxygenase 1 (HO-1), transferrin, glutathione peroxidase (GPx) and heat shock protein 70 (HSP70)). Immunohistochemisty evaluation clearly showed elevated CYP1A mRNA expression in primary hepatocytes isolated from BNF-exposed fish. The transcriptional results showed that PCDD exposure resulted in a 311-fold up-regulation of CYP1A and Cd a 1.82-fold increase of MT. Unexpectedly, AhR2 and UGT mRNA levels were not significantly up-regulated in PCDD-exposed cod hepatocytes. HO-1 and transferrin showed a dose-dependent transcriptional response to Cd exposure. Cd appears to act as an endocrine-disrupting metal in exposed primary Atlantic cod hepatocytes. This study demonstrates the use of Atlantic cod primary hepatocyte cultures in toxicological research.