Biomarker protein expression in primary cultures of salmon (Salmo salar L.) hepatocytes exposed to environmental pollutants

Abstract

Primary cultures of salmon (Salmo salar L.) hepatocytes were analysed using ³⁵s-methionine/cysteine incorporation and SDS-PAGE gel electrophoresis (1 and 2-D) and Western blotting after treatment with representative environmental pollutants (benzo(a)pyrene (BaP), 2,3,3′, 4,4′-pentachlorobiphenyl (PCB-105)₁ arsenite (AsO₂-) and cadmium (Cd)). The results demonstrated striking similarities in changes in protein expression after treatment with the different pollutants. Hsp70 (Hsp72/73) proteins were induced after treatment with all the compounds as shown by ³⁵S-methionine/cysteine labelling. However, high background levels of these proteins were shown with Western blotting and an anti-Hsp70 antibody, indicating a slow turnover of these proteins. The Hsp70s in salmon hepatocytes were extremely susceptible to degradation in urea used in 2-D electrophoresis, resulting in peptide fragments of 45–46 kDa. In addition to these Hsp70 fragments, arsenite induced several proteins of 42,38, and in the 30–32 kDa range. CYPlA (58 kDa) and an unidentified protein of 16 kDa were furthermore induced after treatment with the organic xenobiotics (BaP, PCB and the model compound β-naphthoflavone, BNF). CYPlA was expressed in a dose-dependent manner, and was resolved into several protein spots in 2-D Western blotting. Elevated levels of metallothionein and haem oxygenase (HO) were indicated in Western blots after treatment with cadmium or arsenite (only HO). The hepatocytes showed cytoplasmic protrusions after treatment with 35 μM arsenite and 100 μM Cd, indicative of cells entering apoptosis.

Key Words :

• 2-dimensional gel electrophoresis
• stress proteins
• CYPIA
• haem oxygenase
• fish cells