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 (35)s-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 (35)S-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.

Are hsps suitable for indicating stressed states in fish?

“…Indeed, it has been shown that several forms of environmental stressors can induce the hsp response in fish. For example, increased levels of various hsps have been measured in tissues of fish exposed to industrial effluents , polycyclic aromatic hydrocarbons , several metals such as copper, zinc and mercury (Sanders et al, 1995;Williams et al, 1996: Duffy et al, 1999, pesticides (Hassanein et al, 1999) and arsenite (Grosvik and Goksoyr, 1996). These studies and others revealed that the use of hsp as an indicator of stressed states in fish is a very complex issue.…”

Untitled

“…It has been shown that several forms of environmental stressors may induce the Hsp response in fish. For example, increased levels of various Hsps have been measured in tissues of fish exposed to industrial effluents, polycyclic aromatic hydrocarbons , several metals such as copper, zinc and mercury (Sanders, 1993;Williams et al, 1996), pesticides (Hassanein et al, 1999) and arsenite (Grosvik and Goksoy, 1996). These studies and others revealed the use of Hsp as an indicator of stressed states in fish is a complex issue.…”

Physiological Adaptations of Stressed Fish to Polluted Environments: Role of Heat Shock Proteins

“…HSP induction in response to hyperammonemic conditions has been documented in carp (Hernandez et al 1999). Enhanced levels of different HSPs have also been demonstrated in tissues of fish exposed to polycyclic aromatic hydrocarbons ), metals such as copper, zinc, and mercury (Duffy et al 1999;Williams et al 1996), pesticides (Hassanein et al 1999), and arsenite (Grosvik and Goksoyr 1996). Cara et al (2005) reported the impact of fooddeprivation, reduced oxygen levels, and heat shock on the expression of HSP70 and HSP90 in the early life stages of the gilthead sea bream (Sparus aurata), a warmwater aquaculture species.…”