The use of protein biomarkers in ecotoxicology : Studies of oxidative and genotoxic stress in the blue mussel (Mytilus edulis)

Abstract: Many environmental pollutants, heavy metals and polyaromatic hydrocarbons (PAHs) among them, are toxic by oxidative stress. Oxidative stress may be defined as a state in an organism when its inherent capacity to handle oxyradicals is surpassed, and it may result in peroxidation of lipids, and damage to proteins and DNA. Thus, genotoxic stress may follow oxidative stress.Little is known about the effects of genotoxic stress in invertebrates, although the occurrence of tumors has been known for quite some time, in bivalve mollusks at least since the 1960s. Less dramatic manifestations of genotoxic stress may include impaired enzyme functions, altered protein turnover, with possible effects on physiological processes. Keys to a better understanding of genotoxic stress are the proteins involved in the regulation of the cell-cycle and DNA repair. Virtually nothing is known about these proteins in mussels. The work presented in the papers I, II, and IV aimed to give a first insight into how the blue mussel responds to genotoxic stress following exposure to the polyaromatic hydrocarbon (PAH) benzo[a]pyrene (B[a]P), PAHs in petrochemical pollution, copper (Cu) or cadmium (Cd), all substances known to enhance formation of oxyradicals. PAHs may also be genotoxic by formation of DNA adducts. The cell-cycle and DNA repair proteins studied were the proliferating cell nuclear antigen (PCNA) and the retinoblastoma protein 110 (Rb110). The first is involved in DNA repair and cell proliferation; the second at the G1 checkpoint of the cell-cycle. We showed that the PCNA is a potential marker of genotoxic response (DNA repair, and/or cell proliferation) to PAHs (papers I and IV) and cadmium (paper II). Furthermore, cadmium may possibly elicit a cell-cycle stop mediated by the Rb110 (the paper II). Based on these cell-cycle and DNA repair proteins, the genotoxic responses in blue mussels seem to be similar to that in vertebrates.Markers of general stress or oxidative stress were used in all studies. The applicability of the general stress marker heat shock proteins (HSP70 and HSP60) (papers I and IV), the polyglycoprotein (P-gp) (paper I), a marker of exposure to planar organic compounds, and markers of oxidative stress, such as the antioxidant defense enzyme copper/zinc-superoxide dismutase (Cu/Zn-SOD) (paper II), and the oxidative protein modifications protein carbonyls and disulfides (papers III and IV) were tested in various situations. In accordance with results by others, my studies have shown that HSPs, which formerly were considered as universal stress markers, are not always applicable, as their expression is affected by factors that are difficult to control (papers I). I found that the P-gp can be used as a marker of B[a]P exposure in Baltic Sea blue mussels (paper I). In paper II, I reported that the Cu/Zn-SOD was up-regulated in response to Cd exposure, a clear indication of oxidative stress that was also manifested as a response in the studied cell-cycle and DNA repair proteins. Protein carbonyls were successfully used in the papers III and IV to indicate oxidative stress. In paper III, I also investigated the potential of diagonal gels as a tool for identification of protein disulfides in protein expression signatures (PESs). The diagonal gels hold a good potential for identification of novel and prime targets of oxidative stress.My work was performed as short-term laboratory studies, in vivo, in blue mussels from the Baltic Sea, and as a one year long field experiment with Icelandic blue mussels in a tidal system. The idea was to use blue mussels that were already physiologically challenged (by osmotic stress, or alternating aerobic and anaerobic metabolism), to see how they responded to additional stress in the form of toxicants capable of inducing oxidative and genotoxic stress. Most of the work on biochemical endpoints in blue mussels published so far was done on blue mussels from fully marine environments, and without consideration to the effect of tide.