Relationships between tissue concentrations of polycyclic aromatic hydrocarbons and antioxidative responses of marine mussels, Perna viridis
Introduction
In recent years, Hong Kong's population, as well as its concomitant urban and industrial development have expanded rapidly. Over half of Hong Kong's 6.8 million people are located in southern Kowloon and northern Hong Kong Island, and sewage is discharged into Victoria Harbour with very little treatment (Connell et al., 1998). Trace organic contaminants present in the wastewater generally include polychlorinated biphenyls (PCBs), hexachlorobenzene (HCB) and the chlorinated hydrocarbons used in agriculture, principally DDT isomers and metabolites (Phillips, 1989, Connell et al., 1998). Contaminants such as polycyclic aromatic hydrocarbons (PAHs), which can be derived from crude or refined petroleum or combustion sources, are also important. Sediments from Victoria Harbour and other port shelters in Hong Kong have higher total PCB and PAH concentrations than sites less exposed to intensive human activities (Connell et al., 1998).
Green lipped mussels, Perna viridis, have been shown to be useful organisms for monitoring conservative contaminants, including metals and trace organic contaminants, in Hong Kong waters (Phillips, 1985, Phillips, 1989, Tanabe et al., 1987). Although indigenous mussels have been used in the past on a ‘Musselwatch’ basis, more recent studies have utilized transplanted organisms (e.g. Richardson et al., 2000). Such studies have most recently included the use of biomarkers, to enhance understanding of the responses of the organisms to local pollution influences (e.g. Xu et al., 1999).
Upon exposure to pollutants, organisms usually attempt to metabolize and depurate directly, minimizing any cellular damages they cause. Such protective mechanisms often include alterations to enzyme activities, which can be regarded as biomarkers of exposure or effects. Glutathione S transferase (GST) is one such enzyme. GST catalyzes the conjugation of various electrophilic compounds (e.g. epoxides of PAHs) with the tripeptide glutathione, the resulting conjugates being water soluble and thus more easily excretable. In addition, GST serves to reduce the likelihood of electrophilic compounds covalently binding to important cellular macromolecules such as DNA.
Oxidative stress is potentially experienced by all aerobic life when anti-oxidant defenses are overcome by pro-oxidant forces. Hydroxyl radicals, which are extremely potent oxidants, are capable of reacting with critical cellular macromolecules, including DNA and proteins. Hydroxyl radicals are produced in electron-transfer reactions during oxygen reduction metabolism. Oxidative damage results from an imbalance between the production and removal of oxidants, and it has been shown that environmental contaminants may actually enhance oxidative stress in aquatic organisms (Winston, 1991). One example is the production of oxyradicals by the cytochrome P450-dependent oxidative metabolism of aromatic hydrocarbons (Lehtinen, 1990, Palace et al., 1996). Livingstone et al. (1988) showed that NADPH-dependent metabolism of benzo[a]pyrene (B[a]P) in the digestive gland (hepatopancreas) of Mytilus edulis resulted in the production of hydroxyl and superoxide anion radicals. Many substrates in this reaction series are not tightly coupled and a significant proportion of the O2 consumed during metabolism appears as oxygen free radicals. The extent to which oxyradical generation produces biological damage is, in turn, dependent on the effectiveness of antioxidant defenses (Michiels and Remacle, 1988). These protective mechanisms involve a number of antioxidant enzymes: superoxide dismutase (SOD; EC. 1.15.1.1), catalase (CAT; EC. 1.11.1.6), glutathione peroxidase (GPx; EC. 1.11.1.9) and glutathione reductase (GR; EC. 1.6.4.2), NAD(P)H dependent DT-diaphorase (DT-d; EC. 1.6.99.2) and free radical scavengers such as glutathione (Livingstone et al., 1992, Doyotte et al., 1997, Regoli et al., 1997).
Antioxidant enzymes thus play a crucial role in maintaining cell homeostasis. Their induction reflects a specific response to pollutants (Doyotte et al., 1997) and they have been proposed as biomarkers of contaminant-mediated oxidative stress in a variety of marine organisms, including mussels (Livingstone et al., 1992, Gamble et al., 1995, Regoli et al., 1998). Oxidant-mediated toxicities may result if these enzymes are inhibited (Stegeman et al., 1992) and DNA damage (e.g. DNA adduct formation), enzymatic inactivation and lipid peroxidation may occur if the system is impaired (Halliwell and Gutteridge, 1989, Xu et al., 1999).
Previous studies have indicated that DNA adducts have been positively correlated with pollution in both fish (Van der Oost et al., 1996) and mussels (Solé et al., 1996). In Hong Kong, a study of local mussels (P. viridis) showed that DNA adduct levels were correlated with body B[a]P and total PAH concentrations (Xu et al., 1999). In the present study, the primary objective was to investigate the biochemical responses of P. viridis transplanted in Hong Kong waters. Phase II enzyme (GST) and oxyradical scavenger (GSH), antioxidant enzymes (SOD, GR, CAT, GPx, and DT-d) and the extent of oxidative damage (lipid peroxidation) were examined in a field transplant experiment, which also involved the quantitation of PAHs in individual mussels retrieved from the deployment sites after a 30-day exposure.
Section snippets
Study sites
Green lipped mussels (P. viridis) were collected from a reference site, Kat O which is a remote island situated in the north-east of Hong Kong. The sampling criteria and the sites for mussel transplantation followed Xu et al. (1999). The four transplantation sites were located at Kat O, Tsim Sha Tsui, Sai Wan Ho, and Tolo Harbour (Fig. 1) which had average total PAH levels (ng g−1 dry weight in sediment) of 38.5, 49.4, 91.1 and 507.0, respectively (Zheng and Richardson, 1999). At each site, 30
Results
Levels of PAHs in mussel tissue were significantly higher at Tolo Harbour and Tsim Sha Tsui as compared to Sai Wan Ho and Kat O (Kruskal–Wallis test: H=8.27, P<0.05). However, pair-wise comparison revealed a significant difference only between Tolo Harbour and Kat O (Dunn's test: Q=9.00, P<0.05). Standard reference freeze-dried mussel (M. edulis) tissue (SRM2974; National Institute of Standards & Technology, Gaithersburg, USA) was also prepared and analyzed to validate the analytical method.
Discussion
As reported by Kappus (1986) and Di Giulio et al. (1989), antioxidant defenses consist of three general classes including water soluble reductants such as glutathione, fat soluble vitamins such as α-tocopherol and enzymes including glutathione peroxidase and superoxide dismutase. One of the important features of these latter enzymes is their inducibility under conditions of oxidative stress, and such induction can be an important adaptation to pollutant-induced stress.
It is worth noting that
Conclusion
Local mussels, P. viridis, were transplanted from a relatively clean site to various polluted sites in Hong Kong. After a 30-day field exposure, a suite of antioxidant parameters and tissue concentrations of B[a]P and a total of five PAHs with potential carcinogenicity were determined for individual mussels. The results showed that body burden of B[a]P and total PAHs from the same site were highly variable. Most antioxidant parameters were positively correlated with tissue pollutant levels.
Acknowledgements
This research is supported by a Central Allocation Grant (8730011) awarded by the Research Grant Council, Hong Kong Government, HKSAR, and Strategic Research Grants (7000772 and 7001030) of the City University of Hong Kong. We would like to thank the two anonymous referees for their constructive comments which improved the final version of the manuscript.
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