Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology
Effect of cobalt ions on the metabolism of some volatile and polar compounds in the marine invertebrates Mytilus galloprovincialis and Actinia equina
Introduction
Two important groups of compounds in any living organism are the volatile fraction obtained by distillation–extraction and the polar fraction, obtained by n-butanol extraction of defatted extracts. The metabolites from both fractions often possess valuable biological activity. Many of them are semiochemicals, and appear to be of importance for the organism's ecology.
The volatile fractions from plants and insects often contain compounds, which have defensive functions being attractants, repellents, grazing inhibitors, insecticides, etc. (Jiang et al., 1997, Wang et al., 1999). Previously the research has been concentrated almost entirely on the volatile compounds from terrestrial plants, while there was a very limited number of publications on marine algae (Gally et al., 1993, Kamenarska et al., 2000, Mahran et al., 1993). Most of the investigations about the presence of volatile compounds in marine organisms concern the accumulation of carbohydrates due to petrol pollution (Scheuer, 1973, Meyers, 1977, Yan et al., 2002), accumulation of volatile fatty acids during anaerobiosis (Kluytmans et al., 1975), identification of terpenoids (Yasumoto et al., 2000), or carbohydrates (Kohler and Stahel, 1972). Nevertheless, detailed analyses about the volatile profiles in marine invertebrates are limited (De Rosa et al., 2002, Nechev et al., 2002a, Nechev et al., 2002b, Cros et al., 2003, De Rosa et al., 2003, Nechev et al., 2004). In most cases the composition of volatiles from invertebrates was very simple and there was no information about their origin and functions in organism.
Some investigations of volatile compounds in genus Mytilus were focused on the ability of this mussel to accumulate hydrocarbons as a result of oil pollution. Although the presence of data about the content of hydrocarbons in the distinct tissues (Lubet et al., 1985), the use of mussels for biomonitoring makes them suitable for the analyses of polycyclic aromatic hydrocarbons (PAH), which are toxic and carcinogenic to vertebrates. The origin of PAH could be the microbial biodegradation of crude oil (Hamdoun et al., 2002) as well as the incomplete combustion of different fuels (Mix and Schaffer, 1979).
A detailed analysis of volatile compounds of Mytilus galloprovincialis was performed due to its use in food industry (Cros et al., 2003). More than 60 compounds were identified including carbohydrates, ketones, aldehydes, acids, alcohols, sulphur- and nitrogen-containing compounds.
There is no data about the composition of the volatile fraction in Actinia equina.
Polar components, soluble in alcohol and water, predominate in living organisms. Compared to the lipophilic substances, they possess more frequent biological activity, they are presented in higher volumes, and their composition could be used for chemotaxonomical conclusions (Berquist and Wells, 1983, Kubo et al., 1990). Whilst semiochemicals in terrestrial organisms are mainly volatile substances, in marine ecosystems, such compounds must be water-soluble. Despite their interesting properties, polar compounds have rarely been subjected to analyses, due to the complicated composition and the high extent of polarity, that make their separation and purification difficult. For these reasons, GC/MS was the suitable method for investigating polar compounds after derivatization (most often silylation), which increases the volatility of the polar compounds. Little is known about the role of polar compounds in organism. Free amino acids and nucleosides are probably metabolites of protein synthesis or parts from neuropeptides, though some other functions are also possible. A number of polypeptides are being used by marine invertebrates as chemical signals (Zimmer and Butman, 2000). Due to their antibacterial and cytotoxic activity, bromine-containing compounds are thought to possess defensive properties.
The aim of this work is to investigate the composition of volatile and polar constituents in marine invertebrates at different levels of evolution and their changes caused by cobalt ions.
The mussel Mytilus galloprovincialis Lamarck, 1819 (class Bivalvia, phylum Mollusca) is evolutionarily more advanced than Actinia equina Linnaeus, 1758 (class Anthozoa, phylum Cnidaria), which is a more primitive organism. We assume that probably the more advanced invertebrates could develop more efficient mechanisms for adaptation towards a harmful environment. Invertebrates are extensively used in monitoring programs in freshwater (Gundacker, 2000), marine (Frias-Espericueta et al., 1999, Al-Mafda et al., 1998) and antarctic (Kahle and Zauke, 2002) environment due to their ability to concentrate pollutants to several orders of magnitude above ambient levels in water. To the best of our knowledge, no investigation has yet discussed the effect of cobalt ions on the chemical composition of affected organisms.
Cobalt is a relatively rare element (0.0025% (w/v) in the Earth's crust and 4 × 10− 8 % (w/v) in the seawater) that usually exists in association with nickel, silver, lead, copper and iron ores. Cobalt is usually not mined alone, and tends to be produced as a by-product of nickel and copper mining activities. Cobalt is used widely as an alloying ingredient together with nickel, chromium, molybdenum and other elements. Cobalt is an important constituent of magnets and batteries. It is also used as a pigment in glass, ceramics, and paints, as paint drier, as a catalyst for the petroleum and the chemical industries. Many fertilizers are enriched with cobalt, generally in the range of 1 mg/kg to 12 mg/kg in order to amend agricultural soils that are cobalt-deficient (Nagpal, 2004). Due to the high solubility of such products, cobalt ions could pass from the soil to underground or surface waters and thus might be transported at long distances. Other sources of cobalt pollution could be the places for mining and extracting of nickel, copper, silver, lead and iron. Despite the fact that there is no chance for a massive contamination with cobalt, point-source pollution could appear in limited areas, displaying the effect of cobalt; most significant cases had appeared along the coasts and in rivers of Australia, Canada, Russia, Congo and Zambia (World Mineral Statistics, 2002).
Monitoring in the Hungarian aquatory of the river Danube, which runs into the Black Sea, showed the presence of 14 different metals. Four of them (Ag, Cd, Mg and Co) had higher concentrations in periphyton compared to surrounding water and sediments. This ratio was higher for Co, indicating that cobalt is accumulated in the highest extent in periphyton (Oertel, 1991).
According to the Australian National Pollutant Inventory cobalt is more hazardous to environment than Ni, Cu, Pb, As, Mn and is as hazardous as Cd, Hg, Zn. Cobalt persists in higher concentrations in marine waters and accumulates in marine animals (Department of the Environment and Heritage, 2006).
Although cobalt is less frequently encountered in metalloenzymes than the other first-row transition metals, it is nevertheless an important cofactor in vitamin B12-dependent enzymes. To date eight cobalt-containing enzymes have been isolated (Kobayashi and Shimizu, 1999), some of which are nitrile hydratase (Brennan et al., 1996), aldehyde decarbonylase (Dennis and Kolattukudy, 1992), bromoperoxidase (Itoh et al., 1994), etc.
Despite metal ions play a variety of roles in natural proteins, the functions of cobalt have rarely been studied. Cobalt is essential in trace amounts for humans and other mammals. It is also an essential element for the growth of many algal species (Bruland et al., 1991). In higher concentrations cobalt ions are toxic towards humans, terrestrial and aquatic animals, and plants. Independently of the low concentrations of cobalt in the environment, some trees and mosses can incorporate significant amounts of cobalt (Popov and Stancheva, 2004).
There is very limited data about the harmful biological activity of cobalt. It interacts with sulphydryl groups to impair thiol–enzyme activities (Alexander, 1972). In vitro studies state that cobalt causes DNA damage and induces the formation of reactive oxygen species in the presence of hydrogen peroxide (Beyersmann and Hartwig, 1992).
Cobalt is immunogenic and shows myocardial toxicity and severe mitochondrial damage (Sandusky et al., 1981). Cobalt salts were more soluble than salts of other metals (Pb, Cu, W, Ta, etc.) in lung cytosol, plasma, serum, synovial fluid, alveolar fluid, gastric and intestinal juice (Stopford et al., 2003). In marine water, cobalt is normally present as Co2+ (Hamilton, 1994).
There are no data about the effect of cobalt ions on the metabolism of marine invertebrates, but some investigators (Amiard, 1976, Smith and Carson, 1981) showed the accumulation of cobalt in marine animals. The majority of the marine toxicity data for cobalt is from a study by Amiard (1976) in which acute (96 h) and chronic (216 h) toxicity tests were conducted using several species of diatoms, crustaceans, cephalopods and fish. The results of this study indicate that the sensitivity of adult crustaceans and adult fish to cobalt exposure is similar with LC50 concentrations ranging from 225 mg/L to 675 mg/L. Investigations of Smith and Carson (1981) confirmed those of Amiard – marine shellfish were found to have up to tens of thousands times higher concentrations of cobalt than its concentrations in the surrounding water. Other determinations of LC50 to cobalt exposure have also been performed for marine animals: fish (Krishnakumari et al., 1983), crustaceans – Copepoda (Bengtsson, 1978) and Isopoda (El-Nady and Atta, 1996), Nematoda (Vranken et al., 1991). These investigations also confirmed the accumulation of cobalt in marine benthic invertebrates. The concentrations of cobalt were up to 40,000 times higher than the concentration presented in the ambient water.
Our recent research (Nechev et al., 2006) on the lipid and sterol composition of M. galloprovincialis and A. equina showed that the concentrations of the lipid and sterol compounds strongly depended on the presence and concentration of the cobalt ions and that the observed changes probably had adaptive value. If there are similar changes in the volatile and polar fractions, the data could be of complementary value for the influence of cobalt to marine benthic invertebrates' metabolism.
Section snippets
Sample collection
The mussel M. galloprovincialis and the actinia A. equina were collected during August 2003 at depths between 0 and 2 m, near Russalka resort, located in the northern Bulgarian shore of the Black Sea. The identification of the samples was performed by Dr. St. Andreev and voucher specimens were deposited in the National Museum of Natural History in Sofia, Bulgaria. The invertebrates (at least 20 organisms/sample) were placed in 3 aquaria, 5 l each, with marine water for 5 days. Aeration,
Volatile fraction
In M. galloprovincialis the volatile compounds possess a simple composition (Table 2). In the control sample, we identified a number of fatty acid methyl esters, containing 14–20 carbon atoms, which were absent in the other samples, treated with cobalt ions. The concentrations of the esters were relative high, especially for these containing 16 and 18 carbon atoms. Contrary to the analogous ethyl esters, which we suppose to be artifacts derived from the ethanol used in the lipid extraction of
Acknowledgements
The authors are grateful to the National Council for Scientific Research in Bulgaria for the partial financial support under contract X-1101.
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