Effect of cobalt ions on lipid and sterol metabolism in the marine invertebrates Mytilus galloprovincialis and Actinia equina

Comparative Biochemistry and Physiology, Part A 144 (2006) 112 – 118 www.elsevier.com/locate/cbpa

Effect of cobalt ions on lipid and sterol metabolism in the marine invertebrates Mytilus galloprovincialis and Actinia equina Jordan Nechev ⁎, Kamen Stefanov, Simeon Popov Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences, Sofia, Bulgaria Received 4 November 2005; received in revised form 3 February 2006; accepted 12 February 2006 Available online 28 February 2006

Abstract The lipid and sterol fractions of the mussel Mytilus galloprovincialis and the anemone Actinia equina were analyzed before and after incubation with cobalt ions. There were significant changes in the lipid and sterol composition, strongly depending on the cobalt ions concentrations. The changes in the lipid composition of both invertebrates were different for neutral lipids and phospholipids. These changes concern the length of the fatty acid chains as well as their unsaturation. The cobalt ions inhibited the oxidation of sterols and their alkylation at C-24. Because of these results we can assume that some of the changes caused by cobalt ions might possess adaptive value. © 2006 Elsevier Inc. All rights reserved. Keywords: Actinia equina; Cobalt; Fatty acids; Lipids; Mytilus galloprovincialis; Sterols

1. Introduction The Black Sea is a brackish, non-tidal and semi-enclosed sea. The relative ions concentrations of heavy metals are often greater than in the Mediterranean Sea (Windom et al., 1999; Birkun, 2002). Heavy metals are among the major anthropogenic contaminants of water and all of them are toxic above certain threshold concentrations and can cause physiological stress in marine organisms (Biesinger and Christensen, 1972; Frias-Espericueta et al., 1999; Kahle and Zauke, 2002). The metabolic changes in the living organisms are due mainly to stress induced degradation processes as well as to changes leading to a better adaptation towards the harmful environment. The lipid cell membranes are important for this adaptation, because one of the effects of the stress impact is to perturb the physical properties of the cell membranes by changing their chemical composition and biophysical organization. In such a case the adequate response of the cells would be a series of biochemical modifications of the lipophylic

⁎ Corresponding author. Postal address: Institute of Organic Chemistry, ul. “Acad. G. Bonchev”, No 9, Sofia-1113, Bulgaria. Tel.: +359 898218998; fax: +359 2 8700 225. E-mail address: [email protected] (J. Nechev). 1095-6433/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpa.2006.02.022

composition of the cell membranes in order to recover their initial organization. The aim of this paper is the investigation of lipid and sterol changes due to the effect of cobalt ions on two widespread Black Sea invertebrates at different levels of evolution. The mussel Mytillus galloprovincialis Lamarck, 1819 (class Bivalvia, phylum Mollusca) is evolutinary 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 and could tolerate higher concentrations of heavy metals. Invertebrates are extensively used for monitoring programs in freshwater (Gundacker, 2000), marine (al-Mafda et al., 1998; FriasEspericueta et al., 1999) and antarctic (Kahle and Zauke, 2002) environment, due to their ability to concentrate pollutants to several orders of magnitude above the ambient levels in water. To the best of our knowledge, no investigations discuss the effect of cobalt ions on the lipid and sterol metabolism 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.

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Cobalt is used widely as an alloying ingredient together with nickel, chromium, molybdenum and other elements. These alloys are utilized in jet aircrafts, turbines, other equipment and high tech tools, operating at high temperatures and extreme conditions. 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 industry. Many fertilizers are enriched with cobalt, generally in the range of 1 to 12 mg/kg in order to amend agricultural soils that are cobalt-deficient (Nagpal, 2004). 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. Although 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, as it is an integral component of the vitamin B12 complex. It is also an essential element for the growth of many algal species (Bruland et al., 1991), but in higher concentrations is toxic towards humans and 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). Despite there is no chance for a massive contamination with cobalt, point source pollution could appear in limited areas, displaying the effect of cobalt. There is limited data about the harmful biological activity of cobalt. It interacts with sulphydryl groups to impair thiol-enzyme activities (Alexander, 1972). In in vitro studies 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, shows myocardial toxicity and severe mitochondrial damage (Sandusky et al., 1981). Cobalt ions were more soluble than other metals (Pb, Cu, W, Ta etc.) in lung cytosol, plasma, synovial fluid, alveolar fluid, serum, gastric and intestinal juice (Stopford et al., 2003). In marine water, cobalt is normally present as Co2+ (Hamilton, 1994). There is no data about the effect of cobalt ions on the metabolism of marine invertebrates, but some investigations (Amiard, 1976; Smith and Carson, 1981) show 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 225,000 to 675,000 μg/L. Marine shellfish were found to have up to tens of thousands times higher concentrations of cobalt than its concentrations in surrounding water. Some other marine benthic invertebrates had concentrations of

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cobalt ranging to 40,000 times higher than the concentration presented in the ambient water. 2. Materials and methods 2.1. Sample collection The mussel M. galloprovincialis (Mollusca: Mytilidae) and the beadlet anemone A. equina (Cnidaria, Actiniidae) were collected in 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 from each species were separated into three samples (at least 20 organisms per sample) and placed in 3 aquara, 5 l each, with marine water for 5 days. Aeration, temperature (23 ± 2 °C) and natural light (12/12 h day/night period) were used. 2.2. Incubation with Co2+ One of the aquaria was used for control and no cobalt ions were added. In the other two aquaria, solutions of CoCl2·6H2O (0.2 and 0.3 mM Co2+ respectively) were added. After 5 days of incubation the shells of the mussels were removed, all samples were rinsed carefully with seawater to avoid cobalt accumulation on the animal surface, and transported to the laboratory in ethanol. 2.3. Extraction All samples of M. galloprovincialis and A. equina were cut into small pieces and consecutively extracted with ethanol, followed by a mixture of chloroform:ethanol (2:1 v/v). The extracts were combined and diluted with water until two layers were obtained. Table 1 shows the dry weights of the samples and yields of the lipophilic extracts. 2.4. Analysis of the lipids 2.4.1. Separation of neutral lipids and phospholipids The lipophilic extracts were evaporated under reduced pressure at 40 °C. Part of the dry residues (70 mg from each sample) was subjected to preparative TLC on 20 × 20 cm silica gel G (Merck) plates (layer thickness 0.5 mm) to separate the Table 1 Dry masses of the samples and yields of the lipophilic extracts Concentration of Co2+ [mM]

Mytilus galloprovincialis Actinia equina Control 0.2

Dry mass [mg] 3687 Yield of lipophilic 362 extract [mg] Yield of the lipophylic 9.8 extracts from the dry weight [%]

4586 375 8.2

0.3

Control 0.2

0.3

4198 385

2383 182

5992 540

9.2

7.6

4525 490 10.8

9.0

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lipid classes. Chloroform–methanol–acetone–acetic acid mixture (70:14:24:0.4 v/v/v/v) was used as a mobile phase. The two main lipid classes — neutral lipids (NL) and phospholipids (PL) were isolated. To each lipid class, an internal standard of 0.3 mL margarinic acid (C 17:0) in methanol (1.0 mg/mL) was added. 2.4.2. Preparation of fatty acid methyl esters The fatty acids of each lipid class were converted to methyl esters with 5 mL of 15% acetyl chloride in absolute methanol. After 3 h at 55 °C the samples were diluted with water and extracted twice with hexane. The methyl esters were separated from the sterols and purified by TLC on preparative silica gel G plate with hexane–acetone 95:5 (v/v). 2.4.3. Gas chromatography of fatty acid methyl esters GC: Hewlett Packard 5890 (Hewlett Packard, Palo Alto, California, USA), equipped with FID and capillary column HPINNOWAX (30 m × 0.25 mm, 0.25 μm film thickness) was used. The temperature was programmed from 150 to 240 °C at a rate of 4 °C min− 1 and a 10-min hold at 240 C. The temperature of injector and detector was 260 °C; the carrier gas was nitrogen. All procedures of quantitative determination were carried out three times. The quantities of the main lipid classes were obtained by gravimetrical estimation of the amounts of fatty acids methyl esters (FAME), using coefficient of calculation: 1.0 for NL and 1.4 for PL (Elenkov et al., 1993).

2.5. Isolation and analysis of sterols The chloroform extracts were evaporated and part of the dry residues (100 mg) from each sample was subjected to silica gel column chromatography (1:40). The column was eluted with 100 ml hexane, followed by 100 ml hexane:acetone (25:1), 100 ml hexane:acetone (12:1), 100 ml chloroform, 100 ml chloroform:methanol (98:2), chloroform:methanol (9:1), chloroform:methanol (1:1), and 100 ml methanol. Fractions containing sterols (eluted with hexane:acetone (12:1), chloroform and chloroform:methanol (98:2) ) were identified by thin layer chromatography (TLC) on silica gel G. Further purification by preparative TLC on silica gel G (hexane:acetone 10:1) was performed with these fractions and the sterol mixtures isolated were analyzed by gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS). A GC Pye Unicam 304 (Scientific Instrument Company of Philips, Cambridge, England) equipped with a flame ionization detector (FID) and a capillary column SPB-1 (30 m × 0.32 mm, 0.25 μm film thickness, Supelco Park, Bellefonte, PA, USA) was used. The temperature programme was 230–300 °C at 4 °C min− 1 and a 10-min hold. The injector temperature was 300 °C and the detector temperature − 320 °C; the carrier gas was nitrogen. A GC Hewlett Packard 6890 + MS 5973 (Hewlett Packard, Palo Alto, California, USA) with a capillary column SPB-50 (30 m × 0.32 mm, 0.25 μm film thickness, Supelco Park, Bellefonte, PA, USA) was used. The carrier gas was helium and

Table 2 Fatty acid composition of Actinia equina and Mytilus galloprovincialis Concentration of Co2+ [mM] →

Actinia equina

FA

Control

0.2

0.3

Control

0.2

0.3

Control

0.2

0.3

Control

0.2

0.3

1.8 0.8 17.2 1.6 2.6 1.8 – 1.9 16.0 17.0 1.9 0.9 – 0.9 2.6 0.9 4.1 3.9 17.3 – 3.4 2.3 1.1

1.8 1.0 20.3 5.1 2.3 2.5 – 2.5 14.0 21.6 2.2 0.6 – 1.1 3.7 1.0 2.3 1.7 12.9 – 1.8 0.8 0.8

1.4 0.9 21.9 4.7 2.1 3.4 – 2.3 15.4 20.1 1.6 0.5 – 1.0 4.1 0.9 1.9 1.6 13.1 – 1.2 0.5 0.8

Traces 1.5 14.3 3.8 5.3 3.8 6.9 2.2 15.1 11.3 1.4 1.1 – 4.2 1.1 – 2.3 1.2 17.1 – 2.6 Traces 5.0

Traces 0.5 18.2 1.7 4.6 1.7 1.9 2.4 21.1 7.1 1.3 0.8 – 3.2 1.1 0.6 2.3 1.6 23.3 – 4.2 1.6 0.8

Traces Traces 6.7 1.2 1.2 0.8 2.8 1.5 15.8 5.8 0.7 0.5 – 28.6 2.4 0.4 1.6 2.2 24.9 – 1.4 0.5 1.0

1.7 1.2 25.8 6.1 2.1 – – 2.0 8.6 9.6 3.4 2.7 0.7 – 10.2 1.3 5.0 6.4 – 7.0 – 1.2 4.9

2.3 1.6 33.4 7.9 2.5 0.3 – 2.4 9.8 10.0 2.6 1.2 0.3 – 11.0 1.1 2.4 2.1 – 7.3 – 0.3 1.4

2.6 1.5 35.4 7.9 2.8 0.4 – 2.9 11.8 10.4 2.1 0.3 – – 11.8 0.9 1.1 1.0 – 6.8 – 0.1 0.5

1.2 0.6 28.8 2.5 1.9 – 1.2 1.3 9.0 24.3 3.3 0.8 – – 9.5 0.6 1.9 1.9 – 9.7 – 0.5 1.0

0.6 1.0 28.2 2.4 3.2 – 1.5 2.1 8.3 4.5 1.5 5.1 – – 17.5 0.9 3.5 2.6 – 13.8 – 0.4 2.0

0.9 1.3 24.7 3.2 2.1 – 2.1 1.9 9.1 5.2 1.6 0.7 – – 20.1 1.0 4.1 3.0 – 14.8 – 0.7 2.8

14:0 15:0 16:0 16:1 16:2 16:3 16:4 17:0 18:0 18:1 18:2 18:3 18:4 20:0 20:1 20:2 20:4 20:5 22:1 22:2 22:4 22:5 22:6

a

Mytilus galloprovincialis

NL [%]

PL [%]

NL [%]

PL [%]

Values obtained from three parallel measurements; the standard deviations (related to peak proportions of the chromatograms) are as follows: ±0.3 for C16 : 0 and C20 : 5; ± 0.2 for C18 : 0, C20 : 1 and C22 : 6; and ±0.1 for the others. a Percentage of each fatty acid from the corresponding lipid class.

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a temperature programme of 270–290 °C at 4 °C min− 1 and a 20-min hold was used. The ion source was set at 250 °C and the ionisation voltage was 70 eV.

3. Results and discussion

2.6. Identifications

3.1.1. Lipids from M. galloprovincialis The main groups of lipids in the mussel and the sea anemone were NL and PL. We separated the two lipid groups and analyzed the fatty acid compositions of each sample — control and those treated with cobalt ions. The results obtained were summarized in Table 2. In the mussel M. galloprovincialis the effect of cobalt ions on the NL and PL composition showed significant differences. In the NL fraction there was an increase of the concentrations of saturated fatty acids proportional to the cobalt ions concentrations. The same was true for the monoenoic fatty acids, but their increase was biggest at the medium cobalt concentrations. An important group of marine fatty acids are the polyunsaturated fatty acids (PUFA), which are obtained in all marine algae and invertebrates. In the investigated mussel there were three groups of PUFA, containing 18, 20 and 22 carbon atoms. In all the PUFA groups the cobalt ions led to a sharp decrease of their concentrations, proportional to the increase of cobalt ions concentrations. The phospholipid changes appeared to be different. The cobalt ions caused a decrease of the concentrations of saturated fatty acids with even number of carbon atoms. There was some increase only in the concentrations of fatty acids with an odd

The FAME of NL, PL, and sterols, were identified by GC according to their retention times, and confirmed by their fragmentation on GC/MS. The GC/MS investigation was based on the interpretation of the mass spectral fragmentation followed by comparisons of the spectra obtained with those of authentic samples. Computer searches in a HP Mass Spectral Library NIST98 (Hewlett Packard, Palo Alto, California, USA) were also applied in most of the cases. When the spectra of some isomers were very similar and these compounds could not be identified unambiguously, comparisons of the GC retention times, obtained under the same conditions, were used. When there were no suitable authentic samples and spectra for comparison, no identifications were made. Only the unambiguously identified compounds were reported in Tables 2 and 3. The ion currents generated depend on the characteristics of the compound and for this reason are not a true quantitation. The results obtained by GC/MS might be used for characteristics of the biodiversity in the investigated organisms, as well as for quantitative comparisons between different groups of metabolites in them.

3.1. Lipids

Table 3 Sterol composition (% of total sterols) a of A. equina and M. galloprovincialis Sterol

preg-5-en-3β-ol dinor-chol-5-en-3β-ol dinor-chola-5,20(22)-dien-3β-ol 24-nor-chol-5-en-3β-ol 24-nor-cholesta-5,22-dien-3β-ol 24-nor-cholest-22-en-3β-ol 22(Z)-cholest-5,22-dien-3β-ol and/or occelasterol 22(E)-cholest-5,22-dien-3β-ol cholest-22-en-3β-ol 24(28)-methylene-cholest-3β-ol cholest-5-en-3β-ol cholestan-3β-ol cholesta-5,24-dien-3β-ol 24-methyl-cholesta-5,22-dien-3β-ol 24-methyl-cholesta-5,25-dien-3β-ol 24-methyl-cholesta-5,23-dien-3β-ol 24-methyl-cholesta-5,24(28)-dien-3β-ol 24-methyl-cholest-5-en-3β-ol cholest-5-en-3-one cholest-4-en-3-one 24-ethyl-cholesta-5,22-dien-3β-ol 22,23-methylene-cholest-5-en-3β-ol cholest-8-en-3β-ol 5a-24-ethyl-cholesta-7,22-dien-3β-ol 24-ethyl-cholest-5-en-3β-ol 24(E)-ethyliden-cholest-5-en-3β-oltraces 24-ethyl-cholestan-3β-ol 24(Z)-ethyliden-cholest-5-en-3β-oltraces a

Actinia equina

Mytilus galloprovincialis

Control

+0.2 mM Co2+

+0.3 mM Co2+

Control

+0.2 mM Co2+

+0.3 mM Co2+

– – – – 2.1 ± 0.2 traces 1.4 ± 0.1 5.3 ± 0.5 – – 72.9 ± 7.1 traces – 4.1 ± 0.4 traces 1.5 ± 0.1 0.2 ± 0.02 1.5 ± 0.1 – – 0.4 ± 0.03 traces 0.6 ± 0.05 – 1.0 ± 0.1 traces – –

– – – – 2.9 ± 0.2 – 1.7 ± 0.2 5.8 ± 0.5 traces traces 70.2 ± 7.0 traces – 4.3 ± 0.4 0.7 ± 0.07 1.1 ± 0.1 0.3 ± 0.03 1.8 ± 0.2 – – 0.5 ± 0.04 0.5 ± 0.05 1.2 ± 0.1 – 1.6 ± 0.1 traces – –

– – – – 2.4 ± 0.2 – 1.3 ± 0.1 4.8 ± 0.5 0.5 ± 0.04 – 80.9 ± 7.5 – 0.5 ± 0.04 3.8 ± 0.3 0.1 ± 0.01 0.6 ± 0.05 0.6 ± 0.05 1.3 ± 0.1 – – 0.5 ± 0.04 – – – 1.0 ± 0.1 0.4 ± 0.05 – –

0.2 ± 0.03 0.5 ± 0.05 0.1 ± 0.02 0.5 ± 0.04 3.5 ± 0.3 traces 3.0 ± 0.3 7.7 ± 0.6 – – 38.1 ± 3.2 – 1.3 ± 0.1 12.9 ± 1.1 1.7 ± 0.1 1.3 ± 0.1 0.7 ± 0.06 3.4 ± 0.3 0.7 ± 0.09 – 3.3 ± 0.3 1.0 ± 0.1 – – 4.2 ± 0.4 – – –

– – – – 3.9 ± 0.3 traces 3.1 ± 0.3 8.0 ± 0.6 traces – 53.3 ± 4.1 – 0.9 ± 0.6 16.1 ± 1.2 traces 5.2 ± 0.4 0.6 ± 0.06 2.6 ± 0.2 – – 2.2 ± 0.2 – traces – 2.2 ± 0.2 – – –

– – – – 4.7 ± 0.4 0.2 ± 0.03 3.4 ± 0.3 8.5 ± 0.7 0.5 ± 0.04 – 51.8 ± 4.1 – – 16.0 ± 1.2 0.3 ± 0.03 2.2 ± 1.3 2.2 ± 1.4 2.5 ± 0.2 – 0.2 ± 0.03 1.9 ± 0.2 – – 0.1 ± 0.02 1.8 ± 0.2 – traces –

wt.% of total sterols ± standard deviations from three parallel GC-analyses.

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number of carbon atoms. This might be explained with their origin — they are typical bacterial acids and probably the metabolism of bacteria reacts differently to the cobalt ions. Contrary to NL fatty acids, now the concentrations of the fatty acids depended not only on the cobalt ions concentrations, but also on the length of the chain — 18:1 acid concentrations decreased after an incubation with cobalt ions, while these of 16:1 and 20:1 acids increased at the same conditions. The concentrations of PUFA with 18 carbon atoms decreased after the cobalt treatment, while these of PUFA containing 20 and 22 carbon atoms sharply increased at these conditions. Similar changes in membrane lipids occur in invertebrates inhabiting low temperatures. Adaptations of membrane fluidity in marine bivalves from polar waters involve higher lipid unsaturation, which leads to the enhanced risk of oxidation damages (Abele and Puntarulo, 2004). It is possible that cobalt ions provoke some oxidation damages in the mussel. 3.1.2. Lipids from A. equina The changes in the fatty acid composition of A. equina were similar to these in M. galloprovincialis at the same conditions, but the concentration changes were smaller. In some cases the changes in the fatty acid composition of A. equina were more intense at medium cobalt concentrations. In summary, the changes in FA from M. galloprovincialis were greater than those in A. equina. Of special interest were the changes of the main fatty acids caused by cobalt ions. In M. galloprovincialis probably the cobalt ions inhibited the introduction of double bonds in the fatty acids in NL and activated this process in the PL fatty acids. Another explanation could be the eventual selective transport of PUFA from the diet, to PL (Senkal et al., 2005), in order to adjust the fluidity or the metabolic activity of cell membranes. A higher concentration of PUFA is typical of membranes with metabolically active complexes (Hulbert and Else, 2000). 3.2. Sterols 3.2.1. Sterols from M. galloprovincialis The purified by column and thin layer chromatography sterol fractions from M. galloprovincialis (control sample and two samples treated with different concentrations of CoCl2) were subjected to an analysis by GC and GC/MS, and the results obtained summarized in Table 3. We succeeded to identify a series of minor sterols, including the new for the Black Sea invertebrates 24-methyl-cholesta-5,23-dien-3β-ol and 22,23-methylene-cholest-5-en-3β-ol, as well as four short side chain sterols. The last 4 sterols were new for the phylum Mollusca and they were present only in the sample that was not treated with cobalt ions. The identification of the rare sterols, mentioned above, gives some information on the biodiversity and might be applied for investigations on some food chains in the Black Sea. According to our results, Co ions change the sterol metabolism in the mussel M. galloprovincialis and almost all sterols undergo some changes in their concentrations. These changes strongly depend on the concentrations of the cobalt ions.

The sterols with the unusually short side chains, containing 21–23 carbon atoms, were probably a product of biological oxidation (Carlson et al., 1978). We found them only in the control sample. The complete absence of these sterols in the mussels treated with cobalt ions is an indication that cobalt ions suppress some oxidation processes in the mussel. This is in agreement with the identification of another oxidized sterol derivative, cholest-5-en-3-one, only in the control sample. Since benthic invertebrates, particularly from genus Mytilus, have been broadly used for biomonitoring, we propose that sterols, found only in control sample, are suitable for indication of contamination with cobalt. The concentrations of the sterols, containing 26 and 27 carbon atoms, strongly depended on the concentrations of cobalt ions. The sterols from this group, which contain C-22 double bonds showed an increase of their concentrations proportionally to the concentrations of the cobalt ions, while the cholesterol content increased independently to the cobalt concentrations. From this group of sterols only cholest-5,24dien-3β-ol concentrations decreased proportionally to the cobalt ions concentrations. Probably this is due to its function of cholesterol precursor and increasing concentrations of cholesterol consumed a significant part of the cholesta-5,24dien-3β-ol pool. Alkylation of dietary sterols is a process common in mussels (Teshima and Kanazawa, 1974; oysters (Knauer et al., 1998), other bivalves (Voogt, 1975) and many marine invertebrates (Kerr et al., 1992). At the first stage of alkylation (incorporation of C1 unit at C-24) there were different effects of cobalt ions. Some of the C28-sterols (i.e. 24-methyl-cholesta-5,23-dien-3β-ol) increased their concentrations when cobalt concentrations increased, while other sterols (i.e. 24-methyl-cholest-5-en-3β-ol) decreased with the increase of the cobalt ions concentrations. The introduction of C-23 double bond caused the biggest increase of the corresponding sterol concentrations when cobalt ions concentrations increased. This increase reached to a maximum at medium cobalt concentrations. The increased concentrations of the 24-methyl-cholesta-5,24(28)-dien-3β-ol at higher cobalt ions concentrations could be due to its function as a precursor of the C-24 alkylated sterols. The decrease of the alkylation, caused by the cobalt ions and especially at the second stage of the alkylation, leading to C29-sterols might be a reason for the sharp increase of the 24-methyl-cholesta-5,24(28)-dien-3β-ol concentrations in the sample treated with the highest cobalt ions concentrations. This proposal is in agreement with the observed significant decrease of the concentrations of the C29-sterols. This indicates an intensive inhibition of the second phase of the C-24 alkylation by cobalt ions, while the first phase of the alkylation is suppressed less significantly and for some sterols there is even a stimulation of the alkylation. There are two possible explanations of the results obtained. One of them is that the presence of cobalt ions inhibited the enzyme systems, responsible for the C-24 alkylation and especially these, realizing the second stage of the alkylation, leading to C29-sterols. Evidently, the inhibition or stimulation of different biosynthetic processes depended not only on the concentrations of the cobalt ions, but also on the structures of

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the sterols. The obtained changes in the sterol concentrations might also be a result from some adaptation processes, leading to a decrease of the harmful effect of the cobalt ions. This assumption is in agreement with the obtained increase of cholesterol and decrease of 24-alkylated sterols concentrations, since it has been known that cholesterol, as a planar sterol, reduces the cell wall permeability, contrary to other less planar sterols, especially 24-methyl-cholest-5-en-3β-ol and 24-ethylcholest-5-en-3β-ol (Kuiper, 1998). 3.2.2. Sterols from A. equina The purified by column and thin layer chromatography sterol fractions from A. equina (control sample and two samples treated with different concentrations of CoCl2, equal to these, used for M. galloprovincialis) were subjected to an analysis by GC and GC/MS and the results obtained summarized in Table 3. Unlike the mussels, A. equina is a predator, which is in agreement with the differences in the sterol compositions between both investigated invertebrates (Table 3). From the Table 3 it is evident that, analogously to M. galloprovincialis, the treatment with cobalt ions led to significant dependence of the sterol composition from the cobalt ions concentrations. Similarly to the changes of the sterol composition of M. galloprovincialis, in A. equina the cobalt ions caused an increase of the cholesterol concentrations, but only at the highest concentrations of cobalt ions. If there was a double bond at C-22 in sterols containing 27 or 26 carbon atoms, their concentrations showed a significant increase only in the samples with medium concentrations of cobalt ions. Such a maximum of concentrations we obtained for almost all identified sterols, present in A. equina. Contrary to M. galloprovincialis now the C-24-alkylation is stimulated at medium cobalt concentrations and is not significantly inhibited even at higher cobalt concentrations. This was the main difference in the two investigated invertebrates. Probably the enzymes responsible for the C-24 alkylation in A. equina sterols were activated at medium cobalt concentrations but were inhibited at higher concentrations. In such a case the stabilizing effect of the cholesterol increase could be compensated by the destabilizing effect of the increased concentrations of 24-alkylated sterols. Probably in the evolutionary less advanced A. equina, the defensive mechanisms, based on the changes of sterol concentrations and leading to a better control on the cell wall permeability, were not sufficiently developed. Since 24-methyl-cholest-5-en-3β-ol is less planar sterol than cholesterol, its decrease should have a stabilizing affect to the cell membrane. The decrease of 24-methyl-cholest-5-en-3β-ol concentration in both invertebrates was probably due to inhibition of its biosynthesis by the Co ions. This inhibition was displayed differently in each organism. In A. equina there was an increase of cholesta-5,24-dien-3β-ol and of 24-methyl-cholesta-5,24(28)dien-3β-ol which is the intermediate precursor of 24-methylcholest-5-en-3β-ol. This fact could be explained with an inhibition of the alkylation of both precursors of 24-methyl-cholest-5-en3β-ol. In M. galloprovincialis the inhibition was displayed only in the second stage of alkylation, since cholesta-5,24-dien-3β-ol was absent in the highest Co ions concentrations, and 24methyl-cholesta-5,24(28)-dien-3β-ol increased significantly.

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4. Conclusions From the results obtained we could assume that the cobalt ions change the metabolism of the components of the lipid cell membranes — phospholipids and sterols. The changes in the metabolism of mussels were more intensive, which could be explained with their advanced evolutionary position — they might react more intensively to the environmental changes, if these reactions have adaptive value. In the mussel M. galloprovincialis the changes in the lipid and sterol metabolism increased with the increase of the cobalt ion concentrations, whereas in A. equina the changes were more intensive at the medium cobalt concentrations. At the highest cobalt concentrations, the lipid and sterol compositions from A. equina showed small changes. These differences might be explained with the evolutionary differences between the two organisms — the more advanced one might possess stronger enzymatic activity and the changes of its metabolism would correspond to the cobalt ions concentrations. In the evolutionary less advanced organism the enzymatic activity probably was weaker and might be significantly inhibited by the highest cobalt ions concentrations and thus, the composition of the lipid cell membranes did not change. Acknowledgements The authors are grateful to the National Council for Scientific Research in Bulgaria for the partial financial support under contract X-1101. References Abele, D., Puntarulo, S., 2004. Formation of reactive species and induction of antioxidant defence systems in polar and temperate marine invertebrates and fish. Comp. Biochem. Physiol., A 38, 405–415. Alexander, C., 1972. Cobalt-beer cardiomyopathy. A clinical and pathologic study of twenty-eight cases. Am. J. Med. 53, 395–417. al-Mafda, H., Abdel-Moati, M.A., al-Gimaly, F.H., 1998. Pinctada radiata (pearl oyster): a bioindicator for metal pollution monitoring in the Qatari waters (Arabian Gulf). Bull. Environ. Contam. Toxicol. 60, 245–251. Amiard, J., 1976. Etude experimentale de la toxicite aigue de sels de cobalt, d'antimoine, de strontium et d'argent chez quelques crustaces et leurs larves et chez quelques teleosteens. Rev. Int. Oceanogr. Med. Tome XLIII, 79–95. Beyersmann, D., Hartwig, A., 1992. The genetic toxicology of cobalt. Toxicol. Appl. Pharmacol. 115, 137–145. Biesinger, K., Christensen, G., 1972. Effects of various metals on survival, growth, reproduction, and metabolism of Daphnia magna. J. Fish. Res. Board Can. 29, 1691–1700. Birkun Jr., A., 2002. Cetacean habitat loss and degradation in the Black Sea. In: Notarbartolo di Sciara, G. (Ed.), Cetaceans of the Mediterranean and Black Seas: State of Knowledge and Conservation Strategies. A report to the ACCOBAMS Secretariat, Monaco, Section, vol. 8, p. 19. Brennan, B., Alms, G., Nelson, M., Durney, L., Scarrow, R., 1996. Nitrile hydratase from Rhodococcus rhodochrous J1 contains a non-corrin cobalt ion with two sulfur ligands. J. Am. Chem. Soc. 118, 9194–9195. Bruland, K., Donat, J., Hutchins, D., 1991. Interactive influences of bioactive trace metals on biological production in oceanic waters. Limnol. Oceanogr. 36, 1555–1577. Carlson, R., Popov, S., Massey, I., Delseth, C., Ayanoglu, E., Varkony, T., Djerassi, C., 1978. Minor and trace sterols in marine invertebrates. VI. Occurrence and possible origins of sterols possessing unusually short hydrocarbon side chains. Bioorganic Chem. 7, 453–479.

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