Chemosphere 119 (2015) 231–241
Contents lists available at ScienceDirect
Chemosphere journal homepage: www.elsevier.com/locate/chemosphere
Polonium-210 and selenium in tissues and tissue extracts of the mussel Mytilus galloprovincialis (Gulf of Trieste) Urška Kristan a,b, Petra Planinšek a,b, Ljudmila Benedik a,b, Ingrid Falnoga a, Vekoslava Stibilj a,b,⇑ a b
Jozˇef Stefan Institute, Jamova 39, SI-1000 Ljubljana, Slovenia Jozˇef Stefan International Postgraduate School, Jamova 39, SI-1000 Ljubljana, Slovenia
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Po-210 and Se are sequestered by
different cell compartments and/or components. The highest extractability with enzyme at 40 °C, 1 h, up to 100% Po and 76% Se. Extractability could be related to essentiality (Se) and non-essentiality (Po). Se should be available in cells, Po-210 stored in insoluble parts in cell vacuoles. The identified selenium species were SeMet, SeCys2 and one unidentified compound.
a r t i c l e
i n f o
Article history: Received 6 March 2014 Received in revised form 7 May 2014 Accepted 9 May 2014
Handling Editor: J. de Boer Keywords: Polonium-210 Selenium Mussels Extraction Speciation
a b s t r a c t Marine organisms such as mussels and fish take up polonium (Po) and selenium (Se), and distribute them into different cellular components and compartments. Due to its high radiotoxicity and possible biomagnification across the marine food chain Po-210 is potentially hazardous, while selenium is an essential trace element for humans and animals. The aim of this study was to investigate and compare the presence and extractability of the elements in the mussels Mytilus galloprovincialis collected in the Gulf of Trieste. The levels of Po-210 in the samples ranged from 220 to 400 Bq kg1 and of Se from 2.6 to 8.2 mg kg1, both on a dry matter basis. Using various extraction types and conditions in water, buffer or enzymatic media, the best extractability was obtained with enzymatic extraction (Protease XIV, 1 h shaking at 40 °C) and the worst by water extraction (24 h shaking at 37 °C). 90% of Po-210 and 70% of Se was extractable in the first case versus less than 10% of Po-210 and less than 40% of Se in the second. Such evident differences in extractability between the investigated elements point to different metabolic pathways of the two elements. In enzymatic extracts Se speciation revealed three Se compounds (SeCys2, SeMet, one undefined), while Po-210 levels were too low to allow any conclusions about speciation. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction ⇑ Corresponding author at: Jozˇef Stefan Institute, Jamova 39, SI-1000 Ljubljana, Slovenia. Tel.: +386 1 5885352; fax: +386 1 5885346. E-mail address: [email protected] (V. Stibilj). http://dx.doi.org/10.1016/j.chemosphere.2014.05.017 0045-6535/Ó 2014 Elsevier Ltd. All rights reserved.
The Mediterranean mussel, Mytilus galloprovincialis (M. galloprovincialis) is one of the most widely used sentinel organisms for monitoring marine pollution due to its ability to accumulate
232
U. Kristan et al. / Chemosphere 119 (2015) 231–241
high levels of different contaminants including radionuclides (Livingstone et al., 2000; Rozˇmaric´ et al., 2012). It is distributed in many coastal and estuarine areas, adapted to a variety of environmental conditions, and being a filter feeder exposed to ambient seawater lives attached to hard substrates. Mussels are used for monitoring for several reasons: firstly, as given above, they have a high capacity to accumulate different pollutants in their tissues over time, secondly they have slow decontamination kinetics, thirdly their dimensions are suitable for manipulation, and lastly they are easy to identify and collect (Deudero et al., 2009). In Europe, M. galloprovincialis is found in the Mediterranean Sea and the Black Sea, and on Atlantic coasts from Portugal, north to France and the British Isles. Po-210 (t1/2 = 138.4 days) is a naturally occurring radionuclide originating from the U-238 decay chain as the daughter of Pb-210 (t1/2 = 22 years), with no known physiological function (Stewart et al., 2008). The high radiotoxicity of Po-210 is a consequence of the combination of its high specific activity and its relatively good uptake into soft tissues, where the 5.3 MeV alpha particles have a high linear energy transfer. Several aspects of its chemical properties and its radiotoxicity were summarised in a recent review by Ansoborlo et al. (2012), where as one of the most important chemical property its tendency to hydrolyse and form colloids of Po(OH)4, in analogy with most tetravalent elements is mentioned. Although naturally present mostly at very low concentrations in all environmental media, in some cases it can represent a serious risk to human health, particularly due to its biological magnification along marine food chains. According to Wildgust et al., ‘the major contribution to the radiation dosage received by humans via food consumption is derived from Po-210 in fish and shellfish’ (Wildgust et al., 1999). Fisher et al. (1983) and Cherrier et al. (1995), reported that marine and freshwater microorganisms (e.g. algae, bacteria) ‘metabolise’ Po-210 by accumulating it in cellular components, mostly associated with sulphur-containing proteins. Similar accumulation take place in higher organisms such as mussels and fish (Dahlgaard, 1996; Carvalho, 2011). In fact polonium accumulation in marine organisms takes place through binding sites on various proteins, such as metallothioneins (cysteine-rich cellular protective proteins), ferritin and a haemocyanin, as well as selenoproteins (selenocysteine-containing proteins) and zinc metalloproteins (Durand et al., 1999, 2002; Wildgust et al., 1999). Despite the mentioned data, there is still a lack of information about the distribution and binding of polonium inside cells since most studies were focused on its general uptake and accumulation in organisms and organs. Selenium (Se) is an essential element for animals and humans whose beneficial role in human and animal health has been known since the 1960’s, but which has only a narrow concentration window between essentiality and toxicity (Reilly, 1998). Its essentiality is related to its presence in selenoproteins in the form of the amino acid selenocysteine (SeCys). SeCys is incorporated at defined and specific positions into a small group of functional selenoproteins (Stoedter et al., 2010) with selenocysteine at their active centre. Selenoproteins are divided into different families regarding their function or their activity site. For instance, they carry out antioxidant or redox reactions such as in the case of the glutathione peroxidases (GPx) and thioredoxin reductase (TrxR), while the thyroid hormone deiodinase family activates or deactivates thyroid hormones (Suzuki, 2005). Selenoprotein P (SelP) is also an important member as the most abundant selenoprotein found in plasma, where it represents more than half of the plasma selenium content (Burk and Hill, 2005). Unlike all other known selenoproteins which have one SeCys amino acid residue, SelP consists of several SeCys residues, and functions as a seleno-carrier, antioxidant and metal binder. Se in the form of a second amino acid selenomethionine (SeMet) is incorporated into regular proteins
randomly in place of its sulphur analogue methionine. In such a way it represents the Se storage pool from which SeCys can be synthesized inside cells. The principal source of selenium for humans is diet. According to the US Recommended Dietary Allowance (RDA) the minimum Se daily requirement for adults is given as 55 and 70 lg/day for females and males, respectively, while the recommended daily intake of Se for adults set by the German, Austrian, and Swiss Nutrition Associations (DACH) is between 30 and 70 lg/day (Reference Values for Nutrition Intake, 2002). Its bioavailability and toxicity have been found to be dependent mostly on the concentration and chemical forms of Se ingested (Finley, 1998). In animal foods it is generally present in organic forms such as the selenoamino acids SeMet and SeCys (Bierla et al., 2008), while in seafood (fish, molluscs and mussels) it has been identified mostly as SeMet (Quijano et al., 2000). Although Po and Se are in the same group of the periodic table and have some similar chemical properties, they have very different biological impacts: the first is believed to be without any beneficial biological role, while the second is essential for cell survival but also toxic at excess levels. It should also be noted that selenoproteins with their selenol groups (–SeH) can represent important binding sites for Po (like thiol groups). However, in the literature data about polonium and selenium interactions are scarce. The aim of the present study was to investigate the presence and behaviour of both elements in specimens of M. galloprovincialis sampled in the Gulf of Trieste (North Adriatic Sea). We used the three groups of samples from our previous study dealing with the presence of different selenium species after enzymatic extraction with Protease XIV (Kristan and Stibilj, 2014). In the present investigation we compared the behaviour of Po-210 and Se after various extraction procedures using water, buffer and enzymatic extractions (the latter time and temperature dependent). Extractions were performed simultaneously for both elements and the content of both followed in tissues and the tissue fractions obtained (extracts and non-extractable parts i.e. pellets). In some cases the same type of fractionation was performed on fresh and cooked mussel samples to examine how a typical Mediterranean cooking procedure (‘alla buzara’) affects the extractability of both elements. Additionally, Se speciation was performed in selected water and enzymatic extracts. 2. Materials and methods 2.1. Sample collection Mussel samples of the species M. galloprovincialis (family Mitylidae, genus Mytilus) of 5 kg each, consisting of mussels with shells, were purchased on the Slovenian market. They originated from the Italian (Mussel 1) and Slovenian (Mussels 2 and 3) coastline along the Gulf of Trieste (the northern part of the Adriatic Sea). They were sampled in March (Mussels 1 and 2) and July (Mussel 3) in 2012. Mussels were mostly from 5 to 7 cm long, except in sample Mussel 3 where smaller representatives from 3–5 cm were separated and designated as Mussel 3a. Sea water (10 L) was sampled at three different locations along the Slovenian coastline in the Gulf of Trieste. One location matched the Slovenian mussel sampling site (Mussels 2 and 3). 2.2. Sample preparation Samples were divided into two equal parts and prepared using two different approaches. Approach A: the fresh (raw) mussels were dissected by removing the byssus and shells and the whole mussel tissue was strained to remove remaining sea water.
U. Kristan et al. / Chemosphere 119 (2015) 231–241
Approach B: mussels were first cleaned under tap water and open ones discarded; the washed mussels were fried in hot oil for a few minutes, then white wine was added (1 L per 400 g of mussels) and cooked until they opened. This typical cooking procedure ‘alla buzara’ is common along the Adriatic coast. Tissues of fresh and cooked mussels were then dried in a freeze dryer (ALPHA 1-4, Osterode am Herz, Germany) at 46 °C to constant weight, homogenized firstly in an agate mill and later in a Pulverisete 7 mill (Fritsch, Idar-Oberstein, Germany) at a rotational speed of 2600 rpm for 5 min. Homogenized samples were stored in polyethylene bottles at 18 °C and later used for Po-210 and Se determination. Po-210 in filtered (
Contents lists available at ScienceDirect
Chemosphere journal homepage: www.elsevier.com/locate/chemosphere
Polonium-210 and selenium in tissues and tissue extracts of the mussel Mytilus galloprovincialis (Gulf of Trieste) Urška Kristan a,b, Petra Planinšek a,b, Ljudmila Benedik a,b, Ingrid Falnoga a, Vekoslava Stibilj a,b,⇑ a b
Jozˇef Stefan Institute, Jamova 39, SI-1000 Ljubljana, Slovenia Jozˇef Stefan International Postgraduate School, Jamova 39, SI-1000 Ljubljana, Slovenia
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Po-210 and Se are sequestered by
different cell compartments and/or components. The highest extractability with enzyme at 40 °C, 1 h, up to 100% Po and 76% Se. Extractability could be related to essentiality (Se) and non-essentiality (Po). Se should be available in cells, Po-210 stored in insoluble parts in cell vacuoles. The identified selenium species were SeMet, SeCys2 and one unidentified compound.
a r t i c l e
i n f o
Article history: Received 6 March 2014 Received in revised form 7 May 2014 Accepted 9 May 2014
Handling Editor: J. de Boer Keywords: Polonium-210 Selenium Mussels Extraction Speciation
a b s t r a c t Marine organisms such as mussels and fish take up polonium (Po) and selenium (Se), and distribute them into different cellular components and compartments. Due to its high radiotoxicity and possible biomagnification across the marine food chain Po-210 is potentially hazardous, while selenium is an essential trace element for humans and animals. The aim of this study was to investigate and compare the presence and extractability of the elements in the mussels Mytilus galloprovincialis collected in the Gulf of Trieste. The levels of Po-210 in the samples ranged from 220 to 400 Bq kg1 and of Se from 2.6 to 8.2 mg kg1, both on a dry matter basis. Using various extraction types and conditions in water, buffer or enzymatic media, the best extractability was obtained with enzymatic extraction (Protease XIV, 1 h shaking at 40 °C) and the worst by water extraction (24 h shaking at 37 °C). 90% of Po-210 and 70% of Se was extractable in the first case versus less than 10% of Po-210 and less than 40% of Se in the second. Such evident differences in extractability between the investigated elements point to different metabolic pathways of the two elements. In enzymatic extracts Se speciation revealed three Se compounds (SeCys2, SeMet, one undefined), while Po-210 levels were too low to allow any conclusions about speciation. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction ⇑ Corresponding author at: Jozˇef Stefan Institute, Jamova 39, SI-1000 Ljubljana, Slovenia. Tel.: +386 1 5885352; fax: +386 1 5885346. E-mail address: [email protected] (V. Stibilj). http://dx.doi.org/10.1016/j.chemosphere.2014.05.017 0045-6535/Ó 2014 Elsevier Ltd. All rights reserved.
The Mediterranean mussel, Mytilus galloprovincialis (M. galloprovincialis) is one of the most widely used sentinel organisms for monitoring marine pollution due to its ability to accumulate
232
U. Kristan et al. / Chemosphere 119 (2015) 231–241
high levels of different contaminants including radionuclides (Livingstone et al., 2000; Rozˇmaric´ et al., 2012). It is distributed in many coastal and estuarine areas, adapted to a variety of environmental conditions, and being a filter feeder exposed to ambient seawater lives attached to hard substrates. Mussels are used for monitoring for several reasons: firstly, as given above, they have a high capacity to accumulate different pollutants in their tissues over time, secondly they have slow decontamination kinetics, thirdly their dimensions are suitable for manipulation, and lastly they are easy to identify and collect (Deudero et al., 2009). In Europe, M. galloprovincialis is found in the Mediterranean Sea and the Black Sea, and on Atlantic coasts from Portugal, north to France and the British Isles. Po-210 (t1/2 = 138.4 days) is a naturally occurring radionuclide originating from the U-238 decay chain as the daughter of Pb-210 (t1/2 = 22 years), with no known physiological function (Stewart et al., 2008). The high radiotoxicity of Po-210 is a consequence of the combination of its high specific activity and its relatively good uptake into soft tissues, where the 5.3 MeV alpha particles have a high linear energy transfer. Several aspects of its chemical properties and its radiotoxicity were summarised in a recent review by Ansoborlo et al. (2012), where as one of the most important chemical property its tendency to hydrolyse and form colloids of Po(OH)4, in analogy with most tetravalent elements is mentioned. Although naturally present mostly at very low concentrations in all environmental media, in some cases it can represent a serious risk to human health, particularly due to its biological magnification along marine food chains. According to Wildgust et al., ‘the major contribution to the radiation dosage received by humans via food consumption is derived from Po-210 in fish and shellfish’ (Wildgust et al., 1999). Fisher et al. (1983) and Cherrier et al. (1995), reported that marine and freshwater microorganisms (e.g. algae, bacteria) ‘metabolise’ Po-210 by accumulating it in cellular components, mostly associated with sulphur-containing proteins. Similar accumulation take place in higher organisms such as mussels and fish (Dahlgaard, 1996; Carvalho, 2011). In fact polonium accumulation in marine organisms takes place through binding sites on various proteins, such as metallothioneins (cysteine-rich cellular protective proteins), ferritin and a haemocyanin, as well as selenoproteins (selenocysteine-containing proteins) and zinc metalloproteins (Durand et al., 1999, 2002; Wildgust et al., 1999). Despite the mentioned data, there is still a lack of information about the distribution and binding of polonium inside cells since most studies were focused on its general uptake and accumulation in organisms and organs. Selenium (Se) is an essential element for animals and humans whose beneficial role in human and animal health has been known since the 1960’s, but which has only a narrow concentration window between essentiality and toxicity (Reilly, 1998). Its essentiality is related to its presence in selenoproteins in the form of the amino acid selenocysteine (SeCys). SeCys is incorporated at defined and specific positions into a small group of functional selenoproteins (Stoedter et al., 2010) with selenocysteine at their active centre. Selenoproteins are divided into different families regarding their function or their activity site. For instance, they carry out antioxidant or redox reactions such as in the case of the glutathione peroxidases (GPx) and thioredoxin reductase (TrxR), while the thyroid hormone deiodinase family activates or deactivates thyroid hormones (Suzuki, 2005). Selenoprotein P (SelP) is also an important member as the most abundant selenoprotein found in plasma, where it represents more than half of the plasma selenium content (Burk and Hill, 2005). Unlike all other known selenoproteins which have one SeCys amino acid residue, SelP consists of several SeCys residues, and functions as a seleno-carrier, antioxidant and metal binder. Se in the form of a second amino acid selenomethionine (SeMet) is incorporated into regular proteins
randomly in place of its sulphur analogue methionine. In such a way it represents the Se storage pool from which SeCys can be synthesized inside cells. The principal source of selenium for humans is diet. According to the US Recommended Dietary Allowance (RDA) the minimum Se daily requirement for adults is given as 55 and 70 lg/day for females and males, respectively, while the recommended daily intake of Se for adults set by the German, Austrian, and Swiss Nutrition Associations (DACH) is between 30 and 70 lg/day (Reference Values for Nutrition Intake, 2002). Its bioavailability and toxicity have been found to be dependent mostly on the concentration and chemical forms of Se ingested (Finley, 1998). In animal foods it is generally present in organic forms such as the selenoamino acids SeMet and SeCys (Bierla et al., 2008), while in seafood (fish, molluscs and mussels) it has been identified mostly as SeMet (Quijano et al., 2000). Although Po and Se are in the same group of the periodic table and have some similar chemical properties, they have very different biological impacts: the first is believed to be without any beneficial biological role, while the second is essential for cell survival but also toxic at excess levels. It should also be noted that selenoproteins with their selenol groups (–SeH) can represent important binding sites for Po (like thiol groups). However, in the literature data about polonium and selenium interactions are scarce. The aim of the present study was to investigate the presence and behaviour of both elements in specimens of M. galloprovincialis sampled in the Gulf of Trieste (North Adriatic Sea). We used the three groups of samples from our previous study dealing with the presence of different selenium species after enzymatic extraction with Protease XIV (Kristan and Stibilj, 2014). In the present investigation we compared the behaviour of Po-210 and Se after various extraction procedures using water, buffer and enzymatic extractions (the latter time and temperature dependent). Extractions were performed simultaneously for both elements and the content of both followed in tissues and the tissue fractions obtained (extracts and non-extractable parts i.e. pellets). In some cases the same type of fractionation was performed on fresh and cooked mussel samples to examine how a typical Mediterranean cooking procedure (‘alla buzara’) affects the extractability of both elements. Additionally, Se speciation was performed in selected water and enzymatic extracts. 2. Materials and methods 2.1. Sample collection Mussel samples of the species M. galloprovincialis (family Mitylidae, genus Mytilus) of 5 kg each, consisting of mussels with shells, were purchased on the Slovenian market. They originated from the Italian (Mussel 1) and Slovenian (Mussels 2 and 3) coastline along the Gulf of Trieste (the northern part of the Adriatic Sea). They were sampled in March (Mussels 1 and 2) and July (Mussel 3) in 2012. Mussels were mostly from 5 to 7 cm long, except in sample Mussel 3 where smaller representatives from 3–5 cm were separated and designated as Mussel 3a. Sea water (10 L) was sampled at three different locations along the Slovenian coastline in the Gulf of Trieste. One location matched the Slovenian mussel sampling site (Mussels 2 and 3). 2.2. Sample preparation Samples were divided into two equal parts and prepared using two different approaches. Approach A: the fresh (raw) mussels were dissected by removing the byssus and shells and the whole mussel tissue was strained to remove remaining sea water.
U. Kristan et al. / Chemosphere 119 (2015) 231–241
Approach B: mussels were first cleaned under tap water and open ones discarded; the washed mussels were fried in hot oil for a few minutes, then white wine was added (1 L per 400 g of mussels) and cooked until they opened. This typical cooking procedure ‘alla buzara’ is common along the Adriatic coast. Tissues of fresh and cooked mussels were then dried in a freeze dryer (ALPHA 1-4, Osterode am Herz, Germany) at 46 °C to constant weight, homogenized firstly in an agate mill and later in a Pulverisete 7 mill (Fritsch, Idar-Oberstein, Germany) at a rotational speed of 2600 rpm for 5 min. Homogenized samples were stored in polyethylene bottles at 18 °C and later used for Po-210 and Se determination. Po-210 in filtered (
